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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 4, 2004 as doi:10.1096/fj.03-1367fje. |
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Department of Biological Sciences, Rutgers University, Newark, New Jersey, USA
1Correspondence: Rutgers University, Department Biological Sciences, 101 Warren St., Newark, NJ 07102, USA. E-mail: dganea{at}andromeda.rutgers.edu
SPECIFIC AIMS
Structurally related cytokines IL-12 and the recently discovered IL-23 and IL-27 have overlapping but distinct functions. IL-27, IL-12, and IL-23 act sequentially on CD4+ T cells, inducing the differentiation of Th0 into Th1 effectors, and subsequently into memory Th1. The responsiveness of Th0/effector Th1/memory Th1 cells to different members of the IL-12 family is due to differential expression of the specific receptors. IL-23 is a heterodimer consisting of the subunit p40 shared with IL-12 and the unique subunit p19, distantly related to the IL-12p35 subunit. IL-23 emerged recently as one of the most potent proinflammatory cytokines. Transgenic p19 overexpressing mice die before the age of 3 months after systemic inflammation, indicating a prominent proinflammatory role for IL-23. The proinflammatory effect of IL-23 has both beneficial and detrimental consequences. Through its effects on activated/memory T cells, IL-23 contributes to systemic immunity against pathogens and tumors. However, recent studies also show that IL-23 plays an essential role in chronic inflammatory bowel diseases, experimental autoimmune encephalomyelitis, and rheumatoid arthritis.
After signaling through toll-like receptors (TLR), mature dendritic cells (DCs) function as major sources of IL-12/IL-23. It is not known, however, whether immature DCs can be induced to express IL-12 and/or IL-23 or whether this expression is disconnected from the maturation process defined as the increased capacity to stimulate CD4+ T cells through the up-regulation of stimulatory/costimulatory molecules and migration to T cell areas. Lipid mediators such as prostaglandins, particularly PGE2, are generated in inflammatory conditions and regulate cytokine/chemokine production by immune cells. The major aim of the present study was to examine the effects of PGE2 on the expression of p19, p40, and p35 mRNA and on the release of functional IL-23 and IL-12 by immature bone marrow-derived DCs (BM-DCs).
PRINCIPAL FINDINGS
1. PGE2 induces the release of functional IL-23 from BM-DCs
Immature DCs were generated from the bone marrow of B10.A mice in the presence of GM-CSF and purified based on the expression of the CD11c marker. The purified CD11c + DCs were cultured with various PGE2 concentrations in the presence or absence of GM-CSF. The release of functional IL-23 was assayed by its ability to induce IL-17 production in activated CD4 + T cells. Supernatants harvested from DCs cultured in the presence of increasing concentrations of PGE2 induce the production of increasing amounts of IL-17 (Fig. 1
A). PGE2 added directly to the activated T cells does not induce IL-17. We assessed the relationship between IL-17 release from activated T cells and IL-23 presence in the PGE2-treated DC supernatants by using anti-p40 Abs and soluble IL-23R. IL-12 and IL-23 share the p40 subunit, but only IL-23 induces IL-17 production in activated T cells. Activated T cells exposed to supernatants pretreated with anti-p40 Abs or sIL-23R produce significantly less IL-17, confirming that IL-17 production is due to the presence of IL-23 in the PGE2-treated DC supernatants (Fig. 1B
). Similar results were obtained with human rIL-23 with or without sIL-23R. Although PGE2 alone can induce IL-23 in DCs, the presence of GM-CSF substantially increases the effect of PGE2 (Fig. 1C
), suggesting that production of IL-23 is promoted in inflammatory conditions when both PGE2 and GM-CSF are present.
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2. The effect of PGE2 is mediated by the EP2/EP4 receptors
Four different PGE2 receptors (EP1, 2, 3, and 4) are expressed in various tissues. Our previous studies indicated that immature DCs express EP1, EP2, and EP4 and that the EP2/EP4 receptors mediate the inhibitory effects of PGE2 on LPS- and PGN-induced expression of TNF and CCL3/4. We assessed the contribution of the four EP receptors in the induction of IL-23 by using receptor agonists. Sulprostone, an EP1/EP3 agonist, does not induce IL-23. In contrast, butaprost, a specific EP2 agonist, and misoprostol, an EP4/EP2 agonist, induce IL-23 (Fig. 1D
). This suggests that the induction of IL-23 by PGE2 is mediated through EP2, and possibly EP4 receptors.
Since both EP2 and EP4 are coupled to adenylyl cyclase, we explored the involvement of cAMP. Similar to supernatants from PGE2-treated DCs, supernatants from DCs treated with dbcAMP or forskolin induce IL-17 production in activated CD4 + T cells, whereas dbcAMP and forskolin added directly to the T cells do not induce IL-17 (Fig. 1E
).
3. PGE2 induces p19 and p40, but not p35, expression
IL-23 is a heterodimer composed of p19, a subunit specific for IL-23, and p40, a subunit shared with IL-12. We used regular (Fig. 2
A) and real-time RT-PCR to investigate PGE2 induction of p19. Immature DCs cultured in medium express low levels of p19 at 3 h and no p19 at later time points. In contrast, DCs treated with PGE2 express much higher levels of p19 at 3 h and continue to express p19 up to 12 h after treatment with PGE2. The amplified fragment was identified as p19 by cloning and sequencing (Fig. 2B
). The effect of PGE2 on p40 expression was determined by RPA and real-time RT-PCR. The RPA assay indicated an increase in p40 expression and a significant increase in both IL-1ß and IL-6 expression (Fig. 2C
). Real-time PCR confirmed that, similar to p19, PGE2 induces higher p40 expression. In contrast, PGE2 did not affect p35 expression in either RPA or real-time PCR assays. These results indicate that PGE2 induces the early expression of both IL-23 subunits, with no significant effect on p35, and therefore on IL-12, expression. This was confirmed at the protein level by assaying p40 and IL-12p70 (the biologically active IL-12) by ELISA. PGE2 induced higher levels of p40 but not p70 in immature dendritic cells (Fig. 2D
). As a control, LPS induced both p40 and p70; in agreement with previous reports, PGE2 inhibited p70 production in the LPS-treated DCs (mature DCs).
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CONCLUSIONS AND SIGNIFICANCE
Upon antigen capture, DCs initiate an expression program for proinflammatory genes, including cytokines and chemokines; undergo maturation by up-regulating the expression of stimulatory/costimulatory molecules; and migrate to the T cell areas in the lymphoid organs. Lipid mediators such as PGE2, abundant at inflammatory sites, are being investigated as modulators of DC function.
Here we report on the induction of IL-23 expression and release by PGE2 in immature BM-DCs. PGE2 induces the expression of p19 and p40, but not p35, and subsequently the release of IL-23, but not IL-12, from immature DCs. In addition, PGE2 does not induce DC maturation as defined by the up-regulation of stimulatory/costimulatory molecules. Recently, IL-23 emerged as one of the most potent proinflammatory cytokines. The fact that the ubiquitous transgenic expression of p19 leads to multiorgan inflammation and premature death in mice supports the pivotal role of IL-23 in inflammation. Recent studies also showed that IL-23 functions as the essential factor in autoimmune diseases, including rheumatoid arthritis. Since IL-23 appears to be an essential promoter of joint autoimmune inflammation, we propose that PGE2 generated in the affected joint as a result of cellular stress or in response to microbial/viral stimuli plays an essential role in late-stage joint inflammation and destruction (Fig. 3
). Our data indicate that PGE2 induces resident immature DCs to generate IL-23, which in turn induces IL-17, a potent proinflammatory cytokine secreted by activated T cells and synovial cells, and found at high levels in the synovial fluid of RA patients. The data reported here also support a novel mechanism for the beneficial role of the Cox 1/2 inhibitors in rheumatoid arthritis, i.e., the inhibition of local IL-23/IL-17 production mediated through a reduction in endogenous PGE2 release. PGE2 could synergize with other factors/cytokines in the induction of IL-23. Our data support, for example, such a synergistic effect for PGE2 and GM-CSF. Identification of other factors/cytokines acting in conjunction with PGE2 in the induction of IL-23 could lead to the development of new combined therapies for autoimmune diseases such as rheumatoid arthritis and experimental autoimmune encephalomyelitis, where IL-23 has been shown to play an essential role.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1367fje; doi: 10.1096/fj.03-1367fje
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