Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Figure 1. Induction of Th1/Th2 cytokines and cytolytic activities on the PBMC stimulated with OK-PSA

OK-PSA (0 to 1.0 mg/ml) was added into the PBMC culture. Twenty-four h later, cytokines in the supernatants and NK, LAK activities of the PBMC were measured. As shown in Fig.1A and B, The production of IFN-g, TNF-a, and -b (Th1 cytokines) as well as IL-12 (Th1-inducer) by PBMC was significantly increased addition of OK-PSA. IL-2 was not detected in the supernatant derived from the PBMC culture stimulated with OK-PSA. In Th2 cytokines, OK-PSA treatment accelerated only IL-6 production, but not IL-4 or IL-10 (Fig. 2C). Furthermore, the treatment with OK-PSA significantly accelerated both NK and LAK activities of PBMC in a dose dependent manner as compared with untreated controls (Fig. 1D).

Figure 2. Semiquantitative RT-PCR for cytokines

We concerned the time course of the expression of mRNAs for cytokines using semiquantitative RT-PCR method. In all of the cytokines tested, mRNA expression were increased within 6 h after OK-PSA stimulation. The expression of the mRNAs encoding IL-2, IL-10, IL-12p35 and p40 was decreased over 24 h after OK-PSA stimulation, whereas mRNA expression for IFN-g, TNF-a, -b and IL-6 showed almost same levels from 6 to 48 h after stimulation (Fig. 2A, B and C). In additioon, mRNA expression of Fas ligand was also enhanced from 12 h after stimulation (Fig. 2D). The expression of GAPDH mRNA, used as an internal control, was approximately the same in all times after stimulation.

Figure 3. Th1 cytokine secretion by each fraction of human PBMC stimulated with OK-PSA

To clarify the subpopulations which elicits cytokine-inducing activity after the stimulation of OK-PSA, PBMC were fractionated to monocytes, CD4+T, CD8+T and NK cells. Isolated monocytes, CD4+T, CD8+T and NK cells were 89, 94, 94 and 96% pure, respectively, after subsequent analysis by FACS. Cells (1 x 105) of each fraction separated were cultured for 24 h with OK-PSA (0.1 mg/ml), then the supernatants were assayed for cytokines. Although NK cells stimulated by OK-PSA produced IFN-g both in the presence and in the absence of monocytes, the secretion of IFN-g by CD4+ and CD8+T cells was limited in the presence of monocytes (Fig. 3A and B). TNF-a were produced by monocytes or by lymphocytes cocultured with monocytes (Fig. 3C). Although TNF-b were secreted by T and NK cells, TNF-b secretion by T cells was accelerated in the presence of monocytes (Fig. 3D).

Figure 4. IL-10 induction of OK-432 and M-protein isolated from Streptococcus pyogenes on human PBMC

As described above, only few amounts of IL-10 were induced by OK-PSA (Fig. 1C). As shown in Fig. 4A, PBMC treated with OK-432 for 24 h secreted large amounts of IL-10. Moreover, streptococcal M-protein, one of the components of OK-432, is reportedly suggesed to play an important role for cytokine induction. We hypothesized that the molecule which plays a main role on IL-10 inducing activity of OK-432, may be streptococcal M-protein but not OK-PSA, and examined IL-10 inducing activity of streptococcal M-protein. Data were shown in Fig. 4B. Streptococcal M-protein demonstrated significant IL-10 inducing activity on PBMC as compared with untreated control.

To evaluate the effect of IL-6 and IL-10 on IFN-g production and cytolytic activities of PBMC in vitro, we added anti-IL-6 and anti-IL-10 neutralizing antibodies as well as rIL-10 into the PBMC culture in the presence or absence of OK-PSA, then IFN-g in the supernatants and NK, LAK activities of PBMC were analysed. Addition of rIL-10 significantly inhibited IFN-g-inducing activity of OK-PSA (Fig. 5A). Recombinant IL-10 as well as anti-IL-6 antibody significantly decreased both NK and LAK activities augmented by the treatment with OK-PSA as compared with respective controls. Furthermore, NK and LAK activities of the PBMC stimulated with OK-PSA were slightly but significantly increased by addition of anti-IL-10 neutralizing antibodies (Fig. 5B and C).

Figure 5. Effect of anti-IL-6 and anti-IL-10 neutralizing antibodies as well as rIL-10 on IFN-g, NK and LAK activities induced by OK-PSA

Back to the top.