BACKGROUND
rheumatoid arthritis  is a chronic autoimmune disease that is mainly characterized by inflammation of the synovial tissue , leading to cartilage and bone destruction.
influx of different inflammatory cells into the st and enhanced production of cytokines and chemokines are all well known features of ra.
chemokines are small proteins that act as key players in the chemo-attraction of different leucocytes and perform their chemo-attractive task through interaction with their receptor on the target cell.
several chemokines have been shown to be abundantly present in ra st at highly strategic sites  <cit> , which suggests a role for these chemokines in the pathogenesis of ra.
in this respect, chemokines could be regarded as promising therapeutic targets in ra.
this concept has already been translated to the clinic, since the blockade of c chemokine receptor  <dig>  has recently been shown to be clinically effective in the treatment of ra  <cit> .
antigen presenting cells , such as dendritic cells  and macrophages , are generally accepted as critical mediators in the complex pathogenesis of ra  <cit> .
apc produce a multitude of chemokines that attract specific t cell subsets.
such chemokines are likely to play a critical role in the regulation of immune responses, since they orchestrate the spatial and temporal interaction between apc and t cells, which determines the fate and nature of the immune response.
evidence for this conceptual framework came recently from the observation that blocking apc-t cell interactions using ctla4-ig led to a significant reduction of disease activity in ra  <cit> .
several chemokines orchestrate the attraction of t cells toward dc.
it is tempting to speculate that interfering with these chemokines would lead to similar effects on disease activity as the direct blockade of t-cell dc interaction.
of this group of t- cell attracting chemokines, ccl <dig> and cxcl <dig> recently came out as potentially interesting targets in ra from previous research by our group and others  <cit> .
cc chemokine ligand  <dig>  was first identified as a naïve t cell attracting chemokine  <cit> .
next to chemo-attraction, ccl <dig> plays a role in stimulation of collagen production by fibroblasts  <cit> .
despite numerous attempts to identify its receptor, ccl <dig> is still an orphan chemokine.
in vivo, ccl <dig> expression was first found in high quantities in the lung, which is caused by the abundant expression by alveolar macrophages  <cit> .
in vitro, dc and mΦ have been identified as ccl <dig> producers  <cit> .
to date, a substantial amount of data points toward the enrichment of dc and mΦ in the synovial tissue which likely to be responsible for the increased levels of ccl <dig> in ra synovial tissue and synovial fluid  compared with that from healthy individuals  <cit> .
in this line, ccl <dig> has been identified as a clinical marker in gaucher's disease, a condition in which mΦ activation is likely to play a role in the pathogenesis  <cit> .
in addition, a role for ccl <dig> has been suggested in a large variety of diseases, such as systemic sclerosis and acute lymphoblastic leukaemia  <cit> .
in ra, we recently found that circulating ccl <dig> levels are elevated compared with controls and correlated with disease activity .
moreover, ccl <dig> mrna expression by dc from ra patients was shown to be higher than by dc from healthy controls, which could be influenced by blockade of tnf-α  <cit> .
the exact regulation of ccl <dig> protein secretion however is complicated and the studies published thus far have led to controversial results  <cit> , as elegantly reviewed by schutyser et al  <cit> .
in order to clarify the mechanism of ccl <dig> expression and secretion in ra, we investigated the role of a large panel of inflammatory mediators known to play a role in the disease process on ccl <dig> secretion.
here we show that ccl <dig> secretion by monocytes and dc is regulated by synergistic effects between il-4/il- <dig>  il- <dig> and ra sf, whereas pro-inflammatory cytokines and toll-like receptor  ligands did not have any influence on ccl <dig> secretion.
these data add novel information to the puzzle of increased ccl <dig> expression in ra.
RESULTS
il- <dig> strongly enhances ccl <dig> production by modc while maturation and pro-inflammatory mediators do not
first we investigated whether several mediators that are known to be important in ra were able to enhance ccl <dig> production by modc.
in line with previous studies, unstimulated immature dc  produced significant amounts of ccl <dig>  <cit> .
interestingly, incubation with tnf-α, il-1β, il- <dig>  il- <dig>  il- <dig>  il- <dig> and ifn-γ did not stimulate ccl <dig> secretion when added to day  <dig> idc .
in contrast, the anti-inflammatory il- <dig> strongly induced ccl <dig> production by idc  .
next we tested whether factors well known to induce maturation or t cell mimicking could induce ccl <dig> production.
these experiments demonstrated that lps, cd40l and rankl did not enhance ccl <dig> production  .
recent studies demonstrated that other tlr pathways than tlr <dig> are all capable of inducing dc maturation, but have different effects on cytokine production  <cit> .
however, stimulation of tlr <dig> , tlr <dig> ), tlr <dig>  or tlr7/ <dig>  did not sort any effect on ccl <dig> secretion  , whereas they did elicit a potent cytokine response  <cit> .
since il- <dig> is more abundantly present in ra than il- <dig> and since some conflicting results have been published on ccl <dig> production induced by lps when dc were cultured with il- <dig> vs. il- <dig>  we compared these culture methods .
in both the il- <dig> and il- <dig> cultures, il- <dig> strongly induced ccl <dig> , while lps again did not .
in addition, il- <dig> in combination with lps was not significantly different from il- <dig> alone .
also co-stimulation with lps and the cytokines tested  did not sort any effect on ccl <dig> secretion .
il- <dig> acts in synergy with il-4/l- <dig> in promoting ccl <dig> production by monocytes
modc and alternatively activated mΦ   <cit>  are known to produce ccl <dig>  both these cell types originate from cd14+ monocytes and depend on il- <dig> or il- <dig>  for their differentiation.
to determine whether ccl <dig> secretion by myeloid cells is dependent on these cytokines, monocytes were freshly isolated and stimulated with gm-csf, il- <dig>  il- <dig> and il- <dig> alone or in combinations .
even after  <dig> days, unstimulated and gm-csf treated monocytes/macrophages did not secrete ccl <dig>  whereas both il- <dig> and il- <dig> stimulation resulted in a clear secretion of ccl <dig>  which is in line with previous findings on aamΦ  <cit> .
interestingly, stimulation with il- <dig> alone only had a minimal effect on ccl <dig> production by these monocytes/macrophages.
when il- <dig> was provided together with il- <dig> or il- <dig>  this resulted in 3- and 2-fold increase in ccl <dig> secretion respectively .
interestingly, already in low concentrations, il- <dig> had its synergistic effect with il- <dig> .
in order to rule out effects of adherence, we cultured cd14+ monocytes/macrophages for three days in teflon bags  <cit>  and in rotation discs  <cit> .
the morphology of these cells was comparable with freshly isolated monocytes according to their forward/side scatter pattern .
in both cultures, il- <dig> did still induce ccl <dig> production in the same way as the cultures in 24-wells plates .
as a proof of principle, we next tested whether the synergy between il-4/il- <dig> and il- <dig> could already be observed after only  <dig> hours.
intriguingly, we could indeed observe a clear ccl <dig> secretion after  <dig> hours upon stimulation of freshly isolated monocytes with il-4/il- <dig> and il- <dig>  whereas stimulation with il- <dig>  il- <dig> or il- <dig> alone did only result in a minor or even undetectable ccl <dig> secretion  .
since il- <dig> appeared to synergize with il- <dig> and il- <dig>  we investigated whether these cytokines could up-regulate each other's receptors, possibly resulting in enhanced signaling.
this was not the case; il- <dig> did not up regulate either the il-4/il- <dig> common receptor il-4rα <dig> or the specific il-13rα <dig>  furthermore, il- <dig> had no effects on il-10rα .
ra synovial fluid enhances ccl <dig> secretion independently of il- <dig> and il-13
we and others demonstrated ccl <dig> expression in ra st in the lining and the peri-vascular regions  <cit> .
in figure  <dig>  we show a high ccl <dig> expression in ra synovial tissue , which was preferentially located in both the synovial lining layer and the peri-vascular regions.
intriguingly, ccl <dig> was also expressed in control synovial tissue, although not as abundant as in ra st .
notably, some parts of the sections were even negative for ccl <dig>  which is in sharp contrast with ra.
in order to explain the abundant ccl <dig> expression in ra, we tested whether incubation with ra sf could induce ccl <dig> production on monocytes/macrophages.
since ra sf itself contains ccl <dig>  <cit> , we cultured freshly isolated monocytes for  <dig> days in the presence of sf, washed the cells and cultured on for another  <dig> days in the absence of ra sf .
firstly, this pre-incubation with ra sf resulted in marked ccl <dig> production  pg/ml) .
secondly, culture of freshly isolated monocytes in the presence of ra sf, resulted in a 9- and 10-fold increase in ccl <dig> secretion upon stimulation with il4/il- <dig> respectively and a 22-fold increase compared with il- <dig> alone .
intriguingly, this synergistic effect with il- <dig>  il- <dig> and il- <dig> could still be observed after  <dig> days of culture in the complete absence of ra sf , indicating that the cell does not require a continuous stimulation in order to secrete ccl <dig>  to exclude intrinsic differences between monocytes/macrophages from ra patients and controls may contribute to the effects on ccl <dig> secretion, we tested whether monocytes/macrophages from ra patients  responded differently to combinations of il- <dig>  il- <dig>  il- <dig> and sf.
no difference in the ccl <dig> secretion pattern was observed between monocytes/macrophages of healthy controls and ra patients upon these stimuli , ruling out intrinsic differences in monocytes in ra that affect ccl <dig> secretion.
since the synergy caused by ra sf appeared similar to the synergy between cytokines we already observed , we tested whether il- <dig> and/or il- <dig>  both present in ra sf, were responsible for this phenomenon by blocking these cytokines with neutralizing antibodies.
the potency of these antibodies was first tested by determining their ability to inhibit the synergistic ccl <dig> secretion upon stimulation with a combination of il- <dig> and il-4/il- <dig>  addition of anti-il- <dig> resulted in a 73% inhibition of the synergy between il- <dig> and il- <dig> and anti il- <dig> completely abrogated the synergistic effect of il- <dig> with il- <dig> .
unexpectedly however, blockade of il- <dig> or il- <dig> in sf in the presence of il- <dig> or il- <dig> and il- <dig> respectively did not inhibit the synergy between these cytokines and sf , suggesting that sf contains a yet unidentified factor that triggers ccl <dig> secretion.
discussion
in this study, we add new pieces to the complicated puzzle of ccl <dig> regulation in ra.
firstly, we demonstrate that ccl <dig> production can be induced by il- <dig>  il- <dig> and il- <dig> in monocyte derived cells.
secondly, we show that a large panel of pro-inflammatory stimuli and tlr mediated signals leading to dc maturation are of no influence on ccl <dig> production.
thirdly, il- <dig> only induces a minor ccl <dig> secretion, but acts in synergy with both il- <dig> and il- <dig> on monocytes and monocyte derived cells.
finally, we provide evidence that ra sf is able to induce ccl <dig> secretion in strong synergy with il- <dig>  il- <dig> and il- <dig>  which could not be inhibited by a blockade of il- <dig> and il- <dig>
ccl <dig> can be produced by modc as well as by certain types of mΦ.
often these cell types are considered to be totally different cells.
however, the differences between modc and aamΦ are not that large, since monocyte derived macrophages are cultured in the presence of gm-csf by some groups  <cit>  and both cells require the presence of il- <dig> or il- <dig>  penna and co workers demonstrated that several in vivo dc subtypes were not able to produce ccl <dig>  <cit> , which is in contrast with previous findings, where ccl <dig> mrna expression was found on cd11c+ myeloid blood dc  <cit> .
moreover, in vitro cultured modc have been identified as potent ccl <dig> producers  <cit> .
these data suggest that a cd14+ monocyte origin in combination with a stimulation by il-4/il- <dig> is critical for ccl <dig> secretion.
this hypothesis is strengthened our data, demonstrating that non-adherent monocytes/macrophages were able to produce ccl <dig> under the influence of il- <dig>  in addition, the synergistic effects of il-4/il- <dig> and il- <dig> on ccl <dig> secretion by freshly isolated monocytes were already clearly visible after  <dig> hours.
this indicates that a full differentiation into dc or mΦ is not essential for ccl <dig> production as has been suggested previously for ccl <dig> mrna expression  <cit> .
thus monocytes rapidly secrete ccl <dig> upon triggering with the right stimuli.
in the literature there is still some controversy regarding the effect of dc maturation on ccl <dig> production.
vulcano and co workers suggested that dc down regulate their ccl <dig> secretion upon maturation  <cit> .
this is in contrast with results from other studies, where maturation caused an increased mrna expression  <cit> .
a similar contrast between protein and mrna was found on blood dc  <cit> .
the reason for these differences between mrna expression and protein secretion patterns still needs to be investigated in detail.
recently, we already provided evidence that dc maturation does not influence ccl <dig> protein secretion  <cit> , which is further strengthened by the data from the present study, in which different tlr stimulatory pathways did not induce ccl <dig> production, whereas full dc maturation was achieved  <cit> .
also tnf-α and cd40l, both well appreciated inducers of dc maturation  <cit> , did not enhance ccl <dig> production.
perhaps the discrepancy between the different reports is hidden in subtle differences in culture conditions, which are difficult to trace in the published data.
intriguingly, stimulation with il- <dig> alone only lead to a marginal induction of ccl <dig> secretion by monocytes/macrophages, but did act in a strong synergy with il- <dig> or il- <dig>  the latter is not caused by an up regulation of the receptors il-10rα, il-4rα or il-13rα <dig> .
probably intracellular pathways direct the synergy between these cytokines, which is an interesting topic that warrants further investigation.
we showed that ra sf induces ccl <dig> production and strongly synergizes with il- <dig>  il- <dig> and il- <dig>  blocking studies revealed that neither il- <dig> nor il- <dig> in sf were responsible for this effect.
this suggests the presence of another, yet unidentified ccl <dig> inducing factor in ra sf.
another explanation for this fact might be the presence of inhibiting factors in sf that counter-regulate the effects of il- <dig> and il- <dig>  the identification of the factor in sf that drives the effects on ccl <dig> secretion may provide important new insights to the pro-vs. anti inflammatory balance in ra.
in order to find this factor in a complex fluid like sf, more knowledge on the pathways of ccl <dig> regulation is critical.
another intriguing observation from our study is the finding that pre-incubation with sf lead to a sustained synergistic ccl <dig> secretion upon stimulation with il- <dig>  il- <dig> and il- <dig>  this could be regarded as an "imprinting effect", meaning that the cell's previous environment determines the nature of response to stimuli, even when the cell is no longer in such an environment.
results from previous studies, in which we showed that modc from ra patients differ in phenotype and cytokine response from control dc after  <dig> days in culture might also be explained by such a phenomenon  <cit> .
upon the encounter of an antigen, dc normally mature and migrate to lymphoid tissues in order to perform their task of antigen presentation to t cells.
immature dc or mΦ can also encounter naïve t cells in the periphery, which subsequently might result in tolerance  <cit> .
this peripheral tolerance is a critical mechanism to prevent auto-immunity.
a role for ccl <dig> in this part might explain the high expression of ccl <dig> by alveolar mΦ  <cit> , which are located at a site where the maintenance of tolerance to non-pathogenic antigens, that are constantly present, is crucial.
also the synergistic effect on ccl <dig> secretion that we found with il- <dig>  a cytokine that is well appreciated as a pivotal regulator of the immune system, fits in this picture.
the synovial lining in the joints has similarities with the alveolar lining in the lung.
they both consist of mΦ-like cells and both form a barrier to a site in which self- and non-pathogenic antigens are constantly present.
the disease process in ra is considered to be driven by pro inflammatory cytokines such as il-1β, tnf-α, il- <dig> and il- <dig>  <cit> , whereas ccl <dig> is regulated by il- <dig>  il- <dig> and il- <dig>  it is therefore tempting to speculate that the high ccl <dig> expression in ra is designed to uphold peripheral tolerance, which however seems to fail.
this failure might be explained in two ways.
the first explanation might be that the skewing in the balance towards th <dig> is still present despite the upregulation of anti inflammatory mediators.
secondly, mature dc are present in the synovial tissue in perivascular regions and secondary lymphoid organs  <cit> , which is in sharp contrast with healthy synovial tissue.
therefore an explanation for the ongoing immune process might be that these mature dc direct naïve t cells towards a phenotype that drives the pro-inflammatory immune response in the synovial tissue.
CONCLUSIONS
in summary, we provide evidence that monocyte derived cells produce ccl <dig> under the influence of il- <dig> and il- <dig>  il- <dig> acts in strong synergy with il- <dig> and il- <dig> as a key regulator of ccl <dig> production by monocytes, which indicates that ccl <dig> secretion is not confined to fully developed dc and mΦ.
in addition, the effects of il- <dig>  il- <dig> and il- <dig> are strongly enhanced by rasf, which is due to yet unidentified factors.
both the in vivo expression pattern and the contributing factors to its regulation in vitro are suggestive for a role for ccl <dig> in the regulation of the immune system, both in health and auto-immune diseases such as ra.

 rheumatoid arthritis is a chronic autoimmune disease that is mainly characterized by inflammation of the synovial tissue, leading to cartilage and bone destruction. influx of different inflammatory cells into the st and enhanced production of cytokines and chemokines are all well known features of ra. chemokines are small proteins that act as key players in the chemo-attraction of different leucocytes and perform their chemo-attractive task through interaction with their receptor on the target cell. several chemokines have been shown to be abundantly present in ra st at highly strategic sites, which suggests a role for these chemokines in the pathogenesis of ra. in this respect, chemokines could be regarded as promising therapeutic targets in ra. this concept has already been translated to the clinic, since the blockade of c chemokine receptor has recently been shown to be clinically effective in the treatment of ra. antigen presenting cells, such as dendritic cells and macrophages, are generally accepted as critical mediators in the complex pathogenesis of ra. apc produce a multitude of chemokines that attract specific t cell subsets. such chemokines are likely to play a critical role in the regulation of immune responses, since they orchestrate the spatial and temporal interaction between apc and t cells, which determines the fate and nature of the immune response. evidence for this conceptual framework came recently from the observation that blocking apc-t cell interactions using ctla4-ig led to a significant reduction of disease activity in ra. several chemokines orchestrate the attraction of t cells toward dc. it is tempting to speculate that interfering with these chemokines would lead to similar effects on disease activity as the direct blockade of t-cell dc interaction. of this group of t- cell attracting chemokines, ccl and cxcl recently came out as potentially interesting targets in ra from previous research by our group and others. cc chemokine ligand was first identified as a naïve t cell attracting chemokine. next to chemo-attraction, ccl plays a role in stimulation of collagen production by fibroblasts. despite numerous attempts to identify its receptor, ccl is still an orphan chemokine. in vivo, ccl expression was first found in high quantities in the lung, which is caused by the abundant expression by alveolar macrophages. in vitro, dc and mΦ have been identified as ccl producers. to date, a substantial amount of data points toward the enrichment of dc and mΦ in the synovial tissue which likely to be responsible for the increased levels of ccl in ra synovial tissue and synovial fluid compared with that from healthy individuals. in this line, ccl has been identified as a clinical marker in gaucher's disease, a condition in which mΦ activation is likely to play a role in the pathogenesis. in addition, a role for ccl has been suggested in a large variety of diseases, such as systemic sclerosis and acute lymphoblastic leukaemia. in ra, we recently found that circulating ccl levels are elevated compared with controls and correlated with disease activity. moreover, ccl mrna expression by dc from ra patients was shown to be higher than by dc from healthy controls, which could be influenced by blockade of tnf-α. the exact regulation of ccl protein secretion however is complicated and the studies published thus far have led to controversial results, as elegantly reviewed by schutyser et al. in order to clarify the mechanism of ccl expression and secretion in ra, we investigated the role of a large panel of inflammatory mediators known to play a role in the disease process on ccl secretion. here we show that ccl secretion by monocytes and dc is regulated by synergistic effects between il-4/il- il- and ra sf, whereas pro-inflammatory cytokines and toll-like receptor ligands did not have any influence on ccl secretion. these data add novel information to the puzzle of increased ccl expression in ra.  il- strongly enhances ccl production by modc while maturation and pro-inflammatory mediators do not first we investigated whether several mediators that are known to be important in ra were able to enhance ccl production by modc. in line with previous studies, unstimulated immature dc produced significant amounts of ccl. interestingly, incubation with tnf-α, il-1β, il- il- il- il- and ifn-γ did not stimulate ccl secretion when added to day idc. in contrast, the anti-inflammatory il- strongly induced ccl production by idc. next we tested whether factors well known to induce maturation or t cell mimicking could induce ccl production. these experiments demonstrated that lps, cd40l and rankl did not enhance ccl production. recent studies demonstrated that other tlr pathways than tlr are all capable of inducing dc maturation, but have different effects on cytokine production. however, stimulation of tlr, tlr ), tlr or tlr7/ did not sort any effect on ccl secretion, whereas they did elicit a potent cytokine response. since il- is more abundantly present in ra than il- and since some conflicting results have been published on ccl production induced by lps when dc were cultured with il- vs. il- we compared these culture methods. in both the il- and il- cultures, il- strongly induced ccl, while lps again did not. in addition, il- in combination with lps was not significantly different from il- alone. also co-stimulation with lps and the cytokines tested did not sort any effect on ccl secretion. il- acts in synergy with il-4/l- in promoting ccl production by monocytes modc and alternatively activated mΦ are known to produce ccl both these cell types originate from cd14+ monocytes and depend on il- or il- for their differentiation. to determine whether ccl secretion by myeloid cells is dependent on these cytokines, monocytes were freshly isolated and stimulated with gm-csf, il- il- and il- alone or in combinations. even after days, unstimulated and gm-csf treated monocytes/macrophages did not secrete ccl whereas both il- and il- stimulation resulted in a clear secretion of ccl which is in line with previous findings on aamΦ. interestingly, stimulation with il- alone only had a minimal effect on ccl production by these monocytes/macrophages. when il- was provided together with il- or il- this resulted in 3- and 2-fold increase in ccl secretion respectively. interestingly, already in low concentrations, il- had its synergistic effect with il-. in order to rule out effects of adherence, we cultured cd14+ monocytes/macrophages for three days in teflon bags and in rotation discs. the morphology of these cells was comparable with freshly isolated monocytes according to their forward/side scatter pattern. in both cultures, il- did still induce ccl production in the same way as the cultures in 24-wells plates. as a proof of principle, we next tested whether the synergy between il-4/il- and il- could already be observed after only hours. intriguingly, we could indeed observe a clear ccl secretion after hours upon stimulation of freshly isolated monocytes with il-4/il- and il- whereas stimulation with il- il- or il- alone did only result in a minor or even undetectable ccl secretion. since il- appeared to synergize with il- and il- we investigated whether these cytokines could up-regulate each other's receptors, possibly resulting in enhanced signaling. this was not the case; il- did not up regulate either the il-4/il- common receptor il-4rα or the specific il-13rα furthermore, il- had no effects on il-10rα. ra synovial fluid enhances ccl secretion independently of il- and il-13 we and others demonstrated ccl expression in ra st in the lining and the peri-vascular regions. in figure we show a high ccl expression in ra synovial tissue, which was preferentially located in both the synovial lining layer and the peri-vascular regions. intriguingly, ccl was also expressed in control synovial tissue, although not as abundant as in ra st. notably, some parts of the sections were even negative for ccl which is in sharp contrast with ra. in order to explain the abundant ccl expression in ra, we tested whether incubation with ra sf could induce ccl production on monocytes/macrophages. since ra sf itself contains ccl, we cultured freshly isolated monocytes for days in the presence of sf, washed the cells and cultured on for another days in the absence of ra sf. firstly, this pre-incubation with ra sf resulted in marked ccl production pg/ml). secondly, culture of freshly isolated monocytes in the presence of ra sf, resulted in a 9- and 10-fold increase in ccl secretion upon stimulation with il4/il- respectively and a 22-fold increase compared with il- alone. intriguingly, this synergistic effect with il- il- and il- could still be observed after days of culture in the complete absence of ra sf, indicating that the cell does not require a continuous stimulation in order to secrete ccl to exclude intrinsic differences between monocytes/macrophages from ra patients and controls may contribute to the effects on ccl secretion, we tested whether monocytes/macrophages from ra patients responded differently to combinations of il- il- il- and sf. no difference in the ccl secretion pattern was observed between monocytes/macrophages of healthy controls and ra patients upon these stimuli, ruling out intrinsic differences in monocytes in ra that affect ccl secretion. since the synergy caused by ra sf appeared similar to the synergy between cytokines we already observed, we tested whether il- and/or il- both present in ra sf, were responsible for this phenomenon by blocking these cytokines with neutralizing antibodies. the potency of these antibodies was first tested by determining their ability to inhibit the synergistic ccl secretion upon stimulation with a combination of il- and il-4/il- addition of anti-il- resulted in a 73% inhibition of the synergy between il- and il- and anti il- completely abrogated the synergistic effect of il- with il-. unexpectedly however, blockade of il- or il- in sf in the presence of il- or il- and il- respectively did not inhibit the synergy between these cytokines and sf, suggesting that sf contains a yet unidentified factor that triggers ccl secretion. discussion in this study, we add new pieces to the complicated puzzle of ccl regulation in ra. firstly, we demonstrate that ccl production can be induced by il- il- and il- in monocyte derived cells. secondly, we show that a large panel of pro-inflammatory stimuli and tlr mediated signals leading to dc maturation are of no influence on ccl production. thirdly, il- only induces a minor ccl secretion, but acts in synergy with both il- and il- on monocytes and monocyte derived cells. finally, we provide evidence that ra sf is able to induce ccl secretion in strong synergy with il- il- and il- which could not be inhibited by a blockade of il- and il- ccl can be produced by modc as well as by certain types of mΦ. often these cell types are considered to be totally different cells. however, the differences between modc and aamΦ are not that large, since monocyte derived macrophages are cultured in the presence of gm-csf by some groups and both cells require the presence of il- or il- penna and co workers demonstrated that several in vivo dc subtypes were not able to produce ccl, which is in contrast with previous findings, where ccl mrna expression was found on cd11c+ myeloid blood dc. moreover, in vitro cultured modc have been identified as potent ccl producers. these data suggest that a cd14+ monocyte origin in combination with a stimulation by il-4/il- is critical for ccl secretion. this hypothesis is strengthened our data, demonstrating that non-adherent monocytes/macrophages were able to produce ccl under the influence of il- in addition, the synergistic effects of il-4/il- and il- on ccl secretion by freshly isolated monocytes were already clearly visible after hours. this indicates that a full differentiation into dc or mΦ is not essential for ccl production as has been suggested previously for ccl mrna expression. thus monocytes rapidly secrete ccl upon triggering with the right stimuli. in the literature there is still some controversy regarding the effect of dc maturation on ccl production. vulcano and co workers suggested that dc down regulate their ccl secretion upon maturation. this is in contrast with results from other studies, where maturation caused an increased mrna expression. a similar contrast between protein and mrna was found on blood dc. the reason for these differences between mrna expression and protein secretion patterns still needs to be investigated in detail. recently, we already provided evidence that dc maturation does not influence ccl protein secretion, which is further strengthened by the data from the present study, in which different tlr stimulatory pathways did not induce ccl production, whereas full dc maturation was achieved. also tnf-α and cd40l, both well appreciated inducers of dc maturation, did not enhance ccl production. perhaps the discrepancy between the different reports is hidden in subtle differences in culture conditions, which are difficult to trace in the published data. intriguingly, stimulation with il- alone only lead to a marginal induction of ccl secretion by monocytes/macrophages, but did act in a strong synergy with il- or il- the latter is not caused by an up regulation of the receptors il-10rα, il-4rα or il-13rα. probably intracellular pathways direct the synergy between these cytokines, which is an interesting topic that warrants further investigation. we showed that ra sf induces ccl production and strongly synergizes with il- il- and il- blocking studies revealed that neither il- nor il- in sf were responsible for this effect. this suggests the presence of another, yet unidentified ccl inducing factor in ra sf. another explanation for this fact might be the presence of inhibiting factors in sf that counter-regulate the effects of il- and il- the identification of the factor in sf that drives the effects on ccl secretion may provide important new insights to the pro-vs. anti inflammatory balance in ra. in order to find this factor in a complex fluid like sf, more knowledge on the pathways of ccl regulation is critical. another intriguing observation from our study is the finding that pre-incubation with sf lead to a sustained synergistic ccl secretion upon stimulation with il- il- and il- this could be regarded as an "imprinting effect", meaning that the cell's previous environment determines the nature of response to stimuli, even when the cell is no longer in such an environment. results from previous studies, in which we showed that modc from ra patients differ in phenotype and cytokine response from control dc after days in culture might also be explained by such a phenomenon. upon the encounter of an antigen, dc normally mature and migrate to lymphoid tissues in order to perform their task of antigen presentation to t cells. immature dc or mΦ can also encounter naïve t cells in the periphery, which subsequently might result in tolerance. this peripheral tolerance is a critical mechanism to prevent auto-immunity. a role for ccl in this part might explain the high expression of ccl by alveolar mΦ, which are located at a site where the maintenance of tolerance to non-pathogenic antigens, that are constantly present, is crucial. also the synergistic effect on ccl secretion that we found with il- a cytokine that is well appreciated as a pivotal regulator of the immune system, fits in this picture. the synovial lining in the joints has similarities with the alveolar lining in the lung. they both consist of mΦ-like cells and both form a barrier to a site in which self- and non-pathogenic antigens are constantly present. the disease process in ra is considered to be driven by pro inflammatory cytokines such as il-1β, tnf-α, il- and il-, whereas ccl is regulated by il- il- and il- it is therefore tempting to speculate that the high ccl expression in ra is designed to uphold peripheral tolerance, which however seems to fail. this failure might be explained in two ways. the first explanation might be that the skewing in the balance towards th is still present despite the upregulation of anti inflammatory mediators. secondly, mature dc are present in the synovial tissue in perivascular regions and secondary lymphoid organs, which is in sharp contrast with healthy synovial tissue. therefore an explanation for the ongoing immune process might be that these mature dc direct naïve t cells towards a phenotype that drives the pro-inflammatory immune response in the synovial tissue.  in summary, we provide evidence that monocyte derived cells produce ccl under the influence of il- and il- il- acts in strong synergy with il- and il- as a key regulator of ccl production by monocytes, which indicates that ccl secretion is not confined to fully developed dc and mΦ. in addition, the effects of il- il- and il- are strongly enhanced by rasf, which is due to yet unidentified factors. both the in vivo expression pattern and the contributing factors to its regulation in vitro are suggestive for a role for ccl in the regulation of the immune system, both in health and auto-immune diseases such as ra. 
in contrast to a large panel of pro-inflammatory stimuli, t cell mimicking molecules or tlr driven maturation, the anti-inflammatory il- strongly stimulated dc to secrete ccl on freshly isolated monocytes, ccl secretion was induced by il- and il- in strong synergy with il- this synergistic effect could already be observed after only hours, indicating that not only macrophages and dendritic cells, but also monocytes secrete ccl under these stimulatory conditions. the t cell attracting chemokine ccl is produced by antigen presenting cells and a role for ccl has been suggested in the pathogenesis of a variety of diseases. a high ccl expression was detected in ra synovial tissue and incubation of monocytes with synovial fluid from ra patients clearly enhanced the effects of il- il- and il- surprisingly, the effect of synovial fluid was not driven by il- of il- suggesting the presence of another ccl inducing factor in synovial fluid. rheumatoid arthritis is one of these conditions, in which abundant ccl production is present. the effects of il- il- and il- are strongly enhanced by synovial fluid. although th cytokines and il- are known to have an effect on ccl production, there are several gaps in our knowledge regarding the exact regulation of ccl secretion, both in general and in ra. in this study we provide new insights in the regulation of ccl secretion by monocytes and dendritic cells.
 the t cell attracting chemokine ccl is produced by antigen presenting cells and a role for ccl has been suggested in the pathogenesis of a variety of diseases. rheumatoid arthritis is one of these conditions, in which abundant ccl production is present. although th cytokines and il- are known to have an effect on ccl production, there are several gaps in our knowledge regarding the exact regulation of ccl secretion, both in general and in ra. in this study we provide new insights in the regulation of ccl secretion by monocytes and dendritic cells.  in contrast to a large panel of pro-inflammatory stimuli, t cell mimicking molecules or tlr driven maturation, the anti-inflammatory il- strongly stimulated dc to secrete ccl on freshly isolated monocytes, ccl secretion was induced by il- and il- in strong synergy with il- this synergistic effect could already be observed after only hours, indicating that not only macrophages and dendritic cells, but also monocytes secrete ccl under these stimulatory conditions. a high ccl expression was detected in ra synovial tissue and incubation of monocytes with synovial fluid from ra patients clearly enhanced the effects of il- il- and il- surprisingly, the effect of synovial fluid was not driven by il- of il- suggesting the presence of another ccl inducing factor in synovial fluid.  in summary, il- synergistically induces ccl secretion in combination with il- of il- on monocytes and monocyte derived cells. the effects of il- il- and il- are strongly enhanced by synovial fluid. this synergy may contribute to the high ccl expression in ra. 