BACKGROUND
β-1→3-d-glucans occur as a principal component of microbial cell walls or can be secreted from both, non-pathogenic and pathogenic fungi such as s. cerevisae and c. albicans  <cit> .
these β-1→3-d-linked glucose polymers are characterized as a fungal pathogen-associated molecular pattern   <cit> .
the primary cellular recognition of β-1→3-d-glucans is mediated by several β-1→3-d-glucan receptors on phagocytes  <cit>  and other cells  <cit> .
human as well as murine dectin- <dig> has been demonstrated to be the major pattern recognition receptor  for intact yeast and β-1→3-d-glucan-containing particles  on monocytes/macrophages as well as neutrophils and on primary cells  <cit> .
in the murine system, binding of zymosan to dectin- <dig> resulted in production of tnfα through toll-like receptor  <dig> and the adaptor protein myd <dig>  <cit> .
another water-soluble β-1→3-d-glucan  has been described to activate nfκb and nfil- <dig> in murine cell lines  <cit> .
similarly, it has been shown that β-1→3-d-glucans activate nfκb in a human monocyte-like cell line  <cit>  and in human polymorphonuclear neutrophils , in the latter case without secretion of pro-inflammatory cytokines   <cit> .
one study proposed that the production of the anti-inflammatory il-1ra, but not il- <dig> by human monocytes may be a potentially protective mechanism induced by β-1→3-d-glucan  <cit> .
three other investigations have reported that human leukocytes and human vascular endothelial cells produce il- <dig> in response to zymosan  <cit>  or a water-soluble β-1→3-d-glucan  <cit> .
in addition, β-1→3-d-glucans seem to be able to modify the response to pro-inflammatory stimuli or even sepsis.
in a murine polymicrobial sepsis model, β-1→3-d-glucan  <cit>  treatment resulted in decreased septic morbidity and mortality mediated via inhibition of nfκb and stimulation of the phosphoinositide-3-kinase  pathway  <cit> .
these and other animal studies  <cit>  as well as a clinical trial  <cit>  support a protective role of β-1→3-d-glucan in certain pro-inflammatory conditions.
the mechanisms underlying these beneficial effects of β-1→3-d-glucan are only partially resolved, especially in humans.
thus, the aim of this study was to elucidate molecular and cellular mechanisms of β-1→3-d-glucans on human leukocytes in pro-inflammatory conditions with special emphasis on the cytokine profile and its transcriptional regulation.
for this purpose, peripheral blood mononuclear cells  were exposed to a well-defined β-1→3-d-glucan, i.e.
glucan phosphate   <cit> , alone or simultaneously with lps from gram-negative bacteria or the superantigen tsst- <dig> from gram-positive bacteria over  <dig> h. because of the potential effect of β-1→3-d-glucan on cytokine production  <cit> , tnfα, il-1β, il- <dig>  il- <dig> and il-1ra were measured as well as ifnγ, il- <dig>  il- <dig>  il- <dig>  il- <dig> and tgfβ <dig>  correspondingly, four nfκb sites from the tnfα promoter   <cit> , a κ consensus site from the il- <dig> promoter  <cit> , an nfat site from the ifnγ promoter   <cit>  and a consensus nfil- <dig> site from the il- <dig> promoter  <cit>  were examined.
because of the anti-inflammatory role of il-1ra, we focused on binding of transcription factors to the il-1ra promoter.
an inhibitory element and three positive-acting lps-response elements  in the il-1ra promoter, including nfκb, pu.
<dig> and nfil- <dig> sites, have been characterized previously  <cit> .
using computational analysis for homology search  <cit> , we looked for new binding motifs in the il-1ra promoter.
RESULTS
binding activities of nfκb, nfil- <dig> and nfat from human pbmc to the tnfα, il- <dig> and ifnγ promoters following an in vitro stimulation  with lps, tsst- <dig>  gp, gp + lps and gp + tsst-1
gp induced band shifts, indicating binding of nfκb as well as of nfil- <dig> to the corresponding dna oligonucleotides .
the extent of the band shifts induced by gp was not statistically different when compared to lps or tsst- <dig> .
a supershift, conducted for an nfκb consensus site from the il- <dig> promoter, demonstrated a gp-induced predominant binding of nfκb p <dig> and to a lesser degree of p <dig> .
accordingly, an immunoblot of nuclear extracts from gp-treated pbmc showed a strong binding of nfκb p <dig> and a weaker reaction of p <dig>  whereas p <dig> was negative .
simultaneous co-treatment of pbmc with gp did not change the lps-induced nfκb binding to oligos from the tnfα promoter significantly , but substantially decreased the tsst-1-induced nfκb binding when compared to tsst- <dig> or gp  and thus differed completely from the theoretical value of gp/tsst- <dig> calc.
.
gp was also able to induce band shifts indicative of binding of particular variants of nfat to an oligo from the ifnγ promoter .
an immunoblot for two nfat subunits demonstrated a gp-induced binding of nfatc <dig> and to a lesser degree of nfatc <dig> .
.
cytokine profile of human pbmc following a  <dig> h in vitro stimulation with lps, tsst- <dig>  gp, gp + lps and gp + tsst-1
because of the gp-induced band shifts to seven sites from the tnfα, il- <dig>  il- <dig> and ifnγ promoters, it could be assumed that gp treatment of human pbmc would lead to production of tnfα, il- <dig>  il- <dig>  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  <cit> .
corroborating and extending these singular findings in terms of examining pro-  as well as anti-  inflammatory cytokines over time  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 :
il-1β
there was no il-1β production detectable following pbmc treatment with  <dig> nor  <dig> μg gp.
an insignificant up-regulation of il-1β production for gp + lps was observed in comparison with lps.
for gp + tsst- <dig> we found a significant reduction in il-1β from  <dig> h –  <dig> h, for the latter by about 40% when compared to tsst- <dig> or the theoretical value of gp/tsst- <dig> calc.
.
on the whole, gp mediated a reduction of the tsst-1-induced amount of il-1β by about 50% .
il-6
gp induced only a small amount of il- <dig> and there was no significant alteration of the lps-induced il- <dig> production by gp.
on the contrary, we observed a decreased il- <dig> production for gp + tsst- <dig> when compared to tsst- <dig> or gp/tsst- <dig> calc., especially at  <dig> h by about 40% , .
il-8
we found that gp induced a substantial il- <dig> production, in comparison with medium control, especially at  <dig> h .
a combination of gp and lps resulted in a non-significant increase of il- <dig> production when compared to lps or gp/lps calc.
there was no significant alteration in il- <dig> production following gp + tsst- <dig> when compared to tsst- <dig> or gp/tsst- <dig> calc.
.
il-1ra
besides il- <dig>  il-1ra was the only mediator which was produced in significant quantities following gp treatment, especially at  <dig> h .
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- <dig> we found a synergistic increase in il-1ra production from  <dig> h to  <dig> h, when compared to tsst- <dig>  or to the theoretical value of gp/tsst- <dig> calc.
.
over the time course of  <dig> h, gp elevated the tsst-1-induced amount of il-1ra by approximately 200% .
positive correlation between gp-induced il- <dig> and il-1ra productions.
following stimulation with gp , we observed a positive correlation between il- <dig> and il-1ra.
moreover, we found this correlation to be dose-dependent, since  <dig> μg of gp induced larger amounts of il- <dig> and il-1ra than  <dig> μg gp .
tnfα
with respect to tnfα production,  <dig> 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- <dig> or gp/tsst- <dig> calc., gp seemed to exert a synergistic effect on tnfα secretion after  <dig> h , but overall an enhancement of only 10% was observed .
ifnγ
no ifnγ production was detectable following treatment of pbmc with  <dig> μg gp.
there was a minor increase in ifnγ production following gp + lps at  <dig> h, when compared to lps or gp/lps calc., and a slight down-regulation following gp + tsst- <dig> at  <dig> h and  <dig> h, when compared to tsst- <dig> or gp/tsst- <dig> calc.
.
il- <dig>  il- <dig>  il- <dig>  il- <dig>  tgfβ1
the production of il- <dig>  il- <dig>  il- <dig>  il- <dig> and tgfβ  <dig> was not induced by gp.
there were also no significant differences between medium control, lps, tsst- <dig>  gp, gp + lps, gp/lps calc.
and gp + tsst- <dig> or gp/tsst- <dig> calc.
over  <dig> h .
role of nfκb, nfil- <dig> 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- <dig> and nfat sites, but no tnfα, il- <dig> 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  using the program omiga .
the lps responsive κb consensus site of lre- <dig>   <cit>  displayed band shifts, but no significant alterations of its binding activity, irrespective of the treatment .
a new κb <dig> site  within the lre- <dig>  <cit>  showed increased binding induced by tsst- <dig> , but gp co-treatment up-regulated the lps - and the tsst- <dig> -induced binding in a superadditive synergistic fashion, when compared to medium control as well as to lps, tsst- <dig> or gp or the theoretical values of gp/lps calc.
or gp/tsst- <dig> calc.
.
binding to a new κb consensus site  was significantly elevated for gp + tsst- <dig> .
the lps-, tsst-1- and gp + lps-induced binding to the new κb2/ <dig> site  was down-regulated by about 20–35% in comparison with medium control .
the nfil- <dig> site  close to lre- <dig>  <cit>  showed an up-regulation of binding for all compounds and combinations vs. medium control, ranging from 25–110% .
further, gp + tsst- <dig> induced an increased binding to the nfil- <dig> site in comparison with tsst- <dig>  or gp , .
a new binding site for nfatp2/ <dig> was found more distal of the transcription start site, between - <dig> and - <dig>  displaying again a substantial increase of binding for all compounds and combinations, when compared to medium control .
in addition, binding to the nfatp2/ <dig> site following both, gp + lps  and gp + tsst- <dig>  was elevated , but only in an additive synergistic manner, i.e.
the experimental results were not different from the theoretical values .
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  <dig> h. comparing lps vs. gp + lps, we did not find substantial alterations over a time course of  <dig> h in the il-1β/il-1ra ratio, only a – non-significant –  <dig> fold higher ratio at  <dig> h. with respect to tsst- <dig> or the theoretical value of gp/tsst- <dig> calc.
vs. gp + tsst- <dig>  we observed a pronounced anti-inflammatory action of gp, as demonstrated by an about 10– <dig> fold reduced ratio at  <dig> h and  <dig> h of incubation, i.e.
a higher production of il-1ra than of il-1β .
a biological relevance of this result was suggested by a decrease of the il- <dig> dependent release of il- <dig> from murine el- <dig> cells following gp + tsst- <dig> when compared to tsst- <dig> or gp/tsst- <dig> calc.
.
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  <cit>  and cya, nfat via cya and nfil- <dig> via chx  <cit>  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 .
the extent of reduction in nfatp2/ <dig> binding is exemplarily shown in an autoradiogram  and graphically summarized .
the results for the nfκb and nfil- <dig> binding sites were similar .
because the highest il-1ra amount was found at  <dig> h, mrna was examined following  <dig> h inhibition and  <dig> 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 .
in addition, corresponding il-1ra protein levels after  <dig> h of gp are shown in fig.
7c .
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.
discussion
animal models of sepsis and myocardial injury suggest that a β-1→3-d-glucan like gp mediates its protective mechanisms, in part, via a rapid shift from nfκb- to pi3k-signaling  <cit> .
our study provides novel evidence that downstream of recognition and signaling pro-inflammatory transcription factor binding and cytokine expression of human leukocytes is switched to an anti-inflammatory phenotype by gp.
we confirmed and extended previous results  <cit>  indicating that, in the absence of other stimuli, β-1→3-d-glucans induced binding of nfκb-, nfil-6- and nfat-mers to cytokine promoters.
because of the multiple band shifts observed for nfat binding , one could speculate that there is activation of several different nfat isoforms, probably derived from alternative splicing  <cit> .
interestingly, the gp-induced transcription factor binding transformed only into a very limited cytokine response, namely il- <dig> and il-1ra .
hence, our data are in aggreement with the few reports describing a β-1→3-d-glucan-mediated il- <dig>  <cit>  and il-1ra production  <cit> .
in addition, our emsa/supershift and immunoblotting results demonstrated a gp-mediated predominant binding of nfκb p <dig> and to a lesser extent of p <dig> to a κb consensus site of the il- <dig> promoter .
results by schulte and colleagues  <cit>  pointed to an induction of il- <dig> transcription depending on activation via an nfκb p65/ <dig> homodimer, rather than via p65/ <dig> heterodimers, which might be the case for the gp-mediated il- <dig> transcription.
a gp-mediated il- <dig> transcription based upon a cooperation between transactivated nfκb p <dig> and nfil- <dig>  <cit>  or nfatc <dig> dimer binding to the il-8κb site  <cit>  seems also possible.
the il-8κb consensus site exhibits a preferentially p <dig> binding half site and thus differs from the κb half site described for tnfα and il-1β  <cit> , supporting the idea of regulating nfκb binding through combinatorial associations of the subunits and the specific sequence of the decameric κb motif  <cit> .
unlike lps or tsst- <dig>  we found that gp did not induce il-1β, but it strongly induced il-1ra, suggesting an immediate anti-inflammatory potential of gp.
analysing the il-1ra promoter  <cit> , we discovered four new binding sites : an nfκb <dig> site , an nfκb consensus site , another nfκb2/ <dig> site  and a more distal nfatp2/ <dig> site .
our data indicated that gp leads to production of il-1ra primarily via induction of nfatp2/ <dig> and nfil- <dig> dna binding, which might be due to differences in the binding motif or the composition of the activated transcription factors  between the ifnγ, il- <dig> and il-1ra promoter.
the differential decrease of nfκb, nfat and nfil- <dig> binding to sites in the il-1ra promoter as well as of il-1ra mrna and protein induced by selective inhibitors prior to gp treatment might suggest that these steps are linked to each other and necessary for induction of il-1ra .
regarding cellular sources, both, monocytes as well as neutrophils have been reported to produce il-1ra  <cit> .
flow cytometric experiments seemed to confirm that monocytes and neutrophils were able to produce il- <dig> just as il-1ra in response to gp .
this gp-induced cytokine profile was substantially more restricted than that of lps or tsst- <dig>  which is likely due to differences in recognition and signaling between lps, tsst- <dig> and gp.
recognition of lps is mainly mediated through toll-like receptor  <dig> and subsequent signaling via the nfκb pathway, leading to expression of pro-inflammatory mediators like tnfα  <cit> .
a bacterial superantigen like tsst- <dig> acts through binding to mhc-ii molecules and subsequently the t cell receptor, leading to release of mainly ifnγ and tnfα, the latter via both, pi3k and p <dig> mitogen-activated kinase signaling  <cit> .
altogether, the induction of the neutrophil-attracting il- <dig> and the anti-inflammatory il-1ra by fungal carbohydrates  may well fit to a benign pamp response, mounting defensive mechanisms against a possible microbial attack.
potentially even more relevant than the specific cytokine panel induced by gp, we found that, in the presence of other stimuli , gp ameliorated their pro-inflammatory immune reactions, similar to the effects in murine models of sepsis and inflammation  <cit> .
mainly, gp altered the tsst-1-induced il-1β/il-1ra ratio from a pro- to an anti-inflammatory profile via down-regulation of il-1β and il- <dig>  at the same time there was a synergistic up-regulation of il-1ra.
in part, this change may be caused by gp-mediated modulations of the tsst- <dig> induced binding of nfκb, nfil- <dig> and nfat to known and up to now unknown sites within the il- <dig> and il-1ra promoter instead of the tnfα, il-1β, il- <dig> or ifnγ promoter.
accordingly, we found that the tsst-1-induced binding of nfκb to sites from the tnfα promoter negatively correlated with the gp-mediated enhancement of the tsst-1-induced il-1ra production .
of the four examined nfκb sites from the il-1ra promoter, mostly the tsst-1- and lps-induced binding to the nfκb <dig> and the tsst-1-induced binding to the new nfκb consensus site seemed to be increased by gp.
so, while we observed a gp-mediated down-regulation of the lps- and the tsst-1-induced nfκb binding to sites of the tnfα promoter, there was an up-regulation to nfκb <dig> and nfκb consensus sites of the il-1ra promoter.
these seemingly contradictory data could be explained by differences in either nfκb subunits or conserved nucleotides  within the decameric nfκb binding motif between the tnfα and the il-1ra promoter , probably leading to differences in binding  <cit> .
despite the location of the new nfκb consensus site  in the inhibitory element  of the il-1ra promoter  <cit> , we observed no inhibition.
on the other side, we found an inhibitory nfκb2/ <dig> site  towards the end of the inhibitory element, demonstrating down-regulations of the lps- and tsst-1-induced binding, which could not be altered by gp.
in our opinion, this site may therefore represent at least a part of the previoulsy described inhibitory element  <cit> .
the gp-modulated increase in tsst-1-induced binding to the new nfκb <dig> and nfκb consensus site, the nfil- <dig> site  <cit>  as well as to the novel nfatp2/ <dig> site may explain the synergistic up-regulation of the tsst-1-induced il-1ra production.
we think that this gp-modulated activation of transcription was reflected by the decrease of the il-1β/il-1ra ratio following gp + tsst- <dig> .
in this context, it has been postulated that in vitro a 100fold excess of il-1ra over il-1β might control the biological effects of il- <dig>  <cit> .
since, in fact, the il-1β/il-1ra ratio following gp + tsst- <dig> is partially less than  <dig> , it is not unreasonable to assume that il-1β bioactivity is inactivated in our system.
indeed, gp reduced the tsst-1-induced, il-1-dependent il- <dig> production of murine el- <dig> cells .
the weaker modulating effects of gp on the lps-induced immune response observed in this study, may be attributed to delicately balanced differences in signaling pathways between lps and tsst- <dig>  <cit> .
tsst- <dig> has been shown to use the pi3k pathway for signaling  <cit>  and this effect may be sustained by gp treatment  <cit> .
it has been demonstrated that in septic/lps-adapted leukocytes the pi3k pathway selectively controls sil-1ra but not il-1β production  <cit> .
signaling via pi3k has been reported to be involved in the activation of nfat in t cells  <cit> .
activation of nfκb can also take place via pi3k  <cit> , which may offer an explanation for a difference in signaling between gp  and lps .
this idea may be supported by another study showing that despite the use of similar prr, lps and peptidoglycan activated the il-1ra gene through different mechanisms/dna-binding proteins and acted synergistically in combination, suggestive of signals which are not equivalent in all parts  <cit> .
CONCLUSIONS
in summary, our data demonstrated that in vitro glucan phosphate induced a transcription factor binding and a subsequent cytokine profile different from lps and also from tsst- <dig> and, moreover, switched a pro-inflammatory tsst-1-induced response to an il-1ra-mediated anti-inflammatory reaction.
our results also generated new insights into a very complex interplay of transcription factor binding to various known and newly identified sites in the il-1ra promoter , which can be regulated differentially by a fungal carbohydrate.
together with the in vivo studies  <cit> , our findings might support the concept of protective effects mediated by glucan phosphate in pro-inflammatory conditions, especially with a dysregulated il-1β/il-1ra ratio  <cit> .

 β-1→3-d-glucans occur as a principal component of microbial cell walls or can be secreted from both, non-pathogenic and pathogenic fungi such as s. cerevisae and c. albicans. these β-1→3-d-linked glucose polymers are characterized as a fungal pathogen-associated molecular pattern. the primary cellular recognition of β-1→3-d-glucans is mediated by several β-1→3-d-glucan receptors on phagocytes and other cells. human as well as murine dectin- has been demonstrated to be the major pattern recognition receptor for intact yeast and β-1→3-d-glucan-containing particles on monocytes/macrophages as well as neutrophils and on primary cells. in the murine system, binding of zymosan to dectin- resulted in production of tnfα through toll-like receptor and the adaptor protein myd. another water-soluble β-1→3-d-glucan has been described to activate nfκb and nfil- in murine cell lines. similarly, it has been shown that β-1→3-d-glucans activate nfκb in a human monocyte-like cell line and in human polymorphonuclear neutrophils, in the latter case without secretion of pro-inflammatory cytokines. one study proposed that the production of the anti-inflammatory il-1ra, but not il- by human monocytes may be a potentially protective mechanism induced by β-1→3-d-glucan. three other investigations have reported that human leukocytes and human vascular endothelial cells produce il- in response to zymosan or a water-soluble β-1→3-d-glucan. in addition, β-1→3-d-glucans seem to be able to modify the response to pro-inflammatory stimuli or even sepsis. in a murine polymicrobial sepsis model, β-1→3-d-glucan treatment resulted in decreased septic morbidity and mortality mediated via inhibition of nfκb and stimulation of the phosphoinositide-3-kinase pathway. these and other animal studies as well as a clinical trial support a protective role of β-1→3-d-glucan in certain pro-inflammatory conditions. the mechanisms underlying these beneficial effects of β-1→3-d-glucan are only partially resolved, especially in humans. thus, the aim of this study was to elucidate molecular and cellular mechanisms of β-1→3-d-glucans on human leukocytes in pro-inflammatory conditions with special emphasis on the cytokine profile and its transcriptional regulation. for this purpose, peripheral blood mononuclear cells were exposed to a well-defined β-1→3-d-glucan, i.e. glucan phosphate, alone or simultaneously with lps from gram-negative bacteria or the superantigen tsst- from gram-positive bacteria over h. because of the potential effect of β-1→3-d-glucan on cytokine production, tnfα, il-1β, il- il- and il-1ra were measured as well as ifnγ, il- il- il- il- and tgfβ correspondingly, four nfκb sites from the tnfα promoter, a κ consensus site from the il- promoter, an nfat site from the ifnγ promoter and a consensus nfil- site from the il- promoter were examined. because of the anti-inflammatory role of il-1ra, we focused on binding of transcription factors to the il-1ra promoter. an inhibitory element and three positive-acting lps-response elements in the il-1ra promoter, including nfκb, pu. and nfil- sites, have been characterized previously. using computational analysis for homology search, we looked for new binding motifs in the il-1ra promoter.  binding activities of nfκb, nfil- and nfat from human pbmc to the tnfα, il- and ifnγ promoters following an in vitro stimulation with lps, tsst- gp, gp + lps and gp + tsst-1 gp induced band shifts, indicating binding of nfκb as well as of nfil- to the corresponding dna oligonucleotides. the extent of the band shifts induced by gp was not statistically different when compared to lps or tsst-. a supershift, conducted for an nfκb consensus site from the il- promoter, demonstrated a gp-induced predominant binding of nfκb p and to a lesser degree of p. accordingly, an immunoblot of nuclear extracts from gp-treated pbmc showed a strong binding of nfκb p and a weaker reaction of p whereas p was negative. simultaneous co-treatment of pbmc with gp did not change the lps-induced nfκb binding to oligos from the tnfα promoter significantly, but substantially decreased the tsst-1-induced nfκb binding when compared to tsst- or gp and thus differed completely from the theoretical value of gp/tsst- calc.. gp was also able to induce band shifts indicative of binding of particular variants of nfat to an oligo from the ifnγ promoter. an immunoblot for two nfat subunits demonstrated a gp-induced binding of nfatc and to a lesser degree of nfatc.. cytokine profile of human pbmc following a h in vitro stimulation with lps, tsst- gp, gp + lps and gp + tsst-1 because of the gp-induced band shifts to seven sites from the tnfα, il- il- and ifnγ promoters, it could be assumed that gp treatment of human pbmc would lead to production of tnfα, il- il- 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. corroborating and extending these singular findings in terms of examining pro- as well as anti- inflammatory cytokines over time 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: il-1β there was no il-1β production detectable following pbmc treatment with nor μg gp. an insignificant up-regulation of il-1β production for gp + lps was observed in comparison with lps. for gp + tsst- we found a significant reduction in il-1β from h – h, for the latter by about 40% when compared to tsst- or the theoretical value of gp/tsst- calc.. on the whole, gp mediated a reduction of the tsst-1-induced amount of il-1β by about 50%. il-6 gp induced only a small amount of il- and there was no significant alteration of the lps-induced il- production by gp. on the contrary, we observed a decreased il- production for gp + tsst- when compared to tsst- or gp/tsst- calc., especially at h by about 40%,. il-8 we found that gp induced a substantial il- production, in comparison with medium control, especially at h. a combination of gp and lps resulted in a non-significant increase of il- production when compared to lps or gp/lps calc. there was no significant alteration in il- production following gp + tsst- when compared to tsst- or gp/tsst- calc.. il-1ra besides il- il-1ra was the only mediator which was produced in significant quantities following gp treatment, especially at h. 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- we found a synergistic increase in il-1ra production from h to h, when compared to tsst- or to the theoretical value of gp/tsst- calc.. over the time course of h, gp elevated the tsst-1-induced amount of il-1ra by approximately 200%. positive correlation between gp-induced il- and il-1ra productions. following stimulation with gp, we observed a positive correlation between il- and il-1ra. moreover, we found this correlation to be dose-dependent, since μg of gp induced larger amounts of il- and il-1ra than μg gp. tnfα with respect to tnfα production, 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- or gp/tsst- calc., gp seemed to exert a synergistic effect on tnfα secretion after h, but overall an enhancement of only 10% was observed. ifnγ no ifnγ production was detectable following treatment of pbmc with μg gp. there was a minor increase in ifnγ production following gp + lps at h, when compared to lps or gp/lps calc., and a slight down-regulation following gp + tsst- at h and h, when compared to tsst- or gp/tsst- calc.. il- il- il- il- tgfβ1 the production of il- il- il- il- and tgfβ was not induced by gp. there were also no significant differences between medium control, lps, tsst- gp, gp + lps, gp/lps calc. and gp + tsst- or gp/tsst- calc. over h. role of nfκb, nfil- 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- and nfat sites, but no tnfα, il- 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 using the program omiga. the lps responsive κb consensus site of lre- displayed band shifts, but no significant alterations of its binding activity, irrespective of the treatment. a new κb site within the lre- showed increased binding induced by tsst-, but gp co-treatment up-regulated the lps - and the tsst- -induced binding in a superadditive synergistic fashion, when compared to medium control as well as to lps, tsst- or gp or the theoretical values of gp/lps calc. or gp/tsst- calc.. binding to a new κb consensus site was significantly elevated for gp + tsst-. the lps-, tsst-1- and gp + lps-induced binding to the new κb2/ site was down-regulated by about 20–35% in comparison with medium control. the nfil- site close to lre- showed an up-regulation of binding for all compounds and combinations vs. medium control, ranging from 25–110%. further, gp + tsst- induced an increased binding to the nfil- site in comparison with tsst- or gp,. a new binding site for nfatp2/ was found more distal of the transcription start site, between - and - displaying again a substantial increase of binding for all compounds and combinations, when compared to medium control. in addition, binding to the nfatp2/ site following both, gp + lps and gp + tsst- was elevated, but only in an additive synergistic manner, i.e. the experimental results were not different from the theoretical values. 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 h. comparing lps vs. gp + lps, we did not find substantial alterations over a time course of h in the il-1β/il-1ra ratio, only a – non-significant – fold higher ratio at h. with respect to tsst- or the theoretical value of gp/tsst- calc. vs. gp + tsst- we observed a pronounced anti-inflammatory action of gp, as demonstrated by an about 10– fold reduced ratio at h and h of incubation, i.e. a higher production of il-1ra than of il-1β. a biological relevance of this result was suggested by a decrease of the il- dependent release of il- from murine el- cells following gp + tsst- when compared to tsst- or gp/tsst- calc.. 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 and cya, nfat via cya and nfil- via chx 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. the extent of reduction in nfatp2/ binding is exemplarily shown in an autoradiogram and graphically summarized. the results for the nfκb and nfil- binding sites were similar. because the highest il-1ra amount was found at h, mrna was examined following h inhibition and 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. in addition, corresponding il-1ra protein levels after h of gp are shown in fig. 7c. 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. discussion animal models of sepsis and myocardial injury suggest that a β-1→3-d-glucan like gp mediates its protective mechanisms, in part, via a rapid shift from nfκb- to pi3k-signaling. our study provides novel evidence that downstream of recognition and signaling pro-inflammatory transcription factor binding and cytokine expression of human leukocytes is switched to an anti-inflammatory phenotype by gp. we confirmed and extended previous results indicating that, in the absence of other stimuli, β-1→3-d-glucans induced binding of nfκb-, nfil-6- and nfat-mers to cytokine promoters. because of the multiple band shifts observed for nfat binding, one could speculate that there is activation of several different nfat isoforms, probably derived from alternative splicing. interestingly, the gp-induced transcription factor binding transformed only into a very limited cytokine response, namely il- and il-1ra. hence, our data are in aggreement with the few reports describing a β-1→3-d-glucan-mediated il- and il-1ra production. in addition, our emsa/supershift and immunoblotting results demonstrated a gp-mediated predominant binding of nfκb p and to a lesser extent of p to a κb consensus site of the il- promoter. results by schulte and colleagues pointed to an induction of il- transcription depending on activation via an nfκb p65/ homodimer, rather than via p65/ heterodimers, which might be the case for the gp-mediated il- transcription. a gp-mediated il- transcription based upon a cooperation between transactivated nfκb p and nfil- or nfatc dimer binding to the il-8κb site seems also possible. the il-8κb consensus site exhibits a preferentially p binding half site and thus differs from the κb half site described for tnfα and il-1β, supporting the idea of regulating nfκb binding through combinatorial associations of the subunits and the specific sequence of the decameric κb motif. unlike lps or tsst- we found that gp did not induce il-1β, but it strongly induced il-1ra, suggesting an immediate anti-inflammatory potential of gp. analysing the il-1ra promoter, we discovered four new binding sites: an nfκb site, an nfκb consensus site, another nfκb2/ site and a more distal nfatp2/ site. our data indicated that gp leads to production of il-1ra primarily via induction of nfatp2/ and nfil- dna binding, which might be due to differences in the binding motif or the composition of the activated transcription factors between the ifnγ, il- and il-1ra promoter. the differential decrease of nfκb, nfat and nfil- binding to sites in the il-1ra promoter as well as of il-1ra mrna and protein induced by selective inhibitors prior to gp treatment might suggest that these steps are linked to each other and necessary for induction of il-1ra. regarding cellular sources, both, monocytes as well as neutrophils have been reported to produce il-1ra. flow cytometric experiments seemed to confirm that monocytes and neutrophils were able to produce il- just as il-1ra in response to gp. this gp-induced cytokine profile was substantially more restricted than that of lps or tsst- which is likely due to differences in recognition and signaling between lps, tsst- and gp. recognition of lps is mainly mediated through toll-like receptor and subsequent signaling via the nfκb pathway, leading to expression of pro-inflammatory mediators like tnfα. a bacterial superantigen like tsst- acts through binding to mhc-ii molecules and subsequently the t cell receptor, leading to release of mainly ifnγ and tnfα, the latter via both, pi3k and p mitogen-activated kinase signaling. altogether, the induction of the neutrophil-attracting il- and the anti-inflammatory il-1ra by fungal carbohydrates may well fit to a benign pamp response, mounting defensive mechanisms against a possible microbial attack. potentially even more relevant than the specific cytokine panel induced by gp, we found that, in the presence of other stimuli, gp ameliorated their pro-inflammatory immune reactions, similar to the effects in murine models of sepsis and inflammation. mainly, gp altered the tsst-1-induced il-1β/il-1ra ratio from a pro- to an anti-inflammatory profile via down-regulation of il-1β and il- at the same time there was a synergistic up-regulation of il-1ra. in part, this change may be caused by gp-mediated modulations of the tsst- induced binding of nfκb, nfil- and nfat to known and up to now unknown sites within the il- and il-1ra promoter instead of the tnfα, il-1β, il- or ifnγ promoter. accordingly, we found that the tsst-1-induced binding of nfκb to sites from the tnfα promoter negatively correlated with the gp-mediated enhancement of the tsst-1-induced il-1ra production. of the four examined nfκb sites from the il-1ra promoter, mostly the tsst-1- and lps-induced binding to the nfκb and the tsst-1-induced binding to the new nfκb consensus site seemed to be increased by gp. so, while we observed a gp-mediated down-regulation of the lps- and the tsst-1-induced nfκb binding to sites of the tnfα promoter, there was an up-regulation to nfκb and nfκb consensus sites of the il-1ra promoter. these seemingly contradictory data could be explained by differences in either nfκb subunits or conserved nucleotides within the decameric nfκb binding motif between the tnfα and the il-1ra promoter, probably leading to differences in binding. despite the location of the new nfκb consensus site in the inhibitory element of the il-1ra promoter, we observed no inhibition. on the other side, we found an inhibitory nfκb2/ site towards the end of the inhibitory element, demonstrating down-regulations of the lps- and tsst-1-induced binding, which could not be altered by gp. in our opinion, this site may therefore represent at least a part of the previoulsy described inhibitory element. the gp-modulated increase in tsst-1-induced binding to the new nfκb and nfκb consensus site, the nfil- site as well as to the novel nfatp2/ site may explain the synergistic up-regulation of the tsst-1-induced il-1ra production. we think that this gp-modulated activation of transcription was reflected by the decrease of the il-1β/il-1ra ratio following gp + tsst-. in this context, it has been postulated that in vitro a 100fold excess of il-1ra over il-1β might control the biological effects of il-. since, in fact, the il-1β/il-1ra ratio following gp + tsst- is partially less than, it is not unreasonable to assume that il-1β bioactivity is inactivated in our system. indeed, gp reduced the tsst-1-induced, il-1-dependent il- production of murine el- cells. the weaker modulating effects of gp on the lps-induced immune response observed in this study, may be attributed to delicately balanced differences in signaling pathways between lps and tsst-. tsst- has been shown to use the pi3k pathway for signaling and this effect may be sustained by gp treatment. it has been demonstrated that in septic/lps-adapted leukocytes the pi3k pathway selectively controls sil-1ra but not il-1β production. signaling via pi3k has been reported to be involved in the activation of nfat in t cells. activation of nfκb can also take place via pi3k, which may offer an explanation for a difference in signaling between gp and lps. this idea may be supported by another study showing that despite the use of similar prr, lps and peptidoglycan activated the il-1ra gene through different mechanisms/dna-binding proteins and acted synergistically in combination, suggestive of signals which are not equivalent in all parts.  in summary, our data demonstrated that in vitro glucan phosphate induced a transcription factor binding and a subsequent cytokine profile different from lps and also from tsst- and, moreover, switched a pro-inflammatory tsst-1-induced response to an il-1ra-mediated anti-inflammatory reaction. our results also generated new insights into a very complex interplay of transcription factor binding to various known and newly identified sites in the il-1ra promoter, which can be regulated differentially by a fungal carbohydrate. together with the in vivo studies, our findings might support the concept of protective effects mediated by glucan phosphate in pro-inflammatory conditions, especially with a dysregulated il-1β/il-1ra ratio. 
our results also offer new insights into the complex regulation of the il-1ra gene, which can be modulated by a β-1→3-d-glucan. glucan phosphate induced significant binding to a known nfil- site and a new nfat site within the il-1ra promoter, which was confirmed by inhibition experiments. further, glucan phosphate modulated the tsst-1-induced inflammatory response via reduction of il-1β and il- as a consequence, glucan phosphate shifted the tsst-1-induced il-1β/il-1ra ratio towards an anti-inflammatory phenotype. when applied in combination with either lps or tsst- at the same time points, we detected that glucan phosphate elevated the lps- and the tsst-1-induced dna binding of nfκb, nfil- and nfat, leading to a synergistic increase of il-1ra. employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-d-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide or toxic shock syndrome toxin. despite an activation of nuclear factor κb, nfinterleukin- and nfat similar to lps or tsst- we observed no significant production of il-1β, il- tumor necrosis factor α or interferon γ induced by glucan phosphate. glucan phosphate-treated leukocytes induced a substantial amount of il-, likely due to binding of nfκb to a consensus site in the il- promoter. an increase in il-1receptor antagonist production by glucan phosphate-treated cells positively correlated with il- levels. subsequently, glucan phosphate decreased the tsst-1-induced, il-1-dependent production of il- 
 β-1→3-d-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-d-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide or toxic shock syndrome toxin.  despite an activation of nuclear factor κb, nfinterleukin- and nfat similar to lps or tsst- we observed no significant production of il-1β, il- tumor necrosis factor α or interferon γ induced by glucan phosphate. glucan phosphate-treated leukocytes induced a substantial amount of il-, likely due to binding of nfκb to a consensus site in the il- promoter. an increase in il-1receptor antagonist production by glucan phosphate-treated cells positively correlated with il- levels. glucan phosphate induced significant binding to a known nfil- site and a new nfat site within the il-1ra promoter, which was confirmed by inhibition experiments. when applied in combination with either lps or tsst- at the same time points, we detected that glucan phosphate elevated the lps- and the tsst-1-induced dna binding of nfκb, nfil- and nfat, leading to a synergistic increase of il-1ra. further, glucan phosphate modulated the tsst-1-induced inflammatory response via reduction of il-1β and il- as a consequence, glucan phosphate shifted the tsst-1-induced il-1β/il-1ra ratio towards an anti-inflammatory phenotype. subsequently, glucan phosphate decreased the tsst-1-induced, il-1-dependent production of il-  thus, β-1→3-d-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory il-1ra-mediated reaction. our results also offer new insights into the complex regulation of the il-1ra gene, which can be modulated by a β-1→3-d-glucan. 