BACKGROUND
following antigen recognition cd4+ t cells differentiate into one of several types of th cells including th <dig>  th <dig>  th <dig> and treg cells that secrete distinct sets of cytokines  <cit> .
studies have suggested that, in addition to the cytokine milieu, vitamin d is an important determinant in this differentiation of cd4+ t cells  <cit> .
thus, in vitro studies have shown that the active form of vitamin d <dig>  1α,25-dihydroxyvitamin d <dig> 2d3), inhibits production of ifn-γ and augment the production of il- <dig>  thereby limiting th <dig> and promoting th <dig> cell differentiation  <cit> .
furthermore,  <dig> d <dig> inhibits th <dig> cell differentiation and induces differentiation of treg cells  <cit> .
it is therefore generally believed that vitamin d plays an anti-inflammatory role, and accordingly vitamin d deficiency has been associated with increased risk of autoimmune diseases such as type  <dig> diabetes mellitus  <cit> , lupus erythematosus  <cit>  and multiple sclerosis  <cit> .
25-hydroxyvitamin d <dig> d3) is the inactive precursor of  <dig> d <dig> and is considered the best parameter for evaluation of the vitamin d status of a subject.
the normal range of serum 25d <dig> concentrations is 25– <dig> nm  <cit> .
the serum concentration of the active  <dig> d <dig> is approximately 1000-fold lower  and far below the effective concentration of  <dig> d <dig> found in in vitro studies.
thus, in most in vitro studies more than a 100-fold higher concentration of  <dig> d <dig> than found in serum is often required to obtain an effect  <cit> .
it has therefore been suggested that the level of circulating  <dig> d <dig> is too low to affect immune responses in vivo, and that sufficient levels are obtained by local conversion of 25d <dig> to  <dig> d <dig>  <cit> .
in accordance, it has been shown that activated antigen presenting cells  express the 25d-1α-hydroxylase cyp27b <dig> that converts 25d <dig> to  <dig> d <dig>  and that apc can produce  <dig> d <dig> from 25d3in vitro and respond to this through the vitamin d receptor  in an autocrine fashion  <cit> .
elevated levels of  <dig> d <dig> in association with hypercalcemia have been observed in patients with sarcoidosis, tuberculosis, and other infections and inflammatory diseases in which the pathology is characterized by granuloma formation  <cit> , supporting the hypothesis that activated macrophages can produce significant amounts of  <dig> d3in vivo.
like apc, activated t cells express the vdr and cyp27b <dig>  <cit> .
however, whether t cells can convert 25d <dig> to  <dig> d <dig> in physiological relevant concentrations and respond to this in an autocrine fashion is a matter of debate.
most studies on the effect of vitamin d on t cells have not addressed this question as they investigated the direct effects of supra-physiological concentrations of  <dig> d <dig> and not how 25d <dig> affects t cell responses.
one study has shown that isolated t cells have the ability to convert 25d <dig> to  <dig> d <dig> in concentrations that actually affects vitamin d-responsive genes in an autocrine fashion  <cit> .
in agreement, we recently found that purified cd4+ t cells have the ability to produce substantial amounts of  <dig> d <dig> when activated in the presence of 25d <dig>  <cit> .
in contrast, another recent study found that although activated t cells do express cyp27b <dig>  the expression level is not sufficiently high to allow production of  <dig> d <dig> in concentrations that affect vitamin d-responsive genes  <cit> .
the authors found that 25d <dig> only affected t cell responses when apc were present, and suggested that apc locally secrete sufficient amounts of  <dig> d <dig> to directly influence the surrounding t cells in a paracrine fashion.
other important players influencing the bioavailable levels of vitamin d are the vitamin d-binding protein  and albumin.
25d <dig> and  <dig> d <dig> circulate bound to dbp  and albumin  with less than 1% in their free form  <cit> .
studies of dbp knock-out mice have shown that dbp acts as a vitamin d reservoir by protecting 25d <dig> and  <dig> d <dig> from degradation and renal secretion  <cit> .
however, dbp also sequesters 25d <dig> and  <dig> d <dig> and inhibits their action on monocytes, dc and keratinocytes in vitro  <cit> .
how dbp affects t cell responses to 25d <dig> still needs to be determined.
the objectives of this study were to further elucidate whether t cells have the ability to convert 25d <dig> to  <dig> d <dig> in proportions that affect a panel of vitamin d-responsive genes in an autocrine fashion and to investigate how dbp regulates t cell responses to 25d <dig>
RESULTS
activated t cells express cyp27b <dig> and have the capacity to convert 25d <dig> to  <dig> d3
in order to convert 25d <dig> to  <dig> d <dig> cells must express the 25d-1α-hydroxylase cyp27b <dig>  to determine whether naïve cd4+ t cells express cyp27b <dig>  we purified cd45ra+cd4+ cells from the blood of healthy donors.
the resulting cell population contained 95–98% cd4+ t cells of which more than 96% were cd45ra+ .
the purified cells were stimulated with cd3/cd <dig> beads for 0–5 days in serum-free medium and their expression of cyp27b <dig> mrna was subsequently measured.
we found that naïve cd4+ t cells express no or very low levels of cyp27b <dig>  however, the cells started to express cyp27b <dig> mrna shortly after stimulation, and the expression peaked after 2– <dig> days of stimulation .
these results suggested that activated human cd4+ t cells have the capacity to convert 25d <dig> to  <dig> d <dig>  to validate this, we stimulated purified cd4+ t cells in the presence of  <dig> nm 25d <dig> corresponding to physiological concentrations of 25d <dig> in serum and then measured the production of  <dig> d <dig>  we found that activated cd4+ t cells produced  <dig> d <dig> with a kinetic similar to the kinetics of cyp27b <dig> expression in the cells, and that the production peaked after 3 days of stimulation .
finally, to determine whether the cells expressed the receptor for  <dig> d <dig>  we determined the expression of the vdr in cd4+ t cells stimulated for 0–5 days.
we found that vdr expression peaked simultaneously with the peak production of  <dig> d <dig> at day  <dig> .
taken together, these experiments demonstrated that activated human cd4+ t cells express cyp27b <dig>  that they have the capacity to convert 25d <dig> at physiological concentrations to the active  <dig> d <dig>  and that they express the receptor for  <dig> d <dig> figure  <dig>
activated t cells express cyp27b <dig> and have the capacity to convert 25d
3
to  <dig>
2
d
3
.
relative cyp27b <dig> mrna expression in t cells activated for 0– <dig> days normalized to cyp27b <dig> expression in naïve t cells.
values are given as mean + sem from  <dig> independent experiments, *p <  <dig> .
<dig> d <dig> in the medium of t cells activated for 0– <dig> days in the presence of  <dig> nm 25d <dig>  data are given as mean ± sem from  <dig> independent experiments, *p <  <dig> .
representative western blot of vdr and cd3ζ  expression in t cells activated for 0– <dig> days.
activated t cells have the ability to produce  <dig> d <dig> in sufficiently high concentrations to affect vitamin d-responsive genes
having demonstrated that activated cd4+ t cells have the capacity to produce the active form of vitamin d and that they simultaneously express the vdr, we next asked whether the production of  <dig> d <dig> was sufficiently high to induce autocrine activation of vitamin d-responsive genes.
to study this, we took advantage of the finding that the cell surface expression levels of cd <dig> on activated t cells are dependent on the concentration of  <dig> d <dig> present during t cell activation  <cit> .
consequently, we activated cd4+ t cells in serum-free medium in the presence of increasing concentrations of 25d <dig> or  <dig> d <dig> and then measured cd <dig> expression at day  <dig>  in accordance with previous studies, we found that  <dig> d <dig> significantly up-regulated cd <dig> expression in activated t cells in a dose-dependent manner   <cit> .
in addition, we found that 25d <dig> also induced cd <dig> up-regulation, suggesting that activated t cells can produce sufficiently high concentrations of  <dig> d <dig> to affect vitamin d-responsive genes .
we noted that whereas 25d <dig> at a physiological concentration of  <dig> nm induced maximum cd <dig> up-regulation,  <dig> d <dig> was required at a 100-fold higher concentration  than physiological levels  to induce the same effect.
that a supra-physiological concentration of  <dig> d <dig> was required to obtain an effect in our study is in good line with previous studies  <cit> .
however, in contrast to our observations that 25d <dig> at  <dig> nm had a clear effect on t cells, most previous studies on 25d <dig> required at least a 100-fold higher concentration of 25d <dig> than found in serum to induce an effect  <cit> .
one explanation for this discrepancy could be that whereas our experiments were performed in serum-free medium most previous studies have been performed in medium supplemented with serum.
to study whether the presence of serum could explain the discrepancy between the concentrations of 25d <dig> required to obtain an effect on t cells, we activated cd4+ t cells in the presence of increasing concentrations of 25d <dig> and  <dig> d <dig> in medium supplemented with 5% fetal bovine serum  and measured cd <dig> expression at day  <dig>  we found that the presence of serum significantly shifted the concentration of 25d <dig> required to induce cd <dig> expression .
thus, compared to serum-free medium approximately 100-fold higher concentrations of 25d <dig> were required to induce a similar effect on cd <dig> expression in t cells cultured in medium supplemented with serum.
in contrast, the presence of serum did not significantly shift the concentration of  <dig> d <dig> required to induce cd <dig> expression.
to separately analyse the role of dbp and albumin in this serum-mediated inhibition of the effect of 25d <dig> on t cells, we activated cd4+ t cells in the presence of 100 nm 25d <dig> in serum-free medium supplemented with increasing concentrations of either dbp or albumin and measured cd <dig> expression at day  <dig>  we found that dbp at concentrations above 250– <dig> nm completely abolished the effect of 25d <dig> on cd <dig> expression, whereas albumin did not affect the effect of 25d <dig> .figure  <dig>
in the absence of serum activated t cells can produce  <dig>
2
d
3
in concentrations that affect cd <dig> expression.
relative cd <dig> cell surface expression on t cells activated for 3 days in x-vivo  <dig>  or x-vivo  <dig> supplemented with 5% fbs  in the presence of the indicated concentrations of 25d <dig> and  <dig> d <dig> normalized to cd <dig> cell surface expression on t cells activated in the absence of 25d <dig> and  <dig> d <dig>  relative cd <dig> cell surface expression on t cells activated for 3 days in x-vivo  <dig> medium supplemented with increasing concentrations of dbp  or albumin  in the presence of  <dig> nm 25d <dig> normalized to cd <dig> cell surface expression on t cells activated in the absence of 25d <dig>  relative cd <dig> cell surface expression on t cells activated for 3 days in the absence or presence of ketoconazole and the indicated concentrations of 25d3
or  <dig> d3
normalized to cd <dig> cell surface expression on t cells activated in the absence of 25d <dig> or  <dig> d <dig>   mean values ± sem from  <dig> independent experiments are plotted, *p <  <dig> .
although t cells converted 25d <dig> to  <dig> d <dig> when activated in serum-free medium, we could not exclude the possibility that the observed 25d3-induced up-regulation of cd <dig> was caused directly by 25d <dig> and not by  <dig> d <dig>  to exclude this possibility, we determined the effect of the cyp27b <dig> inhibitor ketoconazole on cd <dig> up-regulation.
we activated t cells in the absence or presence of ketoconazole and increasing concentrations of 25d <dig>  we found that ketoconazole efficiently inhibited 25d3-induced cd <dig> up-regulation .
these observations indicated that conversion of 25d <dig> to  <dig> d <dig> is required for 25d3-induced cd <dig> up-regulation.
if this is the case and ketoconazole does not in itself influence cd <dig> expression then ketoconazole should not affect  <dig> d3-induced cd <dig> up-regulation.
consequently, we activated t cells in the absence or presence of ketoconazole and increasing concentrations of  <dig> d <dig>  we found that ketoconazole did not affect  <dig> d3-induced cd <dig> up-regulation .
these experiments indicated that it is not 25d <dig> itself that induces cd <dig> up-regulation, but that the conversion of 25d <dig> to  <dig> d <dig> is required for 25d3-induced cd <dig> up-regulation.
thus, we could conclude that activated t cells have the capacity to produce sufficient  <dig> d <dig> to affect vitamin d-responsive genes as measured by cd <dig> expression when cultured with physiological concentrations of 25d <dig> in serum-free medium.
however, addition of dbp to the medium inhibited the effect of 25d <dig>  in contrast, dbp did not seem to affect the concentration of  <dig> d <dig> required to regulate vitamin d-responsive genes.
dbp inhibits 25d3-induced t cell responses
to determine whether the inhibitory effect of dbp on 25d <dig> was limited to cd <dig> gene expression or was a general phenomenon for vitamin d-responsive genes in t cells, we studied the influence of 25d <dig> and dbp alone or in combination on the expression of molecules known to be encoded by vitamin d-responsive genes such as ctla- <dig>  <cit> , plc-γ <dig>  <cit> , il- <dig>  <cit>  and ifn-γ  <cit>  in addition to cd <dig>  <cit> .
we stimulated cd4+ t cells for 3 days in serum-free medium or serum-free medium supplemented with either 25d <dig> or dbp alone or 25d <dig> plus dbp.
we confirmed that 25d <dig> up-regulates cd <dig> mrna and protein in activated t cells, and that this up-regulation is completely inhibited by dbp .
likewise, we found that the 25d3-induced up-regulation of ctla- <dig>  plc-γ <dig> and il- <dig> and down-regulation of ifn-γ was abolished by dbp .
thus, dbp generally inhibits the effect of 25d <dig> on vitamin d-responsive genes in t cells.
to further elucidate the underlying mechanism behind the inhibition of 25d <dig> in t cells mediated by dbp, we measured the production of active  <dig> d <dig> in the medium from the cultures described above.
we found that in the presence of 25d <dig> activated t cells produced significant amounts of  <dig> d3; however, addition of dbp completely abolished the production of  <dig> d <dig> .
thus, we could conclude that dbp strongly constrains the effect of 25d <dig> on vitamin d-responsive genes in t cells by inhibiting the conversion of 25d <dig> to  <dig> d <dig> figure  <dig>
dbp limits the bioavailability of 25d
3
and inhibits vitamin d-induced gene expression in t cells.
relative cd <dig> cell surface expression   and mrna expression  , ctla- <dig> expression  , plc-γ <dig> expression  , il- <dig> production   and mrna expression  , ifn-γ production   and mrna expression  , and  <dig> d <dig> in the medium   of t cells activated for 3 days in x-vivo  <dig> , or x-vivo  <dig> supplemented with either  <dig> nm 25d <dig> ,  <dig> μm dbp  or the combination of 25d <dig> and dbp .
the mean concentration of il- <dig> and ifn-γ in the supernatants of cells activated in x-vivo  <dig>  was  <dig> pg/ml and  <dig> ng/ml, respectively.
mean + sem from the numbers of independent experiments indicated by n in the legend above.
t cells take up dbp from the environment by macropinocytosis
dbp is internalized by megalin-mediated endocytosis in kidney and mammary cells that express megalin and cubilin  <cit> .
megalin-mediated endocytosis of dbp facilitates uptake and conversion of 25d <dig> to  <dig> d <dig> in these types of cells  <cit> .
to study whether t cells can take up dbp from the environment, we incubated naïve and activated t cells with dbp conjugated to alexa fluor  <dig> .
flow cytometry revealed increased fluorescence of activated t cells compared to naïve t cells, suggesting that activated t cells take up dbp-af <dig> .
to exclude that the increased fluorescence simply was caused by dbp-af <dig> adhering to the cell surface of the activated t cells as suggested in a previous study  <cit> , we analysed the cells by confocal microscopy.
we found that dbp-af <dig> resided in small vesicles in the cytosol and could thus conclude that activated t cells actually take up dbp from the medium .
to determine whether this uptake might be mediated by megalin, we measured megalin and cubilin mrna expression in naïve and activated t cells.
activated t cells up-regulated megalin mrna to a level more than 100-fold higher than in naive t cells, whereas the cubilin mrna levels were low in both naïve and activated t cells .
in kidney and mammary cells megalin-mediated endocytosis of dbp is, in addition to megalin, dependent on the presence of cubilin at the cells surface  <cit> .
to determine whether dbp was taken up by megalin-mediated endocytosis in activated t cells despite their low expression of cubilin, we incubated cells with dbp-af <dig> in the absence or presence of the megalin antagonist receptor-associated protein  as previously described  <cit> .
we found that rap did not influence dbp-af <dig> uptake in the t cells .
furthermore, blocking antibodies against megalin and calcium deprivation, known to inhibit megalin-mediated endocytosis  <cit> , did not affect dbp uptake .
in line with this, high concentrations of dbp did not affect the uptake of dbp-af <dig> .
taken together these experiments indicated that dbp is not taken up by megalin-mediated endocytosis in t cells.figure  <dig>
t cells take up dbp from the environment by macropinocytosis.
representative flow cytometry histograms showing dbp-af <dig> fluorescence of naïve t cells and t cells activated for  <dig> and  <dig> days and subsequently incubated with  <dig> nm dbp-af <dig> for 12 h at 37°c.
representative confocal microscopy images illustrating the uptake of dbp-af <dig> in activated t cells.
dbp-af <dig> are shown in green; cell membrane  in red; nucleus in blue .
relative megalin, cubilin, gapdh and rplp <dig> mrna expression in t cells activated from 0– <dig> days normalized to the respective mrna expression in naïve t cells.
flow cytometry histograms illustrating dbp-af <dig> fluorescence of activated t cells incubated with  <dig> nm dbp-af <dig> in the absence or presence of  rap ,  unlabelled dbp , or  eipa .
tcr internalization in activated t cells re-stimulated with  <dig> nm pdbu for 60 min in the absence or presence of eipa .
tcr surface expression of non-stimulated cells was set to 100%.
cd <dig> expression  and ifn-γ production  of t cells activated for 3 days with the indicated concentrations of dbp in x-vivo  <dig> and x-vivo  <dig> supplemented with either 25d <dig>  or  <dig> d <dig> .
data are shown as mean ± sem from  <dig> independent experiments.
in addition to receptor-mediated endocytosis, cells can take up extracellular molecules by macropinocytosis.
macropinocytosis is characterized by its augmentation by cell activation and that it can be inhibited by amiloride  <cit> .
we found that treatment of the cells with the amiloride analogue 5--amiloride  significantly inhibited uptake of dbp-af <dig> .
to ensure that eipa only blocked macropinocytosis and not receptor-mediated endocytosis, we measured the effect of eipa on t cell receptor  internalization in parallel.
tcr internalization is dependent on binding of the cd3γ di-leucine based internalization motif to adaptor molecules of clathrin-coated pits  <cit>  and hence should not be influenced by eipa.
we found that eipa did not affect tcr internalization .
thus, we could conclude that dbp uptake by cd4+ t cells is not mediated by megalin-mediated endocytosis but most likely by macropinocytosis.
whereas megalin-mediated endocytosis of dbp facilitates uptake and conversion of 25d <dig> to  <dig> d <dig> in kidney and mammary cells  <cit> , macropinocytosis of dbp-25d <dig> complexes did certainly not facilitate conversion of 25d <dig> to  <dig> d <dig> in t cells .
to study whether dbp inhibited the effects of  <dig> d <dig> in the same way as it inhibited the effects of 25d <dig> on t cells, we stimulated cd4+ t cells for 3 days in the presence of either 25d <dig> or  <dig> d <dig> and increasing concentrations of dbp.
after 3 days we measured cd <dig> expression and ifn-γ production.
we found that in the absence of dbp both 25d <dig> and  <dig> d <dig> up-regulated cd <dig> expression and down-regulated ifn-γ production.
however, whereas addition of dbp clearly inhibited the effect of 25d <dig> it did not influence the effect of  <dig> d <dig> on t cell responses .
neither actin, arachidonic acid nor albumin affect the dbp-mediated inhibition of 25d3
in addition to 25d <dig> and  <dig> d <dig>  dbp can bind actin  <cit>  and fatty acids  <cit> .
it has been suggested that binding of fatty acids to dbp decreases the affinity of dbp for 25d <dig>  <cit> , and in theory this could increase the bioavailability of 25d <dig> for immune cells.
to study whether binding of actin or arachidonic acid to dbp affected dbp-mediated inhibition of 25d3-induced t cell responses, we stimulated cd4+ t cells in the absence or presence of dbp plus 25d <dig> and increasing concentrations of actin or arachidonic acid.
we found that neither actin nor arachidonic acid affected 25d3-induced t cell responses as measured by cd <dig> expression .
since albumin binds 25d <dig> with lower affinity than dbp, the ratio between dbp and albumin could in theory also affect the bioavailability of 25d3; however, as for actin and arachidonic acid we found that albumin did not affect the dbp-mediated inhibition of 25d3-induced t cell responses .figure  <dig>
neither actin, arachidonic acid or albumin increases the bioavailability of 25d
3
for t cells.
relative cd <dig> cell surface expression on t cells activated for 3 days in the presence of 25d <dig>  and in the absence or presence of dbp  and the indicated concentrations of  actin,  arachidonic acid or  albumin.
mean values ± sem from  <dig> independent experiments are plotted.
dbp carbonylation impedes dbp-mediated inhibition of 25d3-induced t cell responses
the experiments above demonstrated that activated t cells have the capacity to convert 25d <dig> to  <dig> d <dig> given that 25d <dig> is available in a sufficiently high free concentration but that dbp normally binds and sequesters 25d <dig>  the concentration of dbp relative to 25d <dig> determines the ratio of free to sequestered 25d <dig>  <cit> .
the concentration of dbp in serum is approximately  <dig> μm, i.e.
50– <dig> fold higher than the concentration of 25d <dig>  and the free to sequestered ratio of 25d <dig> is very low  <cit> .
however, primary immune responses are most often initiated in secondary lymphoid organs like lymph nodes where the concentration of dbp is unknown.
it has been reported that the protein concentration in extracellular fluid/peripheral lymph might be as low as 5–10% of that found in serum  <cit> , suggesting that the concentration of dbp in secondary lymphoid organs might be significantly lower than the concentration in serum.
in agreement, by semi-quantitative analyses we found that the concentration of dbp in central lymph from mini-pigs was approximately 25% of the concentration of dbp in serum .figure  <dig>
dbp carbonylation impedes dbp-mediated inhibition of 25d3-induced cd <dig> expression.
western blot of dbp and albumin in serial dilutions of plasma and central lymph.
western blot of non-oxidized  and oxidized  dbp that subsequently had been derivatized with  <dig> -dinitrophenyl hydrazine to detect carbonyl groups  or left untreated .
the membranes were first analysed with anti-dnp antibodies to detect carbonylated dbp.
subsequently, the membranes were stripped and re-blotted with anti-dbp antibodies to detect total dbp.
relative cd <dig> cell surface expression on t cells activated in the presence of 25d <dig>  and the indicated concentrations of dbp or carbonylated dbp .
mean values ± sem from  <dig> independent experiments are plotted.
representative western blot analysis of carbonylated dbp and total dbp  in human serum.
precipitated dbp was either derivatized with  <dig> -dinitrophenyl hydrazine to detect carbonyl groups  or left untreated .
lanes  <dig> and  <dig> present precipitated dbp diluted 1: <dig>  and lanes  <dig> and  <dig> present precipitated dbp diluted 1: <dig>
another mechanism that might decrease the concentration of functional dbp and thereby increase the availability of 25d <dig> is carbonylation of dbp.
carbonylation is a protein modification induced by oxidative stress  <cit> , and increased carbonylation of serum proteins including dbp is seen during inflammatory responses  <cit> .
carbonylation of dbp could lead to a higher concentration of free 25d <dig> due to a reduced affinity of carbonylated dbp for 25d <dig> and/or increased degradation of dbp.
to investigate this, we activated cd4+ t cells in the presence of 25d <dig> and increasing concentrations of either unmodified or carbonylated dbp .
we found that carbonylated dbp did not inhibit the effect of 25d <dig> to the same extent as unmodified dbp as measured by cd <dig> expression .
thus, inflammation-induced carbonylation of dbp might contribute to an increased availability of 25d <dig> during an immune reaction.
interestingly, we observed that a fraction of the non-oxidized purified human dbp was actually carbonylated .
this suggested that carbonylated dbp is found in human serum as recently described for rat serum  <cit> .
to investigate whether carbonylated dbp is found in human serum we immunoprecipitated dbp from freshly drawn human blood and either derivatized it with  <dig> -dinitrophenyl hydrazine to detect carbonylated dbp or left it untreated before western blot analysis with anti-dnp antibodies.
we found that carbonylated dbp is found in human serum .
discussion
this study shows that activated human cd4+ t cells express cyp27b <dig> and produce sufficient amounts of  <dig> d <dig> to affect vitamin d-responsive genes when cultured in the presence of physiological concentrations of 25d <dig> in dbp/serum-free medium.
we found that cyp27b <dig> expression becomes strongly up-regulated in activated cd4+ t cells , and our results thereby confirm and extend previous reports on cyp27b <dig> expression in t cells  <cit> .
however, although activated t cells express cyp27b <dig>  is has been discussed whether they actually have the ability to convert 25d <dig> to  <dig> d <dig>  thus, some studies have found that activated t cells can convert 25d <dig> to  <dig> d <dig>  <cit> , whereas a recent study found that t cells do not have this ability  <cit> .
by measuring  <dig> d <dig> in the medium of t cells activated in the presence of 25d <dig> we found that cyp27b <dig> expressed by the t cells is functional, and that t cells have the ability to produce significant amounts of  <dig> d <dig> .
when determining the capacity of t cells to convert 25d <dig> to  <dig> d <dig> the kinetics of cyp27b <dig> expression is important to take into account.
we and others  <cit>  found that  <dig> d <dig> production is very low  <dig> hours after t cell activation but that it strongly increases  <dig> hours after activation.
we find it plausible that the missing detection of  <dig> d <dig> produced by activated t cells in the study by jeffery et al.
<cit>  was due to the fact that the authors measured  <dig> d <dig> production after only  <dig> hours of activation.
thus, our study clarifies that activated human cd4+ t cells have the capacity to convert 25d <dig> to  <dig> d <dig>  furthermore, we demonstrated that activated t cells have the capacity to produce significantly high amounts of  <dig> d <dig> to affect vitamin d-responsive genes such as cd <dig>  <cit> , ctla- <dig>  <cit> , plc-γ <dig>  <cit> , il- <dig>  <cit>  and ifn-γ  <cit>  .
despite the ability of activated t cells to convert 25d <dig> to  <dig> d <dig>  addition of dbp to the medium inhibited the effect of 25d <dig> on vitamin d-responsive genes in a dose-dependent manner .
interestingly, dbp did not seem to significantly inhibit  <dig> d3-induced t cell responses .
the affinity of dbp for 25d <dig> is significantly higher than for  <dig> d <dig> with a kd of  <dig>  nm and  <dig> nm, respectively  <cit> , and this could be one of the reasons that dbp sequestered 25d <dig> more efficiently than  <dig> d <dig>  megalin-mediated endocytosis of dbp facilitates uptake and conversion of 25d <dig> to  <dig> d <dig> in some types of cells such as renal proximal tubule cells and mammary epithelial cells  <cit> .
we found that activated t cells express megalin and take up dbp.
however, they do not take up dbp by megalin-mediated endocytosis as demonstrated by the lack of effect of rap, blocking anti-megalin antibodies and competition experiments .
in line with this, previous studies have demonstrated that megalin-mediated endocytosis of dbp is dependent of the co-expression of cubilin  <cit> , and we found that cubilin expression was very low in naïve t cells and that it was not up-regulated following t cell activation.
interestingly, we found that eipa, which inhibits macropinocytosis, reduced the dbp up-take.
thus, activated t cells take up dbp, but this up-take is not mediated by megalin-mediated endocytosis but most likely by macropinocytosis.
in contrast to megalin-mediated endocytosis that promotes the conversion of 25d <dig> to  <dig> d <dig> in kidney and mammary cells  <cit> , macropinocytosis of 25d3-dbp did not deliver 25d <dig> for subsequent conversion to  <dig> d <dig> in t cells.
similar results have been found for monocytes that also take up dbp by a megalin-independent mechanism and where dbp inhibits the conversion of 25d <dig> to  <dig> d <dig>  <cit> .
by titrations of 25d <dig> and  <dig> d <dig> in serum-free medium, we found that maximal effect on vitamin d-regulated genes was obtained at  <dig> and  <dig> nm, respectively, as assessed by cd <dig> expression .
addition of serum or purified dbp considerably shifted the concentration of 25d <dig> but not of  <dig> d <dig> required to affect vitamin d-responsive genes .
these results support that the physiological concentration of  <dig> d <dig>  is not sufficiently high to affect t cell responses, and that a significant local production of  <dig> d <dig> is essential to reach concentration  required to affect t cells as previously suggested  <cit> .
furthermore, these results indicate that mechanisms must exist whereby 25d <dig> is released from dbp and becomes available for the conversion to  <dig> d <dig>  given that 25d <dig> affects t cell responses in vivo.
in a search for such mechanisms, we investigated whether actin, arachidonic acid or albumin affected the sequestration of 25d <dig> by dbp, as dbp can bind actin  <cit>  and fatty acids  <cit> , and such binding might affect the affinity of dbp for 25d <dig>  <cit> .
however, neither actin, arachidonic acid nor albumin affected the dbp-mediated inhibition of 25d3-induced t cell responses .
local concentrations and/or modifications of dbp might also affect the availability of 25d <dig> to t cells.
inflammation-induced oxidative stress can result in oxidative modifications of proteins leading to protein carbonylation  <cit> .
protein carbonylation is irreversible and leads to disturbances in protein conformation and function  <cit> .
interestingly, we found evidence that carbonylation of dbp impedes dbp-mediated inhibition of 25d3-induced t cell responses .
thus, inflammation-induced oxidative stress could locally lead to dbp carbonylation and thereby to a higher concentration of free 25d <dig>  finally, the dbp gene is polymorphic, and the three most common dbp isotypes termed gc1s, gc1f and gc <dig> have varying affinities for 25d <dig>  which might also influence the availability and conversion of 25d <dig> to  <dig> d <dig> and thereby the efficiency of 25d3-induced t cell responses  <cit> .
experiments by nature indicate that significant amounts of  <dig> d <dig> actually can be produced locally by the involved immune cells during inflammation/infection in vivo.
thus, elevated systemic levels of  <dig> d <dig> can be observed in patients with granulomatous diseases such as sarcoidosis and tuberculosis  <cit> .
the granulomas are characterized by a central area of activated macrophages surrounded by activated cd4+ t cells.
this suggests that interactions between activated t cells and macrophages might induce mechanisms that allow efficient conversion of 25d <dig> to  <dig> d3in vivo despite the presence of dbp.
this is in good accordance with previous studies which found that treatment of macrophages with ifn-γ or soluble cd40l increases their expression of cyp27b <dig> and their capacity to convert 25d <dig> to  <dig> d <dig>  <cit> .
thus, whether vitamin d actually affects a given t cell response in vivo probably relies on a mixture of factors in addition to the concentration of 25d <dig> such as the isotype, local concentration and degradation rate of dbp and the expression levels of cyp27b <dig>  the vdr and the  <dig> d3-24-hydroxylase cyp24a <dig> of the cells locally involved in the immune response.
CONCLUSIONS
in summary, activated t cells express cyp27b <dig> and can convert 25d <dig> to  <dig> d <dig> in sufficiently high concentrations to affect vitamin d-responsive genes when cultured in serum-free medium.
however, dbp sequesters 25d <dig> and inhibits the production of  <dig> d <dig> in t cells.
to fully exploit the immune-regulatory potential of vitamin d, further studies of the mechanisms that enable the immune system to exploit 25d <dig> and convert it to  <dig> d <dig> are required.

 following antigen recognition cd4+ t cells differentiate into one of several types of th cells including th th th and treg cells that secrete distinct sets of cytokines. studies have suggested that, in addition to the cytokine milieu, vitamin d is an important determinant in this differentiation of cd4+ t cells. thus, in vitro studies have shown that the active form of vitamin d 1α,25-dihydroxyvitamin d 2d3), inhibits production of ifn-γ and augment the production of il- thereby limiting th and promoting th cell differentiation. furthermore, d inhibits th cell differentiation and induces differentiation of treg cells. it is therefore generally believed that vitamin d plays an anti-inflammatory role, and accordingly vitamin d deficiency has been associated with increased risk of autoimmune diseases such as type diabetes mellitus, lupus erythematosus and multiple sclerosis. 25-hydroxyvitamin d d3) is the inactive precursor of d and is considered the best parameter for evaluation of the vitamin d status of a subject. the normal range of serum 25d concentrations is 25– nm. the serum concentration of the active d is approximately 1000-fold lower and far below the effective concentration of d found in in vitro studies. thus, in most in vitro studies more than a 100-fold higher concentration of d than found in serum is often required to obtain an effect. it has therefore been suggested that the level of circulating d is too low to affect immune responses in vivo, and that sufficient levels are obtained by local conversion of 25d to d. in accordance, it has been shown that activated antigen presenting cells express the 25d-1α-hydroxylase cyp27b that converts 25d to d and that apc can produce d from 25d3in vitro and respond to this through the vitamin d receptor in an autocrine fashion. elevated levels of d in association with hypercalcemia have been observed in patients with sarcoidosis, tuberculosis, and other infections and inflammatory diseases in which the pathology is characterized by granuloma formation, supporting the hypothesis that activated macrophages can produce significant amounts of d3in vivo. like apc, activated t cells express the vdr and cyp27b. however, whether t cells can convert 25d to d in physiological relevant concentrations and respond to this in an autocrine fashion is a matter of debate. most studies on the effect of vitamin d on t cells have not addressed this question as they investigated the direct effects of supra-physiological concentrations of d and not how 25d affects t cell responses. one study has shown that isolated t cells have the ability to convert 25d to d in concentrations that actually affects vitamin d-responsive genes in an autocrine fashion. in agreement, we recently found that purified cd4+ t cells have the ability to produce substantial amounts of d when activated in the presence of 25d. in contrast, another recent study found that although activated t cells do express cyp27b the expression level is not sufficiently high to allow production of d in concentrations that affect vitamin d-responsive genes. the authors found that 25d only affected t cell responses when apc were present, and suggested that apc locally secrete sufficient amounts of d to directly influence the surrounding t cells in a paracrine fashion. other important players influencing the bioavailable levels of vitamin d are the vitamin d-binding protein and albumin. 25d and d circulate bound to dbp and albumin with less than 1% in their free form. studies of dbp knock-out mice have shown that dbp acts as a vitamin d reservoir by protecting 25d and d from degradation and renal secretion. however, dbp also sequesters 25d and d and inhibits their action on monocytes, dc and keratinocytes in vitro. how dbp affects t cell responses to 25d still needs to be determined. the objectives of this study were to further elucidate whether t cells have the ability to convert 25d to d in proportions that affect a panel of vitamin d-responsive genes in an autocrine fashion and to investigate how dbp regulates t cell responses to 25d  activated t cells express cyp27b and have the capacity to convert 25d to d3 in order to convert 25d to d cells must express the 25d-1α-hydroxylase cyp27b to determine whether naïve cd4+ t cells express cyp27b we purified cd45ra+cd4+ cells from the blood of healthy donors. the resulting cell population contained 95–98% cd4+ t cells of which more than 96% were cd45ra+. the purified cells were stimulated with cd3/cd beads for 0–5 days in serum-free medium and their expression of cyp27b mrna was subsequently measured. we found that naïve cd4+ t cells express no or very low levels of cyp27b however, the cells started to express cyp27b mrna shortly after stimulation, and the expression peaked after 2– days of stimulation. these results suggested that activated human cd4+ t cells have the capacity to convert 25d to d to validate this, we stimulated purified cd4+ t cells in the presence of nm 25d corresponding to physiological concentrations of 25d in serum and then measured the production of d we found that activated cd4+ t cells produced d with a kinetic similar to the kinetics of cyp27b expression in the cells, and that the production peaked after 3 days of stimulation. finally, to determine whether the cells expressed the receptor for d we determined the expression of the vdr in cd4+ t cells stimulated for 0–5 days. we found that vdr expression peaked simultaneously with the peak production of d at day. taken together, these experiments demonstrated that activated human cd4+ t cells express cyp27b that they have the capacity to convert 25d at physiological concentrations to the active d and that they express the receptor for d figure activated t cells express cyp27b and have the capacity to convert 25d 3 to 2 d 3. relative cyp27b mrna expression in t cells activated for 0– days normalized to cyp27b expression in naïve t cells. values are given as mean + sem from independent experiments, *p < . d in the medium of t cells activated for 0– days in the presence of nm 25d data are given as mean ± sem from independent experiments, *p < . representative western blot of vdr and cd3ζ expression in t cells activated for 0– days. activated t cells have the ability to produce d in sufficiently high concentrations to affect vitamin d-responsive genes having demonstrated that activated cd4+ t cells have the capacity to produce the active form of vitamin d and that they simultaneously express the vdr, we next asked whether the production of d was sufficiently high to induce autocrine activation of vitamin d-responsive genes. to study this, we took advantage of the finding that the cell surface expression levels of cd on activated t cells are dependent on the concentration of d present during t cell activation. consequently, we activated cd4+ t cells in serum-free medium in the presence of increasing concentrations of 25d or d and then measured cd expression at day in accordance with previous studies, we found that d significantly up-regulated cd expression in activated t cells in a dose-dependent manner. in addition, we found that 25d also induced cd up-regulation, suggesting that activated t cells can produce sufficiently high concentrations of d to affect vitamin d-responsive genes. we noted that whereas 25d at a physiological concentration of nm induced maximum cd up-regulation, d was required at a 100-fold higher concentration than physiological levels to induce the same effect. that a supra-physiological concentration of d was required to obtain an effect in our study is in good line with previous studies. however, in contrast to our observations that 25d at nm had a clear effect on t cells, most previous studies on 25d required at least a 100-fold higher concentration of 25d than found in serum to induce an effect. one explanation for this discrepancy could be that whereas our experiments were performed in serum-free medium most previous studies have been performed in medium supplemented with serum. to study whether the presence of serum could explain the discrepancy between the concentrations of 25d required to obtain an effect on t cells, we activated cd4+ t cells in the presence of increasing concentrations of 25d and d in medium supplemented with 5% fetal bovine serum and measured cd expression at day we found that the presence of serum significantly shifted the concentration of 25d required to induce cd expression. thus, compared to serum-free medium approximately 100-fold higher concentrations of 25d were required to induce a similar effect on cd expression in t cells cultured in medium supplemented with serum. in contrast, the presence of serum did not significantly shift the concentration of d required to induce cd expression. to separately analyse the role of dbp and albumin in this serum-mediated inhibition of the effect of 25d on t cells, we activated cd4+ t cells in the presence of 100 nm 25d in serum-free medium supplemented with increasing concentrations of either dbp or albumin and measured cd expression at day we found that dbp at concentrations above 250– nm completely abolished the effect of 25d on cd expression, whereas albumin did not affect the effect of 25d.figure in the absence of serum activated t cells can produce 2 d 3 in concentrations that affect cd expression. relative cd cell surface expression on t cells activated for 3 days in x-vivo or x-vivo supplemented with 5% fbs in the presence of the indicated concentrations of 25d and d normalized to cd cell surface expression on t cells activated in the absence of 25d and d relative cd cell surface expression on t cells activated for 3 days in x-vivo medium supplemented with increasing concentrations of dbp or albumin in the presence of nm 25d normalized to cd cell surface expression on t cells activated in the absence of 25d relative cd cell surface expression on t cells activated for 3 days in the absence or presence of ketoconazole and the indicated concentrations of 25d3 or d3 normalized to cd cell surface expression on t cells activated in the absence of 25d or d mean values ± sem from independent experiments are plotted, *p < . although t cells converted 25d to d when activated in serum-free medium, we could not exclude the possibility that the observed 25d3-induced up-regulation of cd was caused directly by 25d and not by d to exclude this possibility, we determined the effect of the cyp27b inhibitor ketoconazole on cd up-regulation. we activated t cells in the absence or presence of ketoconazole and increasing concentrations of 25d we found that ketoconazole efficiently inhibited 25d3-induced cd up-regulation. these observations indicated that conversion of 25d to d is required for 25d3-induced cd up-regulation. if this is the case and ketoconazole does not in itself influence cd expression then ketoconazole should not affect d3-induced cd up-regulation. consequently, we activated t cells in the absence or presence of ketoconazole and increasing concentrations of d we found that ketoconazole did not affect d3-induced cd up-regulation. these experiments indicated that it is not 25d itself that induces cd up-regulation, but that the conversion of 25d to d is required for 25d3-induced cd up-regulation. thus, we could conclude that activated t cells have the capacity to produce sufficient d to affect vitamin d-responsive genes as measured by cd expression when cultured with physiological concentrations of 25d in serum-free medium. however, addition of dbp to the medium inhibited the effect of 25d in contrast, dbp did not seem to affect the concentration of d required to regulate vitamin d-responsive genes. dbp inhibits 25d3-induced t cell responses to determine whether the inhibitory effect of dbp on 25d was limited to cd gene expression or was a general phenomenon for vitamin d-responsive genes in t cells, we studied the influence of 25d and dbp alone or in combination on the expression of molecules known to be encoded by vitamin d-responsive genes such as ctla-, plc-γ, il- and ifn-γ in addition to cd. we stimulated cd4+ t cells for 3 days in serum-free medium or serum-free medium supplemented with either 25d or dbp alone or 25d plus dbp. we confirmed that 25d up-regulates cd mrna and protein in activated t cells, and that this up-regulation is completely inhibited by dbp. likewise, we found that the 25d3-induced up-regulation of ctla- plc-γ and il- and down-regulation of ifn-γ was abolished by dbp. thus, dbp generally inhibits the effect of 25d on vitamin d-responsive genes in t cells. to further elucidate the underlying mechanism behind the inhibition of 25d in t cells mediated by dbp, we measured the production of active d in the medium from the cultures described above. we found that in the presence of 25d activated t cells produced significant amounts of d3; however, addition of dbp completely abolished the production of d. thus, we could conclude that dbp strongly constrains the effect of 25d on vitamin d-responsive genes in t cells by inhibiting the conversion of 25d to d figure dbp limits the bioavailability of 25d 3 and inhibits vitamin d-induced gene expression in t cells. relative cd cell surface expression and mrna expression, ctla- expression, plc-γ expression, il- production and mrna expression, ifn-γ production and mrna expression, and d in the medium of t cells activated for 3 days in x-vivo, or x-vivo supplemented with either nm 25d, μm dbp or the combination of 25d and dbp. the mean concentration of il- and ifn-γ in the supernatants of cells activated in x-vivo was pg/ml and ng/ml, respectively. mean + sem from the numbers of independent experiments indicated by n in the legend above. t cells take up dbp from the environment by macropinocytosis dbp is internalized by megalin-mediated endocytosis in kidney and mammary cells that express megalin and cubilin. megalin-mediated endocytosis of dbp facilitates uptake and conversion of 25d to d in these types of cells. to study whether t cells can take up dbp from the environment, we incubated naïve and activated t cells with dbp conjugated to alexa fluor. flow cytometry revealed increased fluorescence of activated t cells compared to naïve t cells, suggesting that activated t cells take up dbp-af. to exclude that the increased fluorescence simply was caused by dbp-af adhering to the cell surface of the activated t cells as suggested in a previous study, we analysed the cells by confocal microscopy. we found that dbp-af resided in small vesicles in the cytosol and could thus conclude that activated t cells actually take up dbp from the medium. to determine whether this uptake might be mediated by megalin, we measured megalin and cubilin mrna expression in naïve and activated t cells. activated t cells up-regulated megalin mrna to a level more than 100-fold higher than in naive t cells, whereas the cubilin mrna levels were low in both naïve and activated t cells. in kidney and mammary cells megalin-mediated endocytosis of dbp is, in addition to megalin, dependent on the presence of cubilin at the cells surface. to determine whether dbp was taken up by megalin-mediated endocytosis in activated t cells despite their low expression of cubilin, we incubated cells with dbp-af in the absence or presence of the megalin antagonist receptor-associated protein as previously described. we found that rap did not influence dbp-af uptake in the t cells. furthermore, blocking antibodies against megalin and calcium deprivation, known to inhibit megalin-mediated endocytosis, did not affect dbp uptake. in line with this, high concentrations of dbp did not affect the uptake of dbp-af. taken together these experiments indicated that dbp is not taken up by megalin-mediated endocytosis in t cells.figure t cells take up dbp from the environment by macropinocytosis. representative flow cytometry histograms showing dbp-af fluorescence of naïve t cells and t cells activated for and days and subsequently incubated with nm dbp-af for 12 h at 37°c. representative confocal microscopy images illustrating the uptake of dbp-af in activated t cells. dbp-af are shown in green; cell membrane in red; nucleus in blue. relative megalin, cubilin, gapdh and rplp mrna expression in t cells activated from 0– days normalized to the respective mrna expression in naïve t cells. flow cytometry histograms illustrating dbp-af fluorescence of activated t cells incubated with nm dbp-af in the absence or presence of rap, unlabelled dbp, or eipa. tcr internalization in activated t cells re-stimulated with nm pdbu for 60 min in the absence or presence of eipa. tcr surface expression of non-stimulated cells was set to 100%. cd expression and ifn-γ production of t cells activated for 3 days with the indicated concentrations of dbp in x-vivo and x-vivo supplemented with either 25d or d. data are shown as mean ± sem from independent experiments. in addition to receptor-mediated endocytosis, cells can take up extracellular molecules by macropinocytosis. macropinocytosis is characterized by its augmentation by cell activation and that it can be inhibited by amiloride. we found that treatment of the cells with the amiloride analogue 5--amiloride significantly inhibited uptake of dbp-af. to ensure that eipa only blocked macropinocytosis and not receptor-mediated endocytosis, we measured the effect of eipa on t cell receptor internalization in parallel. tcr internalization is dependent on binding of the cd3γ di-leucine based internalization motif to adaptor molecules of clathrin-coated pits and hence should not be influenced by eipa. we found that eipa did not affect tcr internalization. thus, we could conclude that dbp uptake by cd4+ t cells is not mediated by megalin-mediated endocytosis but most likely by macropinocytosis. whereas megalin-mediated endocytosis of dbp facilitates uptake and conversion of 25d to d in kidney and mammary cells, macropinocytosis of dbp-25d complexes did certainly not facilitate conversion of 25d to d in t cells. to study whether dbp inhibited the effects of d in the same way as it inhibited the effects of 25d on t cells, we stimulated cd4+ t cells for 3 days in the presence of either 25d or d and increasing concentrations of dbp. after 3 days we measured cd expression and ifn-γ production. we found that in the absence of dbp both 25d and d up-regulated cd expression and down-regulated ifn-γ production. however, whereas addition of dbp clearly inhibited the effect of 25d it did not influence the effect of d on t cell responses. neither actin, arachidonic acid nor albumin affect the dbp-mediated inhibition of 25d3 in addition to 25d and d dbp can bind actin and fatty acids. it has been suggested that binding of fatty acids to dbp decreases the affinity of dbp for 25d, and in theory this could increase the bioavailability of 25d for immune cells. to study whether binding of actin or arachidonic acid to dbp affected dbp-mediated inhibition of 25d3-induced t cell responses, we stimulated cd4+ t cells in the absence or presence of dbp plus 25d and increasing concentrations of actin or arachidonic acid. we found that neither actin nor arachidonic acid affected 25d3-induced t cell responses as measured by cd expression. since albumin binds 25d with lower affinity than dbp, the ratio between dbp and albumin could in theory also affect the bioavailability of 25d3; however, as for actin and arachidonic acid we found that albumin did not affect the dbp-mediated inhibition of 25d3-induced t cell responses.figure neither actin, arachidonic acid or albumin increases the bioavailability of 25d 3 for t cells. relative cd cell surface expression on t cells activated for 3 days in the presence of 25d and in the absence or presence of dbp and the indicated concentrations of actin, arachidonic acid or albumin. mean values ± sem from independent experiments are plotted. dbp carbonylation impedes dbp-mediated inhibition of 25d3-induced t cell responses the experiments above demonstrated that activated t cells have the capacity to convert 25d to d given that 25d is available in a sufficiently high free concentration but that dbp normally binds and sequesters 25d the concentration of dbp relative to 25d determines the ratio of free to sequestered 25d. the concentration of dbp in serum is approximately μm, i.e. 50– fold higher than the concentration of 25d and the free to sequestered ratio of 25d is very low. however, primary immune responses are most often initiated in secondary lymphoid organs like lymph nodes where the concentration of dbp is unknown. it has been reported that the protein concentration in extracellular fluid/peripheral lymph might be as low as 5–10% of that found in serum, suggesting that the concentration of dbp in secondary lymphoid organs might be significantly lower than the concentration in serum. in agreement, by semi-quantitative analyses we found that the concentration of dbp in central lymph from mini-pigs was approximately 25% of the concentration of dbp in serum.figure dbp carbonylation impedes dbp-mediated inhibition of 25d3-induced cd expression. western blot of dbp and albumin in serial dilutions of plasma and central lymph. western blot of non-oxidized and oxidized dbp that subsequently had been derivatized with -dinitrophenyl hydrazine to detect carbonyl groups or left untreated. the membranes were first analysed with anti-dnp antibodies to detect carbonylated dbp. subsequently, the membranes were stripped and re-blotted with anti-dbp antibodies to detect total dbp. relative cd cell surface expression on t cells activated in the presence of 25d and the indicated concentrations of dbp or carbonylated dbp. mean values ± sem from independent experiments are plotted. representative western blot analysis of carbonylated dbp and total dbp in human serum. precipitated dbp was either derivatized with -dinitrophenyl hydrazine to detect carbonyl groups or left untreated. lanes and present precipitated dbp diluted 1: and lanes and present precipitated dbp diluted 1: another mechanism that might decrease the concentration of functional dbp and thereby increase the availability of 25d is carbonylation of dbp. carbonylation is a protein modification induced by oxidative stress, and increased carbonylation of serum proteins including dbp is seen during inflammatory responses. carbonylation of dbp could lead to a higher concentration of free 25d due to a reduced affinity of carbonylated dbp for 25d and/or increased degradation of dbp. to investigate this, we activated cd4+ t cells in the presence of 25d and increasing concentrations of either unmodified or carbonylated dbp. we found that carbonylated dbp did not inhibit the effect of 25d to the same extent as unmodified dbp as measured by cd expression. thus, inflammation-induced carbonylation of dbp might contribute to an increased availability of 25d during an immune reaction. interestingly, we observed that a fraction of the non-oxidized purified human dbp was actually carbonylated. this suggested that carbonylated dbp is found in human serum as recently described for rat serum. to investigate whether carbonylated dbp is found in human serum we immunoprecipitated dbp from freshly drawn human blood and either derivatized it with -dinitrophenyl hydrazine to detect carbonylated dbp or left it untreated before western blot analysis with anti-dnp antibodies. we found that carbonylated dbp is found in human serum. discussion this study shows that activated human cd4+ t cells express cyp27b and produce sufficient amounts of d to affect vitamin d-responsive genes when cultured in the presence of physiological concentrations of 25d in dbp/serum-free medium. we found that cyp27b expression becomes strongly up-regulated in activated cd4+ t cells, and our results thereby confirm and extend previous reports on cyp27b expression in t cells. however, although activated t cells express cyp27b is has been discussed whether they actually have the ability to convert 25d to d thus, some studies have found that activated t cells can convert 25d to d, whereas a recent study found that t cells do not have this ability. by measuring d in the medium of t cells activated in the presence of 25d we found that cyp27b expressed by the t cells is functional, and that t cells have the ability to produce significant amounts of d. when determining the capacity of t cells to convert 25d to d the kinetics of cyp27b expression is important to take into account. we and others found that d production is very low hours after t cell activation but that it strongly increases hours after activation. we find it plausible that the missing detection of d produced by activated t cells in the study by jeffery et al. was due to the fact that the authors measured d production after only hours of activation. thus, our study clarifies that activated human cd4+ t cells have the capacity to convert 25d to d furthermore, we demonstrated that activated t cells have the capacity to produce significantly high amounts of d to affect vitamin d-responsive genes such as cd, ctla-, plc-γ, il- and ifn-γ. despite the ability of activated t cells to convert 25d to d addition of dbp to the medium inhibited the effect of 25d on vitamin d-responsive genes in a dose-dependent manner. interestingly, dbp did not seem to significantly inhibit d3-induced t cell responses. the affinity of dbp for 25d is significantly higher than for d with a kd of nm and nm, respectively, and this could be one of the reasons that dbp sequestered 25d more efficiently than d megalin-mediated endocytosis of dbp facilitates uptake and conversion of 25d to d in some types of cells such as renal proximal tubule cells and mammary epithelial cells. we found that activated t cells express megalin and take up dbp. however, they do not take up dbp by megalin-mediated endocytosis as demonstrated by the lack of effect of rap, blocking anti-megalin antibodies and competition experiments. in line with this, previous studies have demonstrated that megalin-mediated endocytosis of dbp is dependent of the co-expression of cubilin, and we found that cubilin expression was very low in naïve t cells and that it was not up-regulated following t cell activation. interestingly, we found that eipa, which inhibits macropinocytosis, reduced the dbp up-take. thus, activated t cells take up dbp, but this up-take is not mediated by megalin-mediated endocytosis but most likely by macropinocytosis. in contrast to megalin-mediated endocytosis that promotes the conversion of 25d to d in kidney and mammary cells, macropinocytosis of 25d3-dbp did not deliver 25d for subsequent conversion to d in t cells. similar results have been found for monocytes that also take up dbp by a megalin-independent mechanism and where dbp inhibits the conversion of 25d to d. by titrations of 25d and d in serum-free medium, we found that maximal effect on vitamin d-regulated genes was obtained at and nm, respectively, as assessed by cd expression. addition of serum or purified dbp considerably shifted the concentration of 25d but not of d required to affect vitamin d-responsive genes. these results support that the physiological concentration of d is not sufficiently high to affect t cell responses, and that a significant local production of d is essential to reach concentration required to affect t cells as previously suggested. furthermore, these results indicate that mechanisms must exist whereby 25d is released from dbp and becomes available for the conversion to d given that 25d affects t cell responses in vivo. in a search for such mechanisms, we investigated whether actin, arachidonic acid or albumin affected the sequestration of 25d by dbp, as dbp can bind actin and fatty acids, and such binding might affect the affinity of dbp for 25d. however, neither actin, arachidonic acid nor albumin affected the dbp-mediated inhibition of 25d3-induced t cell responses. local concentrations and/or modifications of dbp might also affect the availability of 25d to t cells. inflammation-induced oxidative stress can result in oxidative modifications of proteins leading to protein carbonylation. protein carbonylation is irreversible and leads to disturbances in protein conformation and function. interestingly, we found evidence that carbonylation of dbp impedes dbp-mediated inhibition of 25d3-induced t cell responses. thus, inflammation-induced oxidative stress could locally lead to dbp carbonylation and thereby to a higher concentration of free 25d finally, the dbp gene is polymorphic, and the three most common dbp isotypes termed gc1s, gc1f and gc have varying affinities for 25d which might also influence the availability and conversion of 25d to d and thereby the efficiency of 25d3-induced t cell responses. experiments by nature indicate that significant amounts of d actually can be produced locally by the involved immune cells during inflammation/infection in vivo. thus, elevated systemic levels of d can be observed in patients with granulomatous diseases such as sarcoidosis and tuberculosis. the granulomas are characterized by a central area of activated macrophages surrounded by activated cd4+ t cells. this suggests that interactions between activated t cells and macrophages might induce mechanisms that allow efficient conversion of 25d to d3in vivo despite the presence of dbp. this is in good accordance with previous studies which found that treatment of macrophages with ifn-γ or soluble cd40l increases their expression of cyp27b and their capacity to convert 25d to d. thus, whether vitamin d actually affects a given t cell response in vivo probably relies on a mixture of factors in addition to the concentration of 25d such as the isotype, local concentration and degradation rate of dbp and the expression levels of cyp27b the vdr and the d3-24-hydroxylase cyp24a of the cells locally involved in the immune response.  in summary, activated t cells express cyp27b and can convert 25d to d in sufficiently high concentrations to affect vitamin d-responsive genes when cultured in serum-free medium. however, dbp sequesters 25d and inhibits the production of d in t cells. to fully exploit the immune-regulatory potential of vitamin d, further studies of the mechanisms that enable the immune system to exploit 25d and convert it to d are required. 
in vitro studies have shown that the active form of vitamin d 1α,25-dihydroxyvitamin d 2d3), can regulate differentiation of cd4+ t cells by inhibiting th and th cell differentiation and promoting th and treg cell differentiation. however, the serum concentration of d is far below the effective concentration of d found in in vitro studies, and it has been suggested that d must be produced locally from the inactive precursor 25-hydroxyvitamin d d3) to affect ongoing immune responses in vivo. although it has been reported that activated t cells express the 25d-1α-hydroxylase cyp27b that converts 25d to d it is still controversial whether activated t cells have the capacity to produce sufficient amounts of d to affect vitamin d-responsive genes. furthermore, it is not known how the vitamin d-binding protein found in high concentrations in serum affects t cell responses to 25d we found that activated t cells express cyp27b and have the capacity to produce sufficient d to affect vitamin d-responsive genes when cultured with physiological concentrations of 25d in serum-free medium. activated t cells express cyp27b and can convert 25d to d in sufficiently high concentrations to affect vitamin d-responsive genes when cultured in serum-free medium. to fully exploit the immune-regulatory potential of vitamin d, future studies of the mechanisms that enable the immune system to exploit 25d and convert it to d3in vivo are required. t cellst cell activationvitamin dvitamin d-binding proteincyp27b1megalincubilinissue-copyright-statement© the author 2014 in contrast, dbp did not inhibit the effect of exogenous d actin, arachidonic acid and albumin did not affect the sequestration of 25d by dbp, whereas carbonylation of dbp did. however, if the medium was supplemented with serum or purified dbp, dbp strictly inhibited the production of d and 25d3-induced t cell responses. however, dbp sequesters 25d and inhibits the production of d in t cells.
 in vitro studies have shown that the active form of vitamin d 1α,25-dihydroxyvitamin d 2d3), can regulate differentiation of cd4+ t cells by inhibiting th and th cell differentiation and promoting th and treg cell differentiation. however, the serum concentration of d is far below the effective concentration of d found in in vitro studies, and it has been suggested that d must be produced locally from the inactive precursor 25-hydroxyvitamin d d3) to affect ongoing immune responses in vivo. although it has been reported that activated t cells express the 25d-1α-hydroxylase cyp27b that converts 25d to d it is still controversial whether activated t cells have the capacity to produce sufficient amounts of d to affect vitamin d-responsive genes. furthermore, it is not known how the vitamin d-binding protein found in high concentrations in serum affects t cell responses to 25d  we found that activated t cells express cyp27b and have the capacity to produce sufficient d to affect vitamin d-responsive genes when cultured with physiological concentrations of 25d in serum-free medium. however, if the medium was supplemented with serum or purified dbp, dbp strictly inhibited the production of d and 25d3-induced t cell responses. in contrast, dbp did not inhibit the effect of exogenous d actin, arachidonic acid and albumin did not affect the sequestration of 25d by dbp, whereas carbonylation of dbp did.  activated t cells express cyp27b and can convert 25d to d in sufficiently high concentrations to affect vitamin d-responsive genes when cultured in serum-free medium. however, dbp sequesters 25d and inhibits the production of d in t cells. to fully exploit the immune-regulatory potential of vitamin d, future studies of the mechanisms that enable the immune system to exploit 25d and convert it to d3in vivo are required. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords t cellst cell activationvitamin dvitamin d-binding proteincyp27b1megalincubilinissue-copyright-statement© the author 2014 