Dexamethasone restrains ongoing expression of interleukin-23p19 in peripheral blood-derived human macrophages

RESEARCH ARTICLE Open Access

Dexamethasone restrains ongoing expression ofinterleukin-23p19 in peripheral blood-derivedhuman macrophagesLinda Palma*, Carla Sfara, Antonella Antonelli and Mauro Magnani

Abstract

Background: Since its recent discovery, interleukin-23 has been shown to be involved in the pathogenesis ofautoimmune diseases favoring the development of a T cell subset referred to as T helper 17. Glucocorticoids arewidely employed in inflammatory and autoimmune diseases as they inhibit pro-inflammatory signaling andprevent production of inflammation mediators. Very limited information is available about the efficacy of syntheticglucocorticoids in containing the expression of interleukin-23 under cell activation.

Results: We demonstrate here that the glucocorticoid analogue dexamethasone administered to humanmonocyte-derived macrophages is indeed able to restrain the expression of interleukin-23 once it has beentriggered by a pro-inflammatory stimulus. This effect of dexamethasone is here demonstrated being secondary tosuppression of p38 MAPK activity, and involving a protein phosphatase - likely MAPK phosphatase-1 (MKP-1).

Conclusions: Results reported in this paper show that a 10 nanomolar dose of dexamethasone not only preventsinflammatory activation but is also efficacious in confining active inflammation. This effect is here demonstratednot to occur through “canonical” inhibition of the NF-�B transcription factor but through a distinct cascade ofdown-modulation, that underlines the importance of the transactivating activity of glucocorticoid receptor in thecontext of its anti-inflammatory action.

BackgroundIL-23 is a heterodimeric cytokine composed of the IL-12p40 subunit and the recently discovered IL-23p19subunit, related to IL-12p35 [1]. The IL-23 receptorcomplex is present on the surface of APC, NK and acti-vated T cells. Despite IL-23p19 expression in differenttissues and cell types, p19 alone has not been found tohave biological activity and appears to be functionalonly when dimerized with p40, which occurs in acti-vated M� and DC. Production of IL-23 by APC is trig-gered by host immune stimuli as interferons and bybacterial products, such as LPS, that signal through TLR[2-4]. Whereas IL-12 drives the differentiation of Thelper 1 cells (Th1), IL-23 favors the development of aT cells subset with a unique expression profile, charac-terized by prominent production of IL-17, thereforereferred to as Th17 cells [5,6]. IL-17, in turn, induces

pro-inflammatory cytokines that contribute to protectiveresponse during infection [7,8]. IL-23 is commonlybelieved to act on memory CD4+ T cells, enhancing theexpansion of committed Th17 and production of IL-17[1,9], although it was also hypothesized that IL-23 roleis not merely limited to a survival factor. In fact, severalobservations suggest that IL-23 may act earlier duringTh17 commitment, promoting their effector function[10]. However, IL-23 is absolutely required for providingTh17 a pathogenic phenotype, because in its absencethese cells may have regulatory functions. Nonetheless,the IL-23/IL-17 axis is evolutionarily conserved as itprovides a rapid response to catastrophic injuries,caused by certain infective agents (e.g. K. pneumoniae,T. gondii, S. aureus and B. pertussis) especially in gut,lung and skin.Although autoimmune diseases have long been con-

sidered being T cell-mediated, the pleiotropic role ofM� in immunity has lately received more attention, andM� products have been implicated in various

* Correspondence: [email protected] di Scienze Biomolecolari, Università degli Studi di Urbino “CarloBo”, Via A. Saffi, 2, 61029 Urbino, PU, Italy

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

© 2011 Palma et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

autoimmune pathologies. In particular, it is now demon-strated that M�-secreted IL-23, and consequently T cell-derived IL-17, are linked to disease in animal models ofautoimmunity [11-13]. Abnormal IL-23 concentration isfound also in human biological fluids and tissuesaffected by rheumatoid arthritis [14], multiple sclerosis[15,16], psoriasis [17], inflammatory bowel diseases[18,19]. Moreover, the IL-23/IL-17 axis plays a role dur-ing inflammatory process-derived tumor development:IL-23 decreases number and activity of cytotoxic T cellsin transformed tissues [20].GC hormones modulate a wide array of physiologic

functions, especially in the context of immune homeosta-sis. Therefore, synthetic GC are broadly employed fortreatment of inflammatory conditions. Suppression ofinflammation by GC is believed to occur through threemain mechanisms: non-genomic pathways, direct genomiceffects and indirect genomic effects [21]. Ligand-boundGR regulates target genes expression by interaction withGRE. However, genes regulated through this direct inter-action are much less (1%) than genes whose expression isindirectly regulated. In fact, the GR is able to influence -mainly inhibit - the activity of several pro-inflammatorytranscription factors, including NF-�B, AP-1, t-BET,STAT5 [22,23]: in the promoter environment of pro-inflammatory genes, active GR competes for binding toco-activators, affects RNA polymerase II activity, interactswith co-repressors and hystone de-acetylases, specificallymodulating the expression of each gene. Besides affectingtranscription, GC reduce post-transcriptional stability ofparticular sets of mRNA, thus dampening the synthesis ofinflammatory proteins after the inflammation has beenactivated [24-26]. The synthetic GC analog DEX hashigher affinity for the receptor, higher anti-inflammatoryactivity and a longer biological half-life than conventionalsteroids [27]. As for the effects of DEX on IL-23 signaling,it is demonstrated that the GC analogue affects activationof STAT4 induced by IL-23 in PHA/IL-2 T cells [28], anda study by Ma et al. shows that DEX inhibits LPS-inducedIL-12p40 production in human monocytes [29].Given the critical role of deregulated IL-23 production

in pathogenesis, we investigated in vitro the effect thatDEX exerts on expression of IL-23 - in particular, of itsunique p19 subunit - by human M�. Moreover, sinceNF-�B and p38 MAPK are involved in the regulation ofIL-23p19 expression in various cell types of the mono-cyte/M� lineage [30-33], we focused on the mechanismof DEX inhibition on these pathways with respect todown-modulation of IL-23p19.

ResultsDEX reduces IL-23 levels in vitroPrimary cultured human M� were first subjected to stimu-lation with LPS in order to assess the time course of IL-23

expression in response to the engagement of TLR-4.Although distinct subjects respond diversely in terms offold induction, they all show a peak level of IL-23p19mRNA after 8h of persistent stimulation with LPS (Figure1A), which is followed by increase of the protein between8h (lane 2) and 24h (lane 3) (Figure 1B). Therefore, weconsider cultured M� persistently stimulated with LPS for8h a suitable in vitro model of established IL-23 expression.The ability of DEX to restrain ongoing IL-23 expression

was next evaluated adding the drug during the last 2h ofLPS treatment: M� stimulated 6h with LPS were eitherleft untreated or treated with 0.001, 0.01, 0.1 or 1 μMDEX, and maintained at 37°C for additional 2h in the pre-sence of LPS. Relative quantification by RT-PCR revealedthat, while DEX 0.001 μM does not exert any effect on IL-23p19 expression, DEX 0.01 μM induces a significantdecrease in the amount of p19 mRNA compared to DEX-untreated samples (Figure 1C). A similar reduction occurswith 0.1 and 1 μM DEX. On the same samples, the expres-sion of other IL subunits strictly related to IL-23p19 wasevaluated and compared by relative quantification, demon-strating that 0.01 μM is the least DEX concentration ableto significantly down-regulate IL-12p40 and IL-12p35mRNA (Figure 1D and 1E). On the other hand, a consis-tent up-regulation of FKBP5 mRNA was observed withdoses ≥ 0.01 μM, demonstrating the effective activation ofthe GR signaling pathway (Figure 1F).We then attempted at validating IL-23 expression data

also on the protein product, by an IL-23 ELISA on M�supernatants collected at 8, 16 and 24h of LPS stimula-tion, either treated or not with DEX. Unfortunately, theamount of protein was below the limit of sensitivity ofthe kit, and this problem could not be circumvented byconcentrating the samples.However, the effect of DEX on IL-23 protein production

was verified at the intracellular level by western immuno-blotting of the IL-23p19 subunit. Human M� were stimu-lated 6h with LPS and then either added with 0.01 μMDEX or left DEX-untreated for additional 2h, then all thecompounds were removed and total cell lysates were pre-pared at 24h. As shown in Figure 2, DEX added during IL-23p19 gene transcription leads to a significant decrease ofthe protein product (lanes 3, 4), confirming the dataobserved at the transcriptional level and suggesting thatthe drug can be effective in reducing the amount ofsecreted interleukin. Interestingly, DEX was also able toprevent IL23-p19 protein accumulation induced in humanM� by a distinct pro-inflammatory agonist, Zymosan fromSaccharomyces cerevisiae (data not shown).

DEX does not affect nuclear localization or DNA-bindingof active NF-�BTranscription factor NF-�B can be regulated by variousevents and at distinct levels of the activation cascade.

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 2 of 12

One such mechanism consists of cytoplasmic sequestra-tion through interaction with a member of the I�Bfamily. Immunofluorescence directed to the p65 subunitof NF-�B was performed on M� stimulated 6h withLPS and then either added with 0.01 μM DEX or leftDEX-untreated for additional 2h. As shown in Figure

3A (left box), nuclear-cytosolic shuttling of p65 isshifted toward nuclei after 8h of LPS treatment. How-ever, addition of DEX does not produce any significantreduction of the nuclear signal (Figure 3A, right box).Consistent with these results, we observed that none

of the tested concentrations of DEX actually up-

Figure 1 DEX affects IL-23 mRNA levels. (A) Time course of IL-23p19 expression in M� stimulated with 1 μg/ml LPS for the indicated times.Relative quantification was performed by Real Time RT-PCR according to the ΔΔCt comparative method and data are expressed as fold changescompared to the peak sample (LPS 8h). Data reported in diagram are the average of three independent experiments. (B) Time course of IL-23p19protein accumulation in M� after 24h of stimulation with 1 μg/ml LPS. Western immunoblotting were performed on total cellular extracts. Imagesare representative of three independent experiments and data are reported in bar graph as fold changes compared to the peak sample (LPS 24h).IL-23p19 signal (upper panel) was normalized to the actin signal (lower panel). (C),(D),(E) DEX-dependent inhibition of expression of the interleukinsubunits IL-23p19, IL-12/IL-23p40 and IL-12p35, respectively. Real Time RT-PCR was performed on LPS-stimulated M� treated with increasingconcentrations of DEX. Results are reported as raw 2^-(Ct target-Ct reference) to compare the relative abundance of each subunit. Amplificationefficiencies of p19 and p35 were comparable, based on the slopes of their standard curves. n = 3. (F) Induction of FKBP5 by DEX in M� treated as in(C). Real Time RT-PCR data are reported as fold changes compared to the DEX-untreated sample; n = 6. * p < 0.05, ** p < 0.01.

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 3 of 12

regulates I�Ba, either as mRNA (not shown) or as pro-tein, after 8h of LPS stimulation (Figure 3B), indicatingthat the lower IL-23p19 expression is not secondary tocytosolic retention of NF-�B. Nonetheless, it could bethe consequence of an impaired DNA-binding activityof NF-�B, therefore we performed NF-�B EMSA on M�treated as for immunofluorescence (Figure 3C). Weobserved that the DNA-binding activity of NF-�B beforeaddition of the drug (LPS 6h, lane 2) is up to twice (1.8± 0.6, p < 0.01, n = 12) that of basal cells (lane 1); whenIL-23p19 mRNA is on its peak level (LPS 8h, lane 3)NF-�B activation remains indeed high (1.7 ± 0.4, p <0.05, n = 12), but is not inhibited by DEX (lane 4) (1.7± 0.4, p < 0.05, n = 12). Surprisingly, the amount ofI�Ba analyzed on the same cellular extracts (Figure 2D)reflects the transcriptional activity of NF-�B rather thanits inhibition. In fact, after 6h of stimulation (lane 2) theamount of I�Ba is up to five fold that of basal M� (lane1) (4.6 ± 0.7, p < 0.01, n = 4), it remains high at 8h(lane 3) (3.2 ± 2.3, p < 0.01, n = 4) and it is almost unaf-fected by DEX (lane 4) (3.1 ± 1.8, p < 0.05, n = 4).

DEX reduces the transactivation ability of activated NF-�BTo confirm and elucidate the effect of DEX on NF-�B, areporter cell line was employed. MM6 cells transfectedwith the LUC coding sequence under NF-�B transcrip-tional control, were stimulated with LPS over a timecourse of 8h. At 2h LUC mRNA is at peak level,

whereas the maximum of LUC activity is observed at 6-7h (not shown). Since our goal is to evaluate DEX activ-ity on pre-existing inflammatory conditions, we stimu-lated reporter cells 1h with LPS to trigger LUCexpression. Afterward, cells were treated with DEX 0,0.001, 0.01, 0.1 or 1 μM for one more hour in presenceof LPS. Culture medium was then replaced and cellswere left at 37°C for additional 5h (Figure 3E). At theend of treatment the luminescent signal is high com-pared to basal reporter cells, confirming the activationof NF-�B. Whereas DEX 0.001 μM has no effect on thereporter activity, a significant reduction of LUC signal isdetectable with DEX ≥ 0.01 μM.Western immunoblotting for p65 was performed on

nuclear and cytosolic extracts from the same cells, toexclude that the lower level of reporter activity was dueactually to cytosolic sequestration of NF-�B. Asexpected, nuclear NF-�B-p65 increases - and the cytoso-lic decreases - as cells are stimulated with LPS, but theDEX concentrations tested do not modify the sub-cellu-lar localization of p65 to an extent significantly consis-tent with the reduction of reporter activity (Figure 3F).Therefore, we conclude that DEX reduces the transacti-vation potential of active NF-�B, because expression ofits target gene is down-modulated although the tran-scription factor remains localized to the nucleus.

DEX down-modulates p38 MAPK activityBecause p38 MAPK usually promotes NF-�B trans-activity, we speculated that the results above describedcould arise from p38 impairment by DEX. Thus, inLPS-stimulated M�, increasing concentrations of drugwere tested on the extent of p38 MAPK phosphoryla-tion, on that of NF-�B p65 phosphorylation on serineresidue 276 (Ser276), and on the expression of MAPKphosphatase-1 (MKP-1), a negative regulator of MAPK.Western blotting was performed on the same type of

samples employed for analyses of IL-23 expression.Accordingly, the decreased IL-23 expression is accompa-nied by a consistent reduction of p38 MAPK phosphor-ylation. Again, 0.01 μM DEX (Figure 4A, lane 3)promotes a significant dephosphorylation of p38 com-pared to the control sample (lane 1) and 0.1 and 1 μMDEX (lanes 4, 5) yield similar results. Interestingly, thephosphorylation of p65(Ser276) was decreased with anapparent dose-trend relationship, consistent with thereduction of p38 MAPK activity (Figure 4B).MKP-1 levels were evaluated by RT-PCR on M�

under the same experimental conditions as above. Asexpected, LPS promotes a five-fold increase of MKP-1mRNA compared to basal M�, and DEX administeredto the cells during the last 2h of stimulation furtherenhances MKP-1 expression. In particular, consistentwith what above described, at least 0.01 μM DEX is

Figure 2 DEX affects IL-23 protein levels. DEX (0.01 μM) reducesIL-23p19 protein production. IL-23p19 protein was analyzed bywestern immunoblotting of M� stimulated with LPS (upper panel)for the times indicated. Data obtained from densitometric analyseswere normalized on respective actin signals (lower panel). Imagesare representative of three independent experiments and data arereported in bar graphs as fold changes relative to the peak samples;n = 3. *p < 0.05

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 4 of 12

Figure 3 Mechanism of NF-�B activity restraint by DEX. (A) Immunofluorescence analyses of NF-�B-p65 sub-cellular localization. FITC picturesshow p65 signal in two representative fields of M� stimulated 8h with LPS (left box) or of M� stimulated 8h with LPS and treated with 0.01 μMDEX (right box). DAPI:FITC pictures were obtained by overlay of the FITC and DAPI signals, and show the nuclear localization of p65. (B). LPS-stimulated M� (lane 1) were treated with increasing concentrations of DEX (lanes 2-5) and the amount of I�Ba was analyzed by westernimmunoblotting (upper panel), normalized to the actin signal (lower panel). Image is representative of three independent experiments. (C). M�

(lane 1, 0h) were subjected to NF-�B EMSA after LPS stimulation for 6h (lane 2) and 8h, in presence (lane 4) or absence (lane 3) of DEX 0.01 μM. Theimage is representative of four independent sets of samples, each run in triplicate. (D) I�Ba western immunoblotting on the same samplesemployed for EMSA, representative of four independent experiments. (E) NF-�B reporter assay on MM6 cells. NF-�B-driven transcription wastriggered by LPS 1 μg/ml for 1h, followed by treatment with indicated DEX concentrations. Data of relative light units/μg of proteins are reportedin bar graph as fold changes compared to the DEX-untreated sample (+LPS, 0 DEX); n = 12. (F) NF-�B p65 western immunoblotting on cytosolicand nuclear extracts from the same cells as in (E). Densitometric analysis was followed by normalization on the internal control represented byactin. Data are reported in bar graphs as fold changes relative to the DEX-untreated sample (lane 2); n = 6. * p < 0.05, ** p < 0.01.

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 5 of 12

needed to raise significantly the amount of MKP-1transcript over the level of the DEX-untreated sample,and this induction is maintained by DEX 0.1 and 1 μM(Figure 4C). Furthermore, the involvement of a phos-phatase activity in the reduction of NF-�B transactiva-tion potential by DEX was demonstrated on MM6cells using the phosphatase inhibitor Na3VO4. MM6reporter cells were stimulated 1h with LPS and then

treated with DEX 0, 0.01, 0.1 or 1 μM, added alone oralong with Na3VO4 for 1h more, in presence of LPS.The medium was then replaced and cells were left at37°C for additional 5h. Increasing concentrations ofDEX promote a significant reduction of the lumines-cent signal compared to DEX-untreated cells, whereasthe phosphatase inhibitor counteracts the trend ofsuch DEX-mediated decrease, as cells treated with

Figure 4 DEX down-modulates p38 MAPK activity. (A) DEX-dependent inhibition of p38 MAPK was measured in LPS-stimulated M� bywestern immunoblotting of the phosphorylated protein (phospho Thr180/Tyr182), normalized on the total amount of p38. Data are reported inbar graph as fold changes relative to the DEX-untreated sample (lane 1); n = 3. (B) Inhibition of NF-�B-p65 transactivity by DEX was measured inLPS-stimulated M� by western immunoblotting of p65 phosphorylation on Ser276, normalized on the total amount of p65. Data are reported inbar graph as fold changes relative to the DEX-untreated sample (lane 1); n = 3. (C) DEX-induced increase of MKP-1 mRNA determined by RT-PCRon the same samples as in (A). Data are reported as fold changes relative to the DEX-untreated sample (lane 1); n = 6. (D) Bar graph shows datafrom luciferase assays performed on MM6 reporter cells, stimulated with LPS and treated with increasing concentrations of DEX, administeredalone or with sodium orthovanadate. Data are reported as fold changes relative to the DEX-untreated sample (+LPS, 0 DEX); n = 15. (E) Bargraph shows RT-PCR data for the IL-23p19 transcript from M� stimulated with LPS and then treated or not with DEX 0.01 μM alone or alongwith sodium orthovanadate. Data are reported as fold changes relative to the DEX-untreated sample (8 hLPS, - DEX, -Na3VO4); n = 6. * p < 0.05,** p < 0.01.

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 6 of 12

Na3VO4 maintain a higher signal compared to cellstreated with DEX alone (Figure 4D).Similarly, the link between phosphatase induction

and IL-23p19 down-modulation was assessed in M�,administering Na3VO4 along with DEX. M� were sti-mulated 6h with LPS and then either left untreated ortreated with DEX 0.01 μM in the presence or absenceof Na3VO4 for additional 2h. RT-PCR revealed thatDEX produces a decrease of IL-23p19 consistent withthat previously observed, compared to DEX-untreatedM�, whereas addition of the Na3VO4 along with DEXimpedes such decrease, yielding an amount of tran-script significantly more abundant not only than thatof DEX-treated M� but also of control (p < 0.05, n =6) (Figure 4E).

DiscussionThe anti-inflammatory and immunosuppressive proper-ties of GC are attributed to their ability of down-regulat-ing pro-inflammatory pathways, such as those of MAPK,AP-1 and NF-�B, and preventing production and/orrelease of several pro-inflammatory factors by a varietyof cell types challenged with different stimuli [29,34,35].During the last decade numerous studies have demon-

strated that aberrant expression of IL-23 and of itsdownstream partner IL-17 underlie development andmaintenance of autoimmunity. The requirement of IL-23 for Th17 cells to acquire a pathogenic phenotypelinks unquestionably IL-23 to disease but, to our knowl-edge, the outcome of DEX treatment on the mechan-isms of IL-23 expression is as yet unexplored. Many invitro studies on the effects of DEX perform either adrug treatment -spanning between 30 min to 2h- beforeactivation of inflammatory pathways, or a co-treatmentwith DEX and pro-inflammatory agonists [29,35-37].Instead, we reasoned that the performance of DEXcould be different if administered after the inflammationhas been triggered; therefore, in a therapeutic perspec-tive, we considered more interesting to verify the effi-cacy of the drug in restraining - rather than preventing- IL-23 expression under active pro-inflammatory sig-naling. Thus, in our model IL-23p19 expression is sti-mulated by LPS (Figure 1A and 1B), then the drug isadded during the phase of maximal accumulation of thetranscript, before its physiological decay. Four loga-rithms of DEX sub-micromolar concentrations (0.001 to1 μM) were tested, and the engagement of the GC sig-naling pathway was verified by the up-regulation ofFKBP5, a GR target gene known to be expressed inresponse to GC as part of the GR auto-regulatory loop[38] (Figure 1F).We show that at least 0.01 μM DEX is necessary to

engage GR (Figure 1F) and to produce a significantreduction of IL-23p19 (Figure 1C). In fact, 0.01-0.1 μM

are DEX concentrations within the physiologically andtherapeutically relevant range for GR-mediated actions[35,39]. However, the results obtained with DEX 0.01μM are not improved by higher doses, and this may ori-ginate from a saturation effect, hypothesis supported bythe evidence that a 0.01 μM plasma concentration ofDEX is sufficient to saturate more than 80% of the GR[40].DEX 0.01 μM reduces the expression of IL-12p40 and

IL-12p35 as well (Figure 1D and 1E, respectively). Actu-ally, high level production of functional IL-12 by humanmonocytes, M� and DC requires interferon-g in additionto microbial stimuli [4,32]. However, this does notexclude that LPS alone may trigger IL-12 expression,though to a lesser extent. In fact, we show that bothp40 and p35 are induced by LPS treatment, but p35 isexpressed at a level two orders of magnitude lower thanp19, based on the respective amplification efficiencies.Unfortunately, since the amplification efficiency of IL-12p40 is not comparable with those of p19 and p35, wecannot make any statement about its relative abundance.However, from these observations we conclude that aphysiologic dose of DEX is able to dampen ongoingexpression of both IL-23 subunits, through inhibition ofthe signaling pathways that govern the transcriptiontriggered by microbial stimuli. Moreover, the transcrip-tional down-modulation of IL-23p19 is here demon-strated to yield a comparable decrease of the proteinamount, leading to the conclusion that DEX effectivelyimpairs the ability of producing IL-23 in human M�(Figure 2). Therefore, we aimed at identifying the mainmolecular mechanisms at the basis of IL-23 reductionby DEX.Consistent with the typical TLR signaling, we have

recently shown that NF-�B activity is required for IL-23expression in LPS-stimulated human M� [33]. Thus, wefocused on the NF-�B signaling pathway, which DEXinhibits at multiple levels. The transcriptional up-regula-tion of I�Ba has long been known to play a major rolein GC-mediated repression of NF-�B, especially in cer-tain cell types [41]. Our research group have demon-strated that in M� DEX up-regulates I�Ba, whichprevents NF-�B triggering by LPS [37]. Instead, whenDEX is added over a pre-existing activation of the path-way, down-modulation of pro-inflammatory transcriptsis not accompanied by a consistent cytosolic sequestra-tion of NF-�B (Figure 3A). In fact, DEX does not affectamount of I�Ba neither at the transcription level nor atthat of translation (Figure 3B). Actually, LPS up-regu-lates I�Ba, which is already up to four times moreabundant than in basal M� when DEX is added to cells(Figure 3D); thus, it is likely that DEX is not able tofurther enhance I�Ba expression. Nonetheless, suchhigh amount of I�Ba seems not to be sufficient or

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 7 of 12

competent for NF-�B inhibition in an environment ofpersistent TLR signaling. In agreement, when NF-�Bactivation is explored by EMSA (Figure 3C), clearlyresults that DEX does not impair transcription factorbinding to target DNA.Although the relevance of I�Ba up-regulation in GC

anti-inflammatory action has been questioned [42,43],our opinion is that it may rather depend on the timingof drug intervention with respect to activation of pro-inflammatory pathways. Accordingly, we verified on acell-based reporter system that expression of a NF-�Breporter gene is indeed down-modulated upon additionof DEX during LPS stimulation (Figure 3E), in a fashionthat does not imply diminished nuclear distribution ofthe transcription factor (Figure 3F) and hence suggeststhat NF-�B transactivation ability is decreased.In synthesis, data from the MM6 cells parallel those

from M� in indicating that 0.01 μM is the least DEXconcentration, among those tested, effective in tetheringNF-�B pro-inflammatory activity triggered by LPS. Inaddition, M� data demonstrate that 0.01 μM DEXdiminishes IL-23 expression while leaving unaffectedNF-�B activity in terms of nuclear localization or DNA-binding; rather, data from the MM6 reporter systemsuggest that, in an ongoing inflammatory state, DEXacts through reduction of NF-�B trans-activity.Besides NF-�B, p38 MAPK regulates inflammation

through both transcriptional and post-transcriptionalmechanisms, and MAPK inhibition has been suggestedas a promising anti-inflammatory approach [44,45].Indeed, p38 MAPK-dependent phosphorylation of pro-teins in the transactivation complex favors NF-�B activ-ity [46]. For example, MSK1 (mitogen- and stress-activated kinase 1), a downstream target of p38 MAPK,phosphorylates serine 276 of p65 enhancing transcrip-tion [36]. In addition, p38 favors inflammation also bypost-transcriptional stabilization of pro-inflammatorymRNAs that contain adenylate/uridylate-rich elementsin the 3’ untranslated region [47-49].We demonstrate that a decrease of p38 MAPK phos-

phorylation takes place in LPS-stimulated M� uponadministration of DEX ≥ 0.01 μM (Figure 4A). Ser276phosphorylation on NF-�B-p65 normally occurs follow-ing activation of p38 MAPK and ERK [50]. As expected,the down-modulation of p38 MAPK is accompanied bya consistent diminution of Ser276 phosphorylation (Fig-ure 4B). Although the role of ERK has not beenexplored in this work, these results strongly support theinvolvement of p38 MAPK inhibition in the mechanismof DEX-induced restraint of NF-�B trans-activity and ofpro-inflammatory gene expression.Recent works have focused on the role of MAP kinase

phosphatase 1 (MKP-1) in the context of GC anti-inflammatory function. MKP-1 is induced by GC, in fact

DEX raises MKP-1 levels in various cell types [51,52].This phosphatase acts on p38, JNK and ERK, therebyterminating their activation. According to the modelhere depicted, we show a DEX-dependent up-regulationof MKP-1 (Figure 4C), consistent with the decrease ofp38 and p65 phosphorylation. Moreover, addition ofNa3VO4, a phosphatase inhibitor, counteracts the effectof DEX on NF-�B transactivation ability, demonstratingthat dephosphorylation of substrates is necessary for theaccomplishment of DEX function (Figure 4D). The samemechanism likely applies to the specific expression ofendogenous IL-23p19 in M�, because the restraintcaused by DEX 0.01 μM is completely prevented byNa3VO4 (Figure 4E).Because we demonstrated that DEX causes inactiva-

tion of p38 MAPK, and p38 favors stability of pro-inflammatory transcripts, we next asked whether thelower amount of IL-23p19 results exclusively fromreduced expression - secondary to reduced NF-�B activ-ity - or is actually due to a combination of bothimpaired synthesis and enhanced degradation. Thedecay of IL-23p19 mRNA, in the presence or absence ofDEX, was analyzed by mRNA decay assays with theinhibitor of gene expression ActD (data not shown). Inour experimental approach ActD is added after 30 minfrom addition of DEX, to allow MKP-1 up-regulation.Unfortunately, due to such particular timing of theassay, we could not conclude whether the rapid (within30 min) decay in presence of DEX can be ascribed alsoto enhanced post-transcriptional destabilization of p19mRNA. A possibility exists that DEX is only capable ofpreventing stabilization of mRNA-protein complexes,but not of destabilizing pre-existing stable complexes[53]. In light of this, the decreased amount of IL-23p19may result from failing stabilization of newly synthesizedmRNA.In sum, we show here for the first time that a 0.01 μM

concentration of DEX reduces the expression of IL-23by restraining NF-�B transactivation ability. We demon-strate that NF-�B trans-activity is reduced by loss ofSer276 phosphorylation on p65, and that this is suffi-cient to switch off the expression of an exogenous NF-�B target gene as well as of the endogenous IL-23p19.We show that this effect requires a phosphatase activity,suggesting that it is mediated by p38 MAPK inhibition,achieved through MKP-1 induction by DEX. From atherapeutic point of view, this latter point is particularlyrelevant in light of the overexpression of p38a by M� ofthe intestinal lamina propria from inflammatory boweldisease patients [54].While this paper was in preparation, it was shown that

expression of IL12p40 is regulated by a ternary NF-�Bcomplex composed of p65, c-Rel and an hypopho-sphorylated form of I�Bb that stabilizes the

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 8 of 12

transcription factor [55]. Therefore, it will be intriguingto verify whether I�Bb plays a role in IL-23p19 expres-sion, and eventually to explore the impact of DEX onthe phosphorylation status of I�Bb.

ConclusionsSince continuous priming and recruitment of new Tcells into the effectors pool underlies chronic autoim-munity and is involved in the relapsing nature of dis-eases, gains of interest the rationale of IL-23neutralization to prevent relapsing-remitting autoimmu-nities [56]. To this regard, the efficacy of DEX treatmentin vitro is here demonstrated, with interesting implica-tions from a therapeutic perspective.Novel GR ligands that selectively promote transrepres-

sion but not transactivation have been proposed tomaintain anti-inflammatory effects while causing fewerside effects; however, our findings show that DEX-engaged GR induces an anti-inflammatory factor, MKP-1, indeed supporting the opinion that dimerization-defi-cient GR ligands might not be effective [57], as anti-inflammatory functions of GR are not independent of itsdimerization and transactivation activity.

MethodsCell cultures and reagentsHuman monocyte-derived M� were obtained fromhealthy blood. Adult volunteers signed an informed con-sent form before donation at the blood collection centreof Hospital S. Maria della Misericordia of Urbino (Italy),and samples were provided as anonymous. M� wereprepared by density gradient separation using Lympho-prep solution (specific density, 1.077; Axis-Shield PoCAS, Oslo, Norway). Cells were resuspended in RPMI-1640 medium supplemented with 10% (v/v) heat-inacti-vated FBS, 100 U/ml penicillin, 100 μg/ml streptomycinand 2 mM L-glutamine. Monocytes were separated byplastic adherence to tissue culture dishes (Sarstedt AG& Co., Nümbrecht, Germany) and/or chamber slides(Nalge Nunc International, Termo Fisher Scientific,Rochester, NY, USA), overnight at 37°C in a humidified5% CO2 atmosphere. Non-adherent cells were removedby repeated washes. Cells were cultured for 7 days, afterwhich > 95% of adherent cells were differentiated M�,as revealed by immunostaining and surface markeranalyses.MM6 cells, derived from human acute monocytic leu-

kemia, were obtained from DMSZ GmbH (Braunsch-weig, Germany) and cultured at a density of 0.5-1 × 106

cells/ml in a 5% CO2 atmosphere at 37°C, in RPMI-1640 supplemented with 10% heat-inactivated FBS, 2mM L-glutamine, 100 μg/ml streptomycin, 100 U/mlpenicillin, 1 × non-essential aminoacids, 9 μg/ml insulin(Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium

pyruvate. All cell culture reagents were from Lonza(Basel, Switzerland).MM6 cells were transfected with a vector containing

the luciferase coding sequence downstream of four tan-dem repeats of the -�B consensus element. Transfectionwas performed with Effectene Transfection Reagent(Qiagen GmbH, Hilden, Germany), according to manu-facturer’s instructions.Approximately 10^6 monocyte-derived M�, or 10^6

MM6 reporter cells, were stimulated with 1 μg/ml ofLPS (serotype 0111:B4, Sigma-Aldrich), for the timesindicated. DEX (Sigma-Aldrich) was resuspended in PBSto 1 mM and working dilutions were dosed spectrofoto-metrically at 239 nm. Na3VO4 (Sigma-Aldrich) was acti-vated according to the standard procedure prior to useat a final concentration of 250 μM.

ImmunofluorescenceThe p65 subunit of NF-�B was detected in human M�by indirect immunofluorescence. 5 × 104 cells werewashed in PBS, fixed with 4% formaldehyde in PBS for20 min at room temperature, and permeabilized with0.5% NP-40 in PBS for 10 min. Samples were blockedwith 1% (w/v) BSA and 0.1% (w/v) gelatin in PBS for1h, then incubated 1h at room temperature with a rab-bit polyclonal anti-p65 (Santa Cruz Biotechnology, SantaCruz, CA, USA), 1:100 in 1% BSA/PBS. Secondary anti-body was a FITC-conjugated goat anti-rabbit IgG(Sigma-Aldrich) 1:300 in 1% BSA/PBS, incubated 1h atroom temperature. DNA was stained with DAPI (Sigma-Aldrich) at a final concentration 0.2 μg/ml. Sampleswere fixed in gelvatol and observed with an OlympusIX51 Fluorescence Microscope. FITC and DAPI imageswere compared by overlay.

RT-PCRTotal RNA from M� was prepared with RNeasy PlusExtraction kit (Qiagen) following the manufacturer’sinstructions. 0.5 μg of RNA was reverse transcribed in areaction mix assembled as described in [58]. Real TimePCR analyses were performed in an ABI PRISM 7700Sequence Detection System 1.9 (Applied Biosystems,Life Technologies, Carlsbad, CA, USA). PCR reactionswere assembled in a 25 μl volume using the Hot RescueReal Time PCR SYBR Green Kit (Diatheva, Fano, Italy)and 0.2 μM of each primer. Primer oligonucleotideswere purchased from Sigma-Aldrich and sequences arereported in Table 1. For each sample three replicateswere run, corresponding to 10 nanograms of total RNA.Thermal cycling was performed as follows: 10 min at95°C; 45 cycles of denaturation at 95°C for 15s, anneal-ing for 30s at the temperatures indicated in Table 1 andextension at 72°C for 30s. At the end of PCR cycles, amelting curve was generated to verify the specificity of

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 9 of 12

PCR products. Relative quantification of selectedmRNAs was performed according to the ΔΔCt com-parative method.

Assay of IL-23 production106 M�, plated as above described, were stimulated for6h with 1 μg/ml LPS, and either treated or not with DEXfor two more hours. The culture medium was collectedat 8h or replaced and collected at 16 or 24h. IL-23 con-centration was measured with the IL-23 HeterodimerImmunoassay kit, according to manufacturer’s instruc-tions (Invitrogen Life Technologies, Carlsbad, CA, USA).

Western immunoblotting and EMSADenatured whole cell extracts for western blotting ana-lyses and native whole cell extracts for EMSA wereprepared as elsewhere described [58] and protein con-centration was determined according to the Lowry [59]and Bradford [60] methods, respectively. Cytosolic andnuclear extracts from MM6 cells were prepared as fol-lows: cells were collected in microfuge tubes and pel-leted, washed with ice-cold PBS and incubated 10 minon ice in buffer A (10 mM Hepes/KOH, pH 7.9, 1.5mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mMPMSF, 0.2 mM EDTA, 10 μg/ml leupeptin, 10 μg/mlpepstatin, 0.1% Nonidet P-40). Lysates were then cen-trifuged 10 min at 12,000 rpm, 4°C. The cytosolic frac-tion was removed to fresh tubes whereas pelletednuclei were resuspended in buffer B (20 mM Hepes/KOH pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mMMgCl2, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF,10 μg/ml leupeptin, 10 μg/ml pepstatin), incubated 20min on ice and centrifuged 10 min at 12,000 rpm, 4°C,finally collecting the supernatants (nuclear extracts) infresh tubes.

Western blotting was performed on cell extracts (15-60 μg, depending on the protein of interest) diluted in 2× sample buffer, boiled, subjected to 10% SDS-polyacry-lamide gel electrophoresis, and electrotransferred to a0.2 μm nitrocellulose membrane (Bio-Rad, Hercules,CA, USA). The membrane was then blocked with 5%non-fat dry milk or 5% BSA in TBS-0.1% Tween-20(Sigma-Aldrich). Antibody against IL-23p19 was amouse monoclonal from BioLegend (San Diego, CA,USA). I�Ba (C-21) and p65 (C-20) Ab were rabbit poly-clonal from Santa Cruz Biotechnology and for a loadingcontrol a rabbit polyclonal anti-actin (Sigma-Aldrich)was utilized. Phosphorylation of p65 was determinedwith a Phospho-Ser276-specific rabbit polyclonal Ab(#3037), whereas that of p38 MAPK was determinedwith a Phospho-Thr180/Tyr182-specific mouse mono-clonal Ab (#9216) and with a p38 rabbit polyclonal Ab(#9212) (Cell Signaling Technology, Boston, MA, USA).Secondary anti-rabbit and anti-mouse HRP-conjugatedAb were from Bio-Rad and ECL reagents were from GEHealthcare (Little Chalfont, UK). Densitometric analyseswere performed with the ChemiDoc System (Bio-Rad).NF-�B EMSA reactions were assembled as in [58] with

10 μg of native whole cell extracts. DNA-protein com-plexes were resolved by electrophoresis on 5% non-dena-turating polyacrilamide gels, in a TBE buffer system, at aconstant voltage of 170 V, for 3 h at 4°C. Gels were thendried and subjected to autoradiography in the GS250Molecular Imager system, followed by densitometric ana-lysis with the Molecular Analyst software (Bio-Rad).

LUC assayLUC assays were performed with the Luciferase AssaySystem (Promega, Madison, WI, USA) according to themanufacturer’s instruction. Briefly, at the end of thetreatment cells were collected, washed in ice cold PBSand lysed in 1 × Reporter Buffer. 10 μl of cleared lysate,corresponding to 10-20 μg of proteins, were read in a96-well plate luminometer (BMG Labtech GmbH,Offenburg, Germany). The luminescent signal was nor-malized to the total amount of proteins per well, deter-mined according to the Bradford method.

Statistical analysisData are reported as the average ± s.d. of at least threeindependent experiments and - unless otherwise stated -are expressed as fold changes relative to the calibratorsample each time indicated. The Friedman statisticaltest was followed by the Dunn’s multiple comparisonand a p value < 0.05 was considered to be significant.

AbbreviationsAb: antibody; ActD: actinomycin D; APC: antigen presenting cell; DC:dendritic cell; DEX: dexamethasone; GC: glucocorticoid; GR: glucocorticoid

Table 1 PCR amplification primers

Target gene Primer sequences Ta(°C)

FKBP5 Fwd 5’-CCCTCGAATGCAACTCTCTT-3’Rev 5’-TCTCCTTTCCGTTTGGTTCT-3’

62

GAPDH Fwd 5’-TGCACCACCAACTGCTTAG-3’Rev 5’-GATGCAGATGATGATGTTC-3’

57

IL-12p35 Fwd 5’-CATGCTGGCAGTTATTGA-3’Rev 5’-AAGTATGCAGAGCTTGATTTT-3’

57

IL-12p40 Fwd 5’-AGGAGAGTCTGCCCATTGAGG-3’Rev 5’-GACCTCCACCTGCCGAGAA-3’

65

IL-23p19 Fwd 5’-CGTCTCCTTCTCCGCTTCA-3’Rev 5’-GTGCCTGGGGTGGTAGATTT-3’

65

MKP-1 Fwd 5’-AGCAGAGGCGAAGCATCATC-3’Rev 5’-CCCAGCCTCTGCCGAAC-3’

61

a) Fwd: forward primer.

b) Rev: reverse primer.

c) Ta: annealing temperature.

d) GAPDH: internal reference gene.

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 10 of 12

receptor; GRE: glucocorticoid response element; IF: immunofluorescence;IκB: inhibitor of -κB; IL-23: interleukin-23; LPS: lipopolysaccharide; LUC:luciferase; M�: macrophage; MKP-1: MAPK phosphatase 1; MM6: monomac6 cells; Na3VO4: sodium orthovanadate; NF-κB: nuclear factor -κB; NK:natural killer cell; PBS: phosphate buffered saline; TLR: Toll-like receptor

AcknowledgementsThe authors declare no financial or commercial conflict of interest.This work was supported by FIRB 2006 funding [RBIP067F9E_007] granted toM. Magnani.Authors state that they have followed the principles outlined in theDeclaration of Helsinki and have obtained institutional review boardapproval for the use of primary cells from human blood in research.

Authors’ contributionsDesign of the study, RT-PCR, western blotting, EMSA, reporter cells were byLP. AA and CS contributed to design of the study and performedmacrophage cultures, immunofluorescence and ELISA. Conception, designand coordination were by principal investigator MM. All authors read andapproved the final manuscript.

Received: 3 May 2011 Accepted: 26 July 2011 Published: 26 July 2011

References1. Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, Vega F, Yu N,

Wang J, Singh K, et al: Novel p19 protein engages IL-12p40 to form acytokine, IL-23, with biological activities similar as well as distinct fromIL-12. Immunity 2000, 13:715-725.

2. Hunter CA: New IL-12-family members: IL-23 and IL-27, cytokines withdivergent functions. Nat Rev Immunol 2005, 5:521-531.

3. Waibler Z, Kalinke U, Will J, Juan MH, Pfeilschifter JM, Radeke HH: TLR-ligand stimulated interleukin-23 subunit expression and assembly isregulated differentially in murine plasmacytoid and myeloid dendriticcells. Mol Immunol 2007, 44:1483-1489.

4. Roses RE, Xu S, Xu M, Koldovsky U, Koski G, Czerniecki BJ: DifferentialProduction of IL-23 and IL-12 by Myeloid-Derived Dendritic Cells inResponse to TLR Agonists. J Immunol 2008, 181:5120-5127.

5. Parham C, Chirica M, Timans J, Vaisberg E, Travis M, Cheung J, Pflanz S,Zhang R, Singh KP, Vega F, et al: A Receptor for the HeterodimericCytokine IL-23 Is Composed of IL-12R{beta}1 and a Novel CytokineReceptor Subunit, IL-23R. J Immunol 2002, 168:5699-5708.

6. McKenzie BS, Kastelein RA, Cua DJ: Understanding the IL-23-IL-17 immunepathway. Trends Immunol 2006, 27:17-23.

7. Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL: Interleukin-23promotes a distinct CD4 T cell activation state characterized by theproduction of interleukin-17. J Biol Chem 2003, 278:1910-1914.

8. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD,McClanahan T, Kastelein RA, Cua DJ: IL-23 drives a pathogenic T cellpopulation that induces autoimmune inflammation. J Exp Med 2005,201:233-240.

9. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B: TGFbeta inthe context of an inflammatory cytokine milieu supports de novodifferentiation of IL-17-producing T cells. Immunity 2006, 24:179-189.

10. McGeachy MJ, Cua DJ: Th17 cell differentiation: the long and windingroad. Immunity 2008, 28:445-453.

11. Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, Lucian L, To W,Kwan S, Churakova T, et al: Interleukin-23 rather than interleukin-12 is thecritical cytokine for autoimmune inflammation of the brain. Nature 2003,421:744-748.

12. Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClanahan T,Kastelein RA, Sedgwick JD, Cua DJ: Divergent pro- and antiinflammatoryroles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med2003, 198:1951-1957.

13. Yen D, Cheung J, Scheerens H, Poulet F, McClanahan T, McKenzie B,Kleinschek MA, Owyang A, Mattson J, Blumenschein W, et al: IL-23 isessential for T cell-mediated colitis and promotes inflammation via IL-17and IL-6. J Clin Invest 2006, 116:1310-1316.

14. Lubberts E: IL-17/Th17 targeting: on the road to prevent chronicdestructive arthritis? Cytokine 2008, 41:84-91.

15. Vaknin-Dembinsky A, Balashov K, Weiner HL: IL-23 Is Increased in DendriticCells in Multiple Sclerosis and Down-Regulation of IL-23 by Antisense

Oligos Increases Dendritic Cell IL-10 Production. J Immunol 2006,176:7768-7774.

16. Li Y, Chu N, Hu A, Gran B, Rostami A, Zhang GX: Increased IL-23p19expression in multiple sclerosis lesions and its induction in microglia.Brain 2007, 130:490-501.

17. Lee E, Trepicchio WL, Oestreicher JL, Pittman D, Wang F, Chamian F,Dhodapkar M, Krueger JG: Increased expression of interleukin 23 p19 andp40 in lesional skin of patients with psoriasis vulgaris. J Exp Med 2004,199:125-130.

18. Duerr RH, Taylor KD, Brant SR, Rioux JD, Silverberg MS, Daly MJ,Steinhart AH, Abraham C, Regueiro M, Griffiths A, et al: A genome-wideassociation study identifies IL23R as an inflammatory bowel diseasegene. Science 2006, 314:1461-1463.

19. Schmidt C, Giese T, Ludwig B, Mueller-Molaian I, Marth T, Zeuzem S,Meuer SC, Stallmach A: Expression of interleukin-12-related cytokinetranscripts in inflammatory bowel disease: elevated interleukin-23p19and interleukin-27p28 in Crohn’s disease but not in ulcerative colitis.Inflamm Bowel Dis 2005, 11:16-23.

20. Langowski JL, Zhang X, Wu L, Mattson JD, Chen T, Smith K, Basham B,McClanahan T, Kastelein RA, Oft M: IL-23 promotes tumour incidence andgrowth. Nature 2006, 442:461-465.

21. Rhen T, Cidlowski JA: Antiinflammatory Action of Glucocorticoids – NewMechanisms for Old Drugs. N Engl J Med 2005, 353:1711-1723.

22. Adcock IM, Ito K, Barnes PJ: Glucocorticoids: Effects on GeneTranscription. Proc Am Thorac Soc 2004, 1:247-254.

23. Barnes PJ: How corticosteroids control inflammation: Quintiles PrizeLecture 2005. Br J Pharmacol 2006, 148:245-254.

24. Newton R, Holden NS: Separating Transrepression and Transactivation: ADistressing Divorce for the Glucocorticoid Receptor? MolecularPharmacology 2007, 72:799-809.

25. De Bosscher K, Van Craenenbroeck K, Meijer OC, Haegeman G: Selectivetransrepression versus transactivation mechanisms by glucocorticoidreceptor modulators in stress and immune systems. Eur J Pharmacol2008, 583:290-302.

26. Barnes PJ, Adcock IM: Glucocorticoid resistance in inflammatory diseases.The Lancet 2009, 373:1905-1917.

27. Yaffe SJ, Aranda JV: Pediatric pharmacology therapeutic principles in practicePhiladelphia: Saunders; 1992.

28. Fahey AJ, Robins RA, Kindle KB, Heery DM, Constantinescu CS: Effects ofglucocorticoids on STAT4 activation in human T cells are stimulus-dependent. J Leukoc Biol 2006, 80:133-144.

29. Ma W, Gee K, Lim W, Chambers K, Angel JB, Kozlowski M, Kumar A:Dexamethasone Inhibits IL-12p40 Production in Lipopolysaccharide-Stimulated Human Monocytic Cells by Down-Regulating the Activity ofc-Jun N-Terminal Kinase, the Activation Protein-1, and NF-{kappa}BTranscription Factors. J Immunol 2004, 172:318-330.

30. Utsugi M, Dobashi K, Ishizuka T, Kawata T, Hisada T, Shimizu Y, Ono A,Mori M: Rac1 negatively regulates lipopolysaccharide-induced IL-23p19 expression in human macrophages and dendritic cells and NF-kappaB p65 trans activation plays a novel role. J Immunol 2006,177:4550-4557.

31. Li Y, Chu N, Hu A, Gran B, Rostami A, Zhang GX: Inducible IL-23p19expression in human microglia via p38 MAPK and NF-kappaB signalpathways. Exp Mol Pathol 2008, 84:1-8.

32. Dobreva ZG, Stanilova SA, Miteva LD: Differences in the inducible geneexpression and protein production of IL-12p40, IL-12p70 and IL-23:involvement of p38 and JNK kinase pathways. Cytokine 2008, 43:76-82.

33. Crinelli R, Carloni E, Menotta M, Giacomini E, Bianchi M, Ambrosi G, Giorgi L,Magnani M: Oxidized ultrashort nanotubes as carbon scaffolds for theconstruction of cell-penetrating NF-kappaB decoy molecules. ACS Nano2010, 4:2791-2803.

34. Lasa M, Brook M, Saklatvala J, Clark AR: Dexamethasone destabilizescyclooxygenase 2 mRNA by inhibiting mitogen-activated protein kinasep38. Mol Cell Biol 2001, 21:771-780.

35. Smoak K, Cidlowski JA: Glucocorticoids regulate tristetraprolin synthesisand posttranscriptionally regulate tumor necrosis factor alphainflammatory signaling. Mol Cell Biol 2006, 26:9126-9135.

36. Beck IM, Vanden Berghe W, Vermeulen L, Bougarne N, Vander Cruyssen B,Haegeman G, De Bosscher K: Altered subcellular distribution of MSK1induced by glucocorticoids contributes to NF-kappaB inhibition. EMBO J2008, 27:1682-1693.

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 11 of 12

37. Crinelli R, Antonelli A, Bianchi M, Gentilini L, Scaramucci S, Magnani M:Selective inhibition of NF-kB activation and TNF-alpha production inmacrophages by red blood cell-mediated delivery of dexamethasone.Blood Cells Mol Dis 2000, 26:211-222.

38. Baughman G, Wiederrecht GJ, Chang F, Martin MM, Bourgeois S: Tissuedistribution and abundance of human FKBP51, and FK506-bindingprotein that can mediate calcineurin inhibition. Biochem Biophys ResCommun 1997, 232:437-443.

39. D’Ascenzo M, Antonelli A, Chiarantini L, Mancini U, Magnani M: In Red bloodcells as glucocorticoids delivery system. In Erythrocytes as drug carriers inmedicine. Edited by: Sprandel U, Way JL. New York: Plenum Press; 1997:.

40. Pierigè F, Serafini S, Rossi L, Magnani M: Cell-based drug delivery. AdvDrug Deliv Rev 2008, 60:286-295.

41. De Bosscher K, Vanden Berghe W, Haegeman G: Mechanisms of anti-inflammatory action and of immunosuppression by glucocorticoids:negative interference of activated glucocorticoid receptor withtranscription factors. Journal of Neuroimmunology 2000, 109:16-22.

42. De Bosscher K, Schmitz ML, Vanden Berghe W, Plaisance S, Fiers W,Haegeman G: Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference withtransactivation. Proc Natl Acad Sci USA 1997, 94:13504-13509.

43. Heck S, Bender K, Kullmann M, Gottlicher M, Herrlich P, Cato AC: I kappaBalpha-independent downregulation of NF-kappaB activity byglucocorticoid receptor. EMBO J 1997, 16:4698-4707.

44. Westra J, Doornbos-van der Meer B, de Boer P, van Leeuwen MA, vanRijswijk MH, Limburg PC: Strong inhibition of TNF-alpha production andinhibition of IL-8 and COX-2 mRNA expression in monocyte-derivedmacrophages by RWJ 67657, a p38 mitogen-activated protein kinase(MAPK) inhibitor. Arthritis Res Ther 2004, 6:R384-R392.

45. Medicherla S, Fitzgerald MF, Spicer D, Woodman P, Ma JY, Kapoun AM,Chakravarty S, Dugar S, Protter AA, Higgins LS: p38alpha-selectivemitogen-activated protein kinase inhibitor SD-282 reduces inflammationin a subchronic model of tobacco smoke-induced airway inflammation.J Pharmacol Exp Ther 2008, 324:921-929.

46. Carter AB, Knudtson KL, Monick MM, Hunninghake GW: The p38 Mitogen-activated Protein Kinase Is Required for NF-kappaB-dependent GeneExpression. Journal of Biological Chemistry 1999, 274:30858-30863.

47. Zhang T, Kruys V, Huez G, Gueydan C: AU-rich element-mediatedtranslational control: complexity and multiple activities of trans-activating factors. Biochem Soc Trans 2002, 30:952-958.

48. Dean JL, Sully G, Clark AR, Saklatvala J: The involvement of AU-richelement-binding proteins in p38 mitogen-activated protein kinasepathway-mediated mRNA stabilisation. Cell Signal 2004, 16:1113-1121.

49. Tudor C, Marchese FP, Hitti E, Aubareda A, Rawlinson L, Gaestel M,Blackshear PJ, Clark AR, Saklatvala J, Dean JL: The p38 MAPK pathwayinhibits tristetraprolin-directed decay of interleukin-10 and pro-inflammatory mediator mRNAs in murine macrophages. FEBS Lett 2009,583:1933-1938.

50. Deak M, Clifton AD, Lucocq LM, Alessi DR: Mitogen- and stress-activatedprotein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38,and may mediate activation of CREB. EMBO J 1998, 17:4426-4441.

51. Kassel O, Sancono A, Kratzschmar J, Kreft B, Stassen M, Cato AC:Glucocorticoids inhibit MAP kinase via increased expression anddecreased degradation of MKP-1. EMBO J 2001, 20:7108-7116.

52. Lasa M, Abraham SM, Boucheron C, Saklatvala J, Clark AR: Dexamethasonecauses sustained expression of mitogen-activated protein kinase (MAPK)phosphatase 1 and phosphatase-mediated inhibition of MAPK p38. MolCell Biol 2002, 22:7802-7811.

53. Bergmann MW, Staples KJ, Smith SJ, Barnes PJ, Newton R: GlucocorticoidInhibition of Granulocyte Macrophage-Colony-Stimulating Factor from Tcells Is Independent of Control by Nuclear Factor-{kappa}B andConserved Lymphokine Element 0. Am J Respir Cell Mol Biol 2004,30:555-563.

54. Waetzig GH, Seegert D, Rosenstiel P, Nikolaus S, Schreiber S: p38 Mitogen-Activated Protein Kinase Is Activated and Linked to TNF-{alpha}Signaling in Inflammatory Bowel Disease. J Immunol 2002, 168:5342-5351.

55. Rao P, Hayden MS, Long M, Scott ML, West AP, Zhang D, Oeckinghaus A,Lynch C, Hoffmann A, Baltimore D, et al: IkappaBbeta acts to inhibit andactivate gene expression during the inflammatory response. Nature 2010,466:1115-1119.

56. Luger D, Silver PB, Tang J, Cua D, Chen Z, Iwakura Y, Bowman EP,Sgambellone NM, Chan CC, Caspi RR: Either a Th17 or a Th1 effectorresponse can drive autoimmunity: conditions of disease induction affectdominant effector category. J Exp Med 2008, 205:799-810.

57. Clark AR, Martins JRS, Tchen CR: Role of Dual Specificity Phosphatases inBiological Responses to Glucocorticoids. Journal of Biological Chemistry2008, 283:25765-25769.

58. Palma L, Crinelli R, Bianchi M, Magnani M: De-ubiquitylation is the mostcritical step in the ubiquitin-mediated homeostatic control of the NF-kappaB/IKK basal activity. Mol Cell Biochem 2009, 331:69-80.

59. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement withthe Folin phenol reagent. J Biol Chem 1951, 193:265-275.

60. Bradford MM: A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem 1976, 72:248-254.

doi:10.1186/1471-2210-11-8Cite this article as: Palma et al.: Dexamethasone restrains ongoingexpression of interleukin-23p19 in peripheral blood-derived humanmacrophages. BMC Pharmacology 2011 11:8.

Submit your next manuscript to BioMed Centraland take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit

Palma et al. BMC Pharmacology 2011, 11:8http://www.biomedcentral.com/1471-2210/11/8

Page 12 of 12