Cytokine profile of human PBMC following a 48 h in vitro stimulation with LPS, TSST-1, GP, GP + LPS and GP + TSST-1
Because of the GP-induced band shifts to seven sites from the TNFα, IL-8, IL-6 and IFNγ promoters, it could be assumed that GP treatment of human PBMC would lead to production of TNFα, IL-8, IL-6, IFNγ and other pro-inflammatory mediators. Previous studies reported that β-1→3-D-glucans induced only a limited cytokine secretion of human blood cells [15-18]. Corroborating and extending these singular findings in terms of examining pro- (eight) as well as anti- (three) inflammatory cytokines over time (48 h) and six additional transcription factor binding sites, our comprehensive analysis revealed the following cytokine profile of human leukocytes in response to a highly purified water soluble β-1→3-D-glucan and in comparison to two pro-inflammatory mediators (LPS, TSST-1):

(i) IL-1β
There was no IL-1β production detectable following PBMC treatment with 1 nor 100 μg GP. An insignificant up-regulation of IL-1β production for GP + LPS was observed in comparison with LPS. For GP + TSST-1 we found a significant reduction in IL-1β from 18 h – 24 h, for the latter by about 40% when compared to TSST-1 or the theoretical value of GP/TSST-1 calc. (n = 6; both p = 0.01). On the whole, GP mediated a reduction of the TSST-1-induced amount of IL-1β by about 50% (Fig. 3A).
Figure 3  A, IL-1β was down-regulated (12 h – 24 h) following GP + TSST-1 when compared to TSST-1. Time course (48 h) of IL-1β production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS. At 18 h and 24 h there was a marked reduction in IL-1β production following GP + TSST-1 when compared to TSST-1 (* = p < 0.05). Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are also shown. IL-1β levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3). B, IL-1RA was exaggerated following GP + TSST-1 (12 h – 48 h) when compared to TSST-1. Time course (48 h) of IL-1RA production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS. Between 18 h and 48 h a simultaneous treatment of PBMC with GP + TSST-1 led to a synergistic effect, i.e. a higher IL-1RA production when compared to an addition of the single values for TSST-1 and GP (* = p < 0.05). Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are included. IL-1RA levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).

(ii) IL-6
GP induced only a small amount of IL-6 and there was no significant alteration of the LPS-induced IL-6 production by GP. On the contrary, we observed a decreased IL-6 production for GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc., especially at 24 h by about 40% (n = 5; both p = 0.02), (data not shown in detail).

(iii) IL-8
We found that GP induced a substantial IL-8 production, in comparison with medium control, especially at 24 h (n = 13; p < 0.01). A combination of GP and LPS resulted in a non-significant increase of IL-8 production when compared to LPS or GP/LPS calc. There was no significant alteration in IL-8 production following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail).

(iv) IL-1RA
Besides IL-8, IL-1RA was the only mediator which was produced in significant quantities following GP treatment, especially at 24 h (n = 14, p < 0.01). When compared to LPS stimulation or GP/LPS calc., GP + LPS did not alter the kinetic course of the IL-1RA production. However, following GP + TSST-1 we found a synergistic increase in IL-1RA production from 18 h to 48 h, when compared to TSST-1 (for instance at 24 h: n = 6; p = 0.01) or to the theoretical value of GP/TSST-1 calc. (from 18 h to 36 h). Over the time course of 48 h, GP elevated the TSST-1-induced amount of IL-1RA by approximately 200% (Fig. 3B).
Positive correlation between GP-induced IL-8 and IL-1RA productions. Following stimulation with GP (24 h), we observed a positive correlation between IL-8 and IL-1RA. Moreover, we found this correlation to be dose-dependent, since 100 μg of GP induced larger amounts of IL-8 and IL-1RA than 1 μg GP (n = 5, p ≤ 0.002, r = 0.9 and 0.98, respectively).

(v) TNFα
With respect to TNFα production, 100 μg of GP yielded minor, statistically not distinguishable amounts, when compared to medium control. GP + LPS did not change the TNFα production when compared to LPS supplementation or GP/LPS calc. When combined with TSST-1 or GP/TSST-1 calc., GP seemed to exert a synergistic effect on TNFα secretion after 36 h (n = 3; p < 0.05), but overall an enhancement of only 10% was observed (p > 0.05; data not shown in detail).

(vi) IFNγ
No IFNγ production was detectable following treatment of PBMC with 100 μg GP. There was a minor increase in IFNγ production following GP + LPS at 36 h, when compared to LPS or GP/LPS calc., and a slight down-regulation following GP + TSST-1 at 24 h and 36 h, when compared to TSST-1 or GP/TSST-1 calc. (data not shown).

(vii) IL-2, IL-12, IL-4, IL-10, TGFβ1
The production of IL-2, IL-12, IL-4, IL-10 and TGFβ 1 was not induced by GP. There were also no significant differences between medium control, LPS, TSST-1, GP, GP + LPS, GP/LPS calc. and GP + TSST-1 or GP/TSST-1 calc. over 48 h (data not shown).