Hydrogen peroxide produced by Aplysia ink toxin kills tumor cells independent of apoptosis via peroxiredoxin I sensitive pathways.

Marine snails of the genus Aplysia possess numerous bioactive substances. We have purified a 60 kDa protein, APIT (Aplysia punctata ink toxin), from the defensive ink of A. punctata that triggers cell death with profound tumor specificity. Tumor cell death induced by APIT is independent of apoptosis but is characterized by the rapid loss of metabolic activity, membrane permeabilization, and shrinkage of nuclei. Proteome analysis of APIT-treated tumor cells indicated a modification of peroxiredoxin I, a cytoplasmic peroxidase involved in the detoxification of peroxides. Interestingly, knockdown of peroxiredoxin I expression by RNA interference sensitized cells for APIT-induced cell death. APIT induced the death of tumor cells via the enzymatic production of H2O2 and catalase completely blocked APITs' activity. Our data suggest that H2O2 induced stress and the modulation of peroxiredoxins might be a promising approach for tumor therapy.

In vitro IgE but not IgG production of canine peripheral blood B cells is inhibited by CD40 ligation.

The aim of this study was to investigate in vitro IgE induction in peripheral canine B cells. CD21(+) B cells were purified from the peripheral blood of beagle dogs by positive selection via magnetic separation to a purity of >/=95%. Subsequently, proliferation, and IgG and IgE production of canine B cells were investigated after stimulation with human recombinant Interleukin-4 (hrIL-4) and human recombinant Interleukin-2 (hrIL-2) in the presence or absence of CD40L-CD8 fusion protein (CD40L) of mouse origin. We could demonstrate that canine B cells react on hrIL-2 alone by proliferation and IgG production but not by IgE secretion, whereas activation with hrIL-4 induced proliferation and mainly IgE production. Together, both cytokines synergistically increased B cell proliferation as well as IgG and IgE production. We could also show that mouse CD40L induces proliferation of dog B cells, which is further enhanced by addition of hrIL-4. Unexpectedly, CD40L led to a dramatic decrease in the IL-4 mediated IgE secretion (82% inhibition on an average). In contrast, IgG production was not affected significantly by CD40L. The same effects of CD40L were observed when B cells were stimulated by a combination of IL-2 and IL-4 and this inhibition could not be abrogated by increasing the amounts of IL-4. In summary, activation of canine B cells from peripheral blood by hrIL-4 in the presence or absence of hrIL-2 led to marked IgE production that is strongly and in a dose-dependent manner inhibited by CD40L. Stimulation of IgG production is not influenced by CD40L.

Th1 development of naive CD4+ T cells is inhibited by co-activation with anti-CD4 monoclonal antibodies.

Co-activation of CD4+ T cells by anti-CD4 mAb strongly enhances the proliferation of these T cells. We have analyzed the influence of CD4-mediated co-activation on Th cell differentiation. Our data demonstrate that activation of high density (HD)-CD4+ T cells by immobilized anti-CD3 mAb in combination with immobilized anti-CD4 mAb led to a strong inhibition of Th1 differentiation and to a variable but significant induction of Th2 development. Priming of highly enriched Mel-14highCD4+ T cells in the presence of anti-CD4 mAb also resulted in a pronounced suppression of secondary IFN-gamma production, indicating that the Th1 development of naive CD4+ T cells is inhibited by co-activation via CD4. In contrast to HD-CD4+ T cells, CD4-induced costimulation of MEL-14highCD4+ T cells did not result in a primary and secondary IL-4 production. Hence, these results suggest that a MEL-14low population within the HD-CD4+ T cell fraction was the source of the endogenous IL-4 and imply, in addition, that co-activation via CD4 inhibits the development of Th1 cells from naive CD4+ T cells independently from endogenous IL-4. This assumption is further corroborated by the fact that neither the application of neutralizing anti-IL-4 mAb nor the use of T cells from IL-4 knockout mice could prevent CD4-mediated inhibition of Th1 development. The Th1-inhibiting effect of anti-CD4 mAb could not be reversed by the application of the Th1 inducer IL-12. On the contrary, the secondary IL-4 production of HD-CD4+ T cells as an indicator of Th2 differentiation, which was promoted by anti-CD4 mAb, was enhanced even in the presence of IL-12. Therefore, our results suggest that co-activation of naive CD4+ T cells by anti-CD4 mAb directly and selectively inhibits Th1 differentiation of naive dense CD4+ T cells.

Co-development of naive CD4+ cells towards T helper type 1 or T helper type 2 cells induced by a combination of IL-12 and IL-4.

Cytokines were found to play a key role in Th cell differentiation. Among them IL-12 was shown to be a potent differentiation factor for Th1 cells, whereas IL-4 is the only known cytokine that promotes the development of Th2 cells. Upon addition of comparable amounts of IL-4 and IL-12 to a primary culture of naive CD4+ T cells activated by immobilized anti-CD3 mAb, it was found that the Th1-inducing capacity of IL-12 is dominated by the Th2-promoting effect of IL-4. However, high amounts of IL-12 (10,000 U/ml) in combination with low amounts of IL-4 (100 U/ml) led to the development of a Th cell population that, upon rechallenge, showed a substantial secondary IFN-gamma (Th1 cytokine) production concomitantly with the production of high amounts of IL-4 (Th2 cytokine). This can be due to the coexistence of Th1 and Th2 cells or to the development of Th0 cells producing a mixed pattern of cytokines. Immunofluorescence double staining of intracellular IL-4 and IFN-gamma in combination with flow cytometry (FACS) revealed that most of the emerging Th cells produced either IL-4 or IFN-gamma. Only a few double producers could be detected. This finding indicates that individual naive CD4+ T cells can differentiate under the same conditions towards Th1 or Th2 cells and implicates that the development of Th1 and Th2 cells is not necessarily mutually exclusive.

Opposing effects of TGF-beta 2 on the Th1 cell development of naive CD4+ T cells isolated from different mouse strains.

The development of naive dense CD4+ T cells from different mouse strains toward Th1 cells, as monitored by measuring secondary IFN-gamma production, was affected by TGF-beta 2 in a differential way. Th1 cell development of naive CD4+ T cells from strains C57Bl/6, BALB/c, and NMRI primed by immobilized anti-CD3 mAb was strongly inhibited in the presence of TGF-beta 2. Even when the Th1 cell-inducer IL-12 was added, the same effect of TGF-beta 2 was observed. In contrast, Th1 development was substantially promoted by TGF-beta 2 with T cells from C3H/He and CBA/J mice. Further analyses using CD4+ T cells from (C57Bl/6xCBA/J)F1 hybrids or DBA/1 mice showed that Th1 development was inhibited by TGF-beta 2 if the T cells were activated by anti-CD3 mAb, but it was enhanced upon costimulation with anti-CD28 mAb. Determination of primary IL-2 production revealed that T cells from (C57Bl/6xCBA/J)F1 and DBA/1 mice produced low amounts of IL-2 following stimulation by anti-CD3 mAb alone and comparatively high amounts after coactivation by anti-CD28 mAb. In the presence of TGF-beta 2, the production of IL-2 was completely suppressed if such T cells were activated solely by anti-CD3 mAb, but it was only partially inhibited after costimulation by anti-CD28 mAb. Furthermore, TGF-beta 2-promoted Th1 development of such T cells was strongly inhibited after neutralization of endogenously produced IL-2 and completely restored by the addition of human IL-2. Thus, our results indicate that the TGF-beta 2-mediated stimulation of Th1 cell development requires the presence of relatively high concentrations of IL-2. Therefore, the opposing effect of TGF-beta 2 on the Th1 cell development of naive CD4+ T cells from different mouse strains appears to be the result of the variable potency of the respective CD4+ T cells to produce IL-2 in the presence of TGF-beta 2.

Co-activation of naive CD4+ T cells and bone marrow-derived mast cells results in the development of Th2 cells.

Activation of naive dense CD4+ T cells by plate-bound anti-CD3 antibodies favors the development of Th1 cells which, upon re-stimulation, produce significant amounts of IFN-gamma but no IL-4. However, co-activation of such naive T cells in the presence of IgE [anti-dinitrophenyl (DNP)]-loaded bone marrow-derived mast cells (BMMC) on plates coated with anti-CD3 antibodies and DNP-BSA led to the development of IL-4-producing Th2 cells. The same result could be observed if irradiated (800 rad) BMMC were applied as co-stimulators. Moreover, BMMC could be replaced by the supernatant of IgE-activated BMMC suggesting that a soluble mediator, presumably IL-4, was responsible for this effect. This assumption was substantiated using neutralizing anti-IL-4 antibodies which abolished the BMMC-mediated Th2 development in all cases. Addition of IL-12, a cytokine that was shown to antagonize the Th2-promoting effect of IL-4 in vivo, could not inhibit the development of IL-4-producing T cells, but gave rise to a T cell population which produced relatively high amounts of IL-4 and IFN-gamma. Since BMMC represent the in vitro equivalent of mucosal mast cells these data suggest that IgE-activated mucosal mast cells can bias an emerging T cell dependent immune response towards a Th2 dominated reaction by the initial production of IL-4.

IL-9 production of naive CD4+ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-beta and IL-4, and is inhibited by IFN-gamma.

Dense CD4+ T cells isolated from naive mice produce only trace amounts of IL-9 when stimulated by immobilized anti-CD3 in combination with anti-CD28 Abs. In this situation, IL-9 production is significantly stimulated by TGF-beta and further enhanced by the addition of IL-4, which, by itself, has only a minimal influence. IFN-gamma was found to inhibit the enhancing effect of IL-4. However, increasing amounts of IL-4 in the presence of a constant concentration of IFN-gamma could overcome the inhibitory activity of IFN-gamma. The application of CD4+ T cells isolated from IL-2 knockout mice unequivocally revealed that IL-2 is essential for the production of IL-9 by T cells. In addition, the use of T cells from IL-4 knockout mice elucidated that the basic (IL-2 + TGF-beta) mediated IL-9 production is independent of IL-4. Therefore, our results demonstrate that optimal IL-9 production of naive dense CD4+ T cells is positively regulated at different levels: 1) by IL-2, which is essential for IL-9 secretion; 2) followed by TGF-beta, which promotes a considerable increase in IL-9 production above the level induced by IL-2; and 3) finally, by IL-4, which requires the presence of IL-2 and TGF-beta to strongly enhance the production of IL-9. IFN-gamma inhibits the production of IL-9 mainly at the level of IL-4 by neutralizing the effect of this cytokine.

T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-gamma and is inhibited by transforming growth factor-beta.

It was observed in vitro and in vivo that both interferon (IFN)-gamma and interleukin (IL)-12 can promote the development of T helper type 1 (TH1) cells. Since IL-12 was shown to be a costimulator for the production of IFN-gamma by T or natural killer (NK) cells, IL-12 might play only an indirect role in TH1 differentiation by providing IFN-gamma which represents the essential differentiation factor. Using anti-CD3 monoclonal antibody (mAb) for activation of naive CD4+ T cells in the absence of accessory cells we could demonstrate that costimulation by IFN-gamma alone results only in marginal TH1 development. Similarly, IL-12 in the absence of IFN-gamma is only a poor costimulator for inducing differentiation towards the TH1 phenotype. Our data indicate that both cytokines are required to allow optimal TH1 development and that IL-12 has a dual role, it promotes differentiation by direct costimulation of the T cells and also enhances the production of IFN-gamma which serves as a second costimulator by an autocrine mechanism. Another cytokine that was reported to favor TH1 differentiation in certain experimental systems is transforming growth factor (TGF)-beta. With naive CD4+ T cells employed in this study TGF-beta strongly inhibited the production of IFN-gamma triggered by IL-12 as well as the IL-12-induced TH1 development. When TGF-beta was combined with anti-IFN-gamma mAb for neutralization of endogenous IFN-gamma the TH1-inducing capacity of IL-12 was completely suppressed.