In the present study, we demonstrate that TH-17 cells are more abundant in RA SF compared with normal PB and RA PB. IL-17 is produced by activated human CD4+CD45RO+ memory T cells, while only low levels of IL-17 are secreted by CD4+CD45RA+ naive T cells [21
]. The increased presence of TH-17 cells in RA SF may be related to an increased percentage of memory CD4+ T cells in RA SF. However, this is not likely to be the entire explanation because about 50% of normal PB and RA PB CD4+ cells are memory cells [23
]. In the absence of IL-23 and P/I, TH-17 cells were 38-fold higher in RA SF compared with normal PB, whereas TH-17 cells were essentially undetectable in RA PB. The reduction of TH-17 cells in RA compared with normal PB may be due to the enrichment of TH-17 cells in the RA joint, although differences in disease activity or treatment may also be responsible. Our observations contrast with a recently published study that observed a reduction of TH-17 cells in RA SF compared with RA PB [24
]. The reason for the difference between that study and other studies concerning IL-17 and TH-17 cells (including the work presented here) is not clear, although it is possible that differences in patient selection and therapy may have contributed, as well as technical differences such as the antibodies used or methods employed for cell fixation or permeabilisation.
Treatment with IL-23 plus P/I resulted in increased numbers of TH-17 cells in RA SF, which were greater than those observed in normal and RA PB. The expression of IL-23R is increased on CD45RO memory T cells, and IL-23 may also induce the expression of its own receptor [21
]. Although our data suggest that RA SF CD4+ cells are capable of responding to IL-23, the role of this cytokine in TH-17 cell polarisation in RA remains uncertain. We detected IL-23 in only four of the 27 RA SFs analysed, while all the RA SFs possessed IL-17. In marked contrast, a recent study showed very high levels of IL-23p19 in RA SF and PB [16
]. The explanation for the very high values [16
] may be technical; however, patient characteristics and therapy at the time of obtaining the SFs may be important. In this regard, a post hoc
examination of the clinical features of the four RA patients in the present study with high SF levels of IL-23 revealed that all four were on anti-TNF treatment, suggesting at least a moderate level of RA burden in order to clinically merit these expensive agents. Further, three of the four had documented lengthy disease duration of between 12 and 25 years (the fourth also had 'longstanding' disease according to the treating physician's narrative notes), and three of the four had a well-documented history of repeated presentation to their treating physician for recurrent and/or unremitting swelling in the knees (which are the joints from which the SF samples in this study were taken) despite being on anti-TNF treatment.
Nonetheless, our observations are consistent with a recent study that also observed low levels of IL-23 in RA SF [25
]. Another study demonstrated detectable levels of IL-23 in about half of RA SFs examined, with only two samples possessing more than 250 pg/ml, which was more consistent with our observations [15
]. Suggesting the importance of therapy in the expression of IL-23, these authors demonstrated a significant correlation between the levels of IL-23 and IL-17 in the RA SF before the initiation of etanercept [15
]. Consistent with these observations, the two patients in the current study who had the greatest increase of TH-17 cells after treatment with P/I plus IL-23 were taking no DMARDs or biological agents. Further, in the RA joint, CD4+ cells may be more responsive to low levels of IL-23, especially in an environment rich in IL-1β and IL-6, as observed in the RA joint.
Even though IL-23 p19/p40 was very low in RA SF, macrophages isolated from RA SF had significantly increased IL-23 p19 mRNA expression (four-fold increase) compared with control macrophages. Consistent with this observation, employing immunohistochemistry, IL-23p19 was expressed abundantly in RA ST [14
]. Further, RA SF macrophages stimulated with PGN expressed significantly higher levels of IL-23 mRNA compared with control macrophages treated similarly. The higher levels of IL-23 mRNA in RA SF versus control macrophages may be due to increased expression of TLR2 on macrophages obtained from the RA joint and the expression of endogenous TLR ligands in the RA joint [19
]. Supporting the relevance of this possibility, TLR ligand-activated monocytes and dendritic cells result in TH-17 polarisation [10
The levels of IL-27 mRNA were also significantly higher in RA SF compared with control macrophages; however, the levels of IL-27 were similar in both groups in the presence of the TLR2 ligation. Since IL-27 may be important in suppressing the differentiation of TH-17 cells [12
], these observations suggest that therapy directed at enhancing the expression of IL-27 in RA may be therapeutically beneficial. Supporting this approach, others have shown that IL-27 ameliorates collagen-induced arthritis [13
], although IL-27 promoted proteoglycan-induced arthritis [27
IFN-γ in RA SF macrophages was also examined because IFN-γ may suppress TH-17 polarisation. IFN-γ mRNA was slightly increased in RA SF, compared with control macrophages. The induction of IFN-γ was significantly greater after TLR2 ligation employing RA SF, compared with control macrophages. The expression of IFN-γ in human alveolar macrophages has been reported in sarcoidosis and after infection with Mycobacterium tuberculosis
]. These results indicate that RA SF contains multiple factors that may modulate the development of TH-17 cells in the RA joint.