Results

Binding activities of NFκB, NFIL-6 and NFAT from human PBMC to the TNFα, IL-6 and IFNγ promoters following an in vitro stimulation (1 h) with LPS, TSST-1, GP, GP + LPS and GP + TSST-1
GP induced band shifts, indicating binding of NFκB as well as of NFIL-6 to the corresponding DNA oligonucleotides (κ consensus, κ1, κ2, κ3 sites from the TNFα promoter; κ consensus from the IL-8 promoter; NFIL-6consensus from the IL-6 promoter; Table 1; Fig. 1A). The extent of the band shifts induced by GP was not statistically different when compared to LPS or TSST-1 (Fig. 1B). A supershift, conducted for an NFκB consensus site from the IL-8 promoter, demonstrated a GP-induced predominant binding of NFκB p65 and to a lesser degree of p50 (Fig. 1A). Accordingly, an immunoblot of nuclear extracts from GP-treated PBMC showed a strong binding of NFκB p65 and a weaker reaction of p50, whereas p52 was negative (Fig. 2). Simultaneous co-treatment of PBMC with GP did not change the LPS-induced NFκB binding to oligos from the TNFα promoter significantly (Fig. 1B), but substantially decreased the TSST-1-induced NFκB binding when compared to TSST-1 or GP (100 μg) and thus differed completely from the theoretical value of GP/TSST-1 calc. (n = 4; p < 0.05 vs. GP/TSST-1 calc. and p = 0.07 vs. TSST-1; Fig. 1B). GP was also able to induce band shifts indicative of binding of particular variants of NFAT to an oligo from the IFNγ promoter (Fig. 1A). An immunoblot for two NFAT subunits demonstrated a GP-induced binding of NFATc2 and to a lesser degree of NFATc1 (Fig. 2).
Table 1  Sense strand of oligonucleotides used in EMSAs.
Oligonucleotidesa  Base sequences (5'- XXX -3')  binding site/cytokine gene promoter (position of the cytokine gene from the TSS)b
NFκBcS  GAT CCT CAG AGG GGA CTT TCCc GAT G  NFκB consensus sequence/TNFα promoter [27]
NFκB1S  GAT CCT GGG ACA GCC CAG  NFκB1 sequence/TNFα promoter [27]
NFκB2S  GAT CCG GGG TAT CCT G  NFκB2 sequence/TNFα promoter [27]
NFκB3S  GAT CCT GGG TTT CTC CG  NFκB3 sequence/TNFα promoter [27]
IL-8κBcS  ATC GTG GAA TTT CCT CTG A  NFκB consensus sequence/IL-8 promoter [28]
NFATP2S  GAT CTA AAA TTT CCA GTC CTT GA  NFATP2 sequence/IFNγ promoter [29]
NFIL-6S  TGC AGA TTG CGC AAT CTG CA  NFIL-6 consensus sequence/IL-6 promoter [13]
NFκBcS  GCG AGG AGG GTA TTT CCG CTT  between -80 and -100 NFκB consensus/IL-1RA promoter [30]
NFκB3S  ACA ACA GCA AGG GTT TCT CTT TTT GGA AAT  between -100 and -130 NFκB3/IL-1RA promoter
NFκBcS  AGT AGG GAG TTT GGT  between -266 and -280 NFκB consensus sequence/IL-1RA promoter
NFκB2/3S  ACT CTG GGT ACC TGT  between -288 and -302 NFκB2/3 sequence/IL-1RA promoter
NFATP2/3S  GGC GCA CAA AAC CTA AAA TAT TTA CTA TCT  between -471 and -500 NFATP2/3 sequence/IL-1RA promoter
NFIL-6S  TTA CAA CAC TCC ATT GCG ACA CTT AGT GGG  between -140 and -170 NFIL-6 sequence/IL-1RA promoter [31]
Oct-1 DNAS  AAT TGC ATT GCC TGC AGG TCG ACT CTA GAG GAT CCA TGC AAA TGG ATC CCC GGG TAC CGA GCT C  exclusion of unspecific bindings, unrelated DNA, (Amersham Pharmacia)
(a: S = sense; b: TSS = transcription start site;c: ___ = main binding motif).
Figure 1  A, GP led to DNA binding of NFAT, NFκB and NFIL-6. Human PBMC were incubated with medium control or GP (1 and 100 μg) for 1 h at 37°C. Nuclear extracts were incubated with a 32P-labeled NFAT, NFκB, NFIL-6 or NFκB-IL-8 oligonucleotide probe corresponding to the IFNγ, TNFα, IL-6 and IL-8 gene promoters (Table 1). Arrows with the black head indicate migrational location of the NFAT-DNA, NFκB-DNA, NFIL-6-DNA or NFκB-IL-8-DNA complex compared to free probe (no shift). Arrow with the open head indicates a supershift of NFκB-IL-8-DNA-anti-p65 and -p50, respectively. An autoradiogram from a representative experiment is shown (n = 7). B, Decreased DNA binding of NFκB following GP + TSST-1, compared to TSST-1, GP, or TSST-1/GP. Human PBMC were incubated with medium control, LPS, TSST-1, GP (100 μg), GP + TSST-1 and GP + LPS for 1 h at 37°C. An autoradiogram from a representative experiment is shown. Schematic representation of NFκB-DNA binding activity following GP (1 and 100 μg) or TSST-1 treatment (250 ng) as well as simultaneous administration of both compounds and the theoretical value (GP/TSST-1 calc.). Medium control levels were set equal to 1± SEM and significances (* = p < 0.05) are shown with respect to medium control (n = 4).
Figure 2  NFκB p65/p50 and NFATc2/c1 were involved in GP-induced DNA binding. In order to determine whether the DNA binding proteins activated by GP were related to NFκB and NFAT, an immuno(dot) blot was performed. A representative dot blot showing staining of negative and positive controls (recombinant proteins NFκB p52, c-rel; nuclear extracts of PMA-treated Jurkat cells positive for NFκB p50, p65 and for NFATc1, c2) in the upper panel and staining of nuclear extracts of GP-treated PBMC (positive for NFκB p65, p50 and NFAT c2, c1) in the lower panel is shown (n = 3).

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).

Role of NFκB, NFIL-6 and NFAT binding in LPS-, TSST-1-, GP-, GP + LPS- and GP + TSST-1-induced IL-1RA expression by human PBMC
Because GP induced band shifts to NFκB, NFIL-6 and NFAT sites, but no TNFα, IL-6 or IFNγ, we hypothesized that the activated transcription factors might bind to sites in the IL-1RA promoter. We investigated previoulsy described and newly discovered transcription factor binding sites of the IL-1RA promoter (Table 1) using the program OMIGA (v.1.1.3, Oxford Molecular, Oxford, UK). The LPS responsive κB consensus site of LRE-1 (between -84 and -93) [30] displayed band shifts, but no significant alterations of its binding activity, irrespective of the treatment (data not shown). A new κB3 site (-100 and -130) within the LRE-1 [30] showed increased binding induced by TSST-1 (+30% vs. control), but GP co-treatment up-regulated the LPS (+65% vs. control)- and the TSST-1 (+110% vs. control)-induced binding in a superadditive synergistic fashion, when compared to medium control as well as to LPS, TSST-1 or GP or the theoretical values of GP/LPS calc. or GP/TSST-1 calc. (all p < 0.05; Fig. 4). Binding to a new κB consensus site (between -266 and -280) was significantly elevated for GP + TSST-1 (p < 0.05; data not shown). The LPS-, TSST-1- and GP + LPS-induced binding to the new κB2/3 site (-288 and -302) was down-regulated by about 20–35% in comparison with medium control (p < 0.05; data not shown). The NFIL-6 site (-147 and -170) close to LRE-2 [31,32] showed an up-regulation of binding for all compounds and combinations vs. medium control, ranging from 25–110% (p < 0.05). Further, GP + TSST-1 induced an increased binding to the NFIL-6 site in comparison with TSST-1 (1.3fold) or GP (1.6fold; p < 0.05), (data not shown). A new binding site for NFATP2/3 was found more distal of the transcription start site, between -471 and -490, displaying again a substantial increase of binding for all compounds and combinations, when compared to medium control (p < 0.05). In addition, binding to the NFATP2/3 site following both, GP + LPS (1.4fold of GP) and GP + TSST-1 (1.5fold of GP, 1.25fold of TSST-1) was elevated (p < 0.05; Fig. 5), but only in an additive synergistic manner, i.e. the experimental results were not different from the theoretical values (calc.).
Figure 4  GP modulated LPS- and TSST-1-induced binding to a new NFκB3 site in the IL-1RA promoter. Binding was enhanced for GP + LPS and GP + TSST-1, when compared to LPS, TSST-1, GP, GP/LPS calc. or GP/TSST-1 calc., respectively (* = p < 0.05; ** = p < 0.01). Medium control levels were set equal to 1 ± SEM (n = 6). DNA binding was assessed by EMSA (see methods).
Figure 5  GP modulated TSST-1-induced DNA binding to a new NFATP2/3 site in the IL-1RA promoter. Binding was up-regulated for GP + LPS and GP + TSST-1, when compared to GP and, in the latter case, to both GP and TSST-1 (* = p < 0.05). Human PBMC were incubated with medium control, LPS, TSST-1, GP, GP + TSST-1 and GP + LPS for 1 h at 37°C. Schematic representation of the elevated NFAT-DNA binding activity to the site in the IL-1RA promoter. Bars depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are added. Medium control levels were set equal to 1 ± SEM (n = 6). An autoradiogram of a representative experiment is shown in the lower panel (lane 1: water; lane 2: medium control; lane 3: GP; lane 4: LPS; lane 5: TSST-1, lane 6: GP + LPS; lane 7: GP + TSST-1).

GP modulated the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype
In a simplified approach, IL-1β and IL-1RA might represent two sides of a coin, i.e. pro- or anti-inflammatory action. Therefore, we applied the IL-1β/IL-1RA ratio as an indicator of the degree of inflammation. Both, LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA at early time points, i.e. up to 24 h. Comparing LPS vs. GP + LPS, we did not find substantial alterations over a time course of 48 h in the IL-1β/IL-1RA ratio, only a – non-significant – 2.5fold higher ratio at 6 h. With respect to TSST-1 or the theoretical value of GP/TSST-1 calc. vs. GP + TSST-1, we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h of incubation, i.e. a higher production of IL-1RA than of IL-1β (n = 3; both p < 0.05; Fig. 6). A biological relevance of this result was suggested by a decrease of the IL-1 dependent release of IL-2 from murine EL-4 cells following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail, n = 4; p < 0.07).
Figure 6  Decreased IL-1β/IL-1RA ratio following GP + TSST-1 when compared to TSST-1. Time course (48 h) of the IL-1β/IL-1RA ratio following incubation of human PBMC with LPS, TSST-1, GP + LPS, GP/LPS calc., GP + TSST-1 or GP/TSST-1 calc. LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA up to 24 h. When comparing LPS or GP/LPS calc. vs GP + LPS we did not find significant alterations in the IL-1β/IL-1RA ratio over 48 h. With respect to TSST-1 or GP/TSST-1 calc. vs GP + TSST-1 we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h, i.e. a higher production of IL-1RA than of IL-1β (* = p < 0.05). Data are shown as mean ± SEM (n = 3).

Inhibition of transcription factors led to reduced IL-1RA levels
Pharmacological inhibitors were used to demonstrate that the transcription factor sites within the IL-1RA promoter are relevant for the induction of IL-1RA by GP. As expected, an inhibition of NFκB via CAPE [35] and CyA, NFAT via CyA and NFIL-6 via CHX [36] could not be overruled by GP, leading to a down-regulated binding activity of the transcription factors to the IL-1RA sites, and a reduction of IL-1RA mRNA and IL-1RA protein levels (Fig. 7). The extent of reduction in NFATP2/3 binding is exemplarily shown in an autoradiogram (Fig. 7A, left side) and graphically summarized (n = 4, Fig. 7A, right side). The results for the NFκB and NFIL-6 binding sites were similar (data not shown). Because the highest IL-1RA amount was found at 24 h, mRNA was examined following 1 h inhibition and 18 h of GP. A representative gel demonstrating a decrease in IL-1RA mRNA after inhibition with CAPE, CyA and CHX is displayed in Fig. 7B (n = 4). In addition, corresponding IL-1RA protein levels after 24 h of GP are shown in Fig. 7C (n = 3). Altogether, NFκB inhibition by CAPE was more pronounced at the binding activity and the transcription, whereas CyA and CHX mainly led to a decreased IL-1RA release.
Figure 7  Inhibition of GP-induced NFκB, NFAT and NFIL-6 DNA binding to the IL-1RA promoter. A, A representative autoradiogram (lane 1: control, lane 2: GP, lane 3: GP + unlabeled mutated NFATP2/3 oligo, lane 4: GP + CAPE, lane 5: GP + CyA, lane 6: GP + CHX, lane 7: GP + unlabeled NFATP2/3 oligo, lane 8: water) as well as a graphical summary for the NFATP2/3 site, depicting a decrease in binding following inhibition when compared to GP is displayed (* = p < 0.05). Data are shown as mean ± SEM (n = 4). B, Inhibition of NFκB, NFAT and NFIL-6 resulted in decreased IL-1RA mRNA following CHX, CAPE or CyA compared to GP. GAPDH is used as housekeeping control (representative gel, n = 4). C, Inhibition of NFκB, NFAT and NFIL-6 significantly reduced the production of IL-1RA by human PBMC following treatment with CHX, CAPE or CyA when compared to GP (* = p < 0.05; ** = p < 0.01). Data are shown as mean ± SEM (n = 3).