Different T cell subsets in the nodule and synovial membraneAbsence of interleukin-17A in rheumatoid nodules.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 58, No. 6, June 2008, pp 1601–1608 DOI 10.1002/art.23455 © 2008, American College of Rheumatology Different T Cell Subsets in the Nodule and Synovial Membrane Absence of Interleukin-17A in Rheumatoid Nodules Lisa K. Stamp,1 Andrea Easson,2 Ulrike Lehnigk,2 John Highton,2 and Paul A. Hessian2 the expression of IFN␥ was 0.67 ⴞ 0.68 ng and that of IL-12 was 0.48 ⴞ 0.23 ng. Conclusion. IL-17 family members are varyingly expressed in rheumatoid nodules. The paucity of IL-17A in nodules suggests an important difference from that observed in the synovium. The expression of IL-23 below a critical threshold level seems the most likely explanation for the virtual absence of IL-17A. The presence of tissue destruction within the nodule despite the absence of IL-17A suggests that IL-17A may be an important amplifier rather than an absolute requirement for inflammation in RA. Objective. To determine gene expression of the interleukin-17 (IL-17) family members (IL-17A–F) in rheumatoid subcutaneous nodules, and to assess the cytokines involved in regulating IL-17A expression. Methods. Total RNA was isolated from 19 nodules obtained from 16 different patients with rheumatoid arthritis (RA). Reverse transcription–polymerase chain reaction (PCR) was used to screen for gene expression of the IL-17 subtypes (IL-17A–F) in all nodules. Quantitative real-time PCR was used to measure the expression of interferon-␥ (IFN␥), IL-6, IL-23, IL-12, and transforming growth factor ␤ (TGF␤), relative to GAPDH as control, in a subset of 10 nodules. Results. IL-17A gene expression was present in only 1 of 19 nodules, IL-17B in 17 of 19 nodules, IL-17C in 18 of 19 nodules, IL-17D in 16 of 19 nodules, and IL-17E in 3 of 19 nodules. IL-17F was absent in all samples. Cytokines that stimulate IL-17A production (IL-6, IL-23) as well as those that inhibit IL-17A production (IL-12, IFN␥, TGF␤) were present in the majority of nodules. Quantitative real-time PCR showed a similar pattern of gene expression for the individual cytokines between the different nodules. The mean ⴞ SD expression of IL-6 relative to GAPDH was 2.28 ⴞ 2.2 ng, and that of TGF␤ was 2.96 ⴞ 1.14 ng. There was a lower relative expression of IL-23 (0.05 ⴞ 0.05 ng), while Rheumatoid arthritis (RA) is a common autoimmune condition, characterized by inflammation of the joint synovial lining and eventual destruction of the joints. Rheumatoid nodules, which are most commonly located at subcutaneous sites overlying bony prominences, are a characteristic extraarticular feature. Nodules are tissue destructive, are typically found in those patients with more severe disease (1), and are a marker for increased mortality (2). Whether the rheumatoid nodule represents the same type of tissue-destructive inflammatory lesion as that found in the synovium or represents a different type of lesion remains unclear. A long-held view has been that the rheumatoid nodule and other extraarticular manifestations of RA result from a different mechanism, namely immune complex deposition (1). However, studies from our own laboratory have shown marked similarities between the nodule and the synovium. Both lesions contain monocyte/macrophages and T cells (3) and putative dendritic cells (4), and both lesions have a broadly Th1 cytokine profile (5). This has led to our hypothesis that the core inflammatory mechanisms in the rheumatoid nodule and in other systemic lesions are essentially the same as those in the synovial lesion. However, it is apparent that although the nodule Supported by the University of Otago, the Royal Australasian College of Physicians, Lottery Health New Zealand, and the New Zealand Health Research Council. 1 Lisa K. Stamp, MBChB, FRACP, PhD: University of Otago, Christchurch, New Zealand; 2Andrea Easson, Ulrike Lehnigk, PhD, John Highton, MD, FRACP, Paul A. Hessian, PhD: University of Otago, Dunedin, New Zealand. Address correspondence and reprint requests to Lisa K. Stamp, MBChB, FRACP, PhD, Department of Medicine, University of Otago, Christchurch, PO Box 4345, Christchurch 8140, New Zealand. E-mail: email@example.com. Submitted for publication October 3, 2007; accepted in revised form February 20, 2008. 1601 1602 STAMP ET AL Table 1. Characteristics of all 16 patients from whom samples of rheumatoid nodules were obtained and of the 10 patients whose nodules were evaluated by quantitative real-time polymerase chain reaction (PCR)* Characteristic Age, mean (range) years Male/female RF positive Radiographic erosions Disease duration, mean (range) years ESR, mean (range) mm/hour CRP, mean (range) mg/dl Taking DMARDs Taking methotrexate Taking prednisone Total (n ⫽ 16) Quantitative real-time PCR (n ⫽ 10) 62.6 (46–76) 1/15 13/16 (81) 16/16 (100) 13.8 (3–30) 61.3 (46–75) 0/10 7/10 (70) 10/10 (100) 16.7 (5–30) 30 (4–66) 19 (5–38) 15/16 (94) 11/16 (69) 3/16 (19) 28 (4–65) 19.8 (5–38) 9/10 (90) 6/10 (60) 0 * Except where indicated otherwise, values are the no./total no. (%) of patients. Comparable values in the 4 patients from whom synovial tissue samples were obtained were as follows: mean age 68 years (range 61–79), 2 male/2 female, all 4 rheumatoid factor (RF) positive, all 4 with erosions, mean disease duration 14.5 years (range 2–25.8), mean erythrocyte sedimentation rate (ESR) 37.5 mm/hour (range 3–85), 3 taking disease-modifying antirheumatic drugs (DMARDs), 2 taking methotrexate, and 3 taking prednisone. and synovial lesions may share similar basic mechanisms, additional features are present in the synovial lesion that are not found in the nodule. This includes the presence of B lymphocytes and lymphoid follicles that are absent from typical subcutaneous nodules. Understanding the similarities and differences in inflammatory mechanisms in the articular and extraarticular features of RA would provide greater insight into the nature of the antigens recognized, would elucidate why patients with extraarticular disease have increased mortality, and would provide guidance on how we should treat such patients (6). The rheumatoid synovium is characterized by infiltration of monocyte/macrophages, T cells, and dendritic cells, which produce a variety of inflammatory cytokines. T cells present within the RA synovium are typically Th1 (CD4⫹) cells expressing the mature memory cell marker CD45RO (7). Despite the abundance of T cells, there is a paucity of T cell cytokines; for example, although interferon-␥ (IFN␥) is present, the levels of this cytokine are low in comparison with that observed in other Th1-mediated diseases (8). Recently, the T cell cytokine interleukin-17A (IL-17A) has been found in rheumatoid synovium and synovial fluid. IL17A has proinflammatory actions, both directly and through synergy with tumor necrosis factor ␣ (TNF␣) and IL-1␤, and has been implicated in the pathogenesis of bone and joint damage in RA (9). The IL-17 family has 6 members (IL-17A–F), of which IL-17A has been associated with RA. IL-17C, IL-17E (also known as IL-25), and IL-17F have also been detected in RA synovial fluid mononuclear cells (10). Although IL-17A is produced by CD4⫹, CD45RO⫹ memory T cells (11), integration of IL-17A into the existing Th1/Th2 paradigm has been difficult. Recently, a specific subset of T cells that produce IL-17A, known as Th17 cells, has been identified in murine models, and these cells are thought to be important in induction of autoimmune disease (12,13). Development of this unique Th17 cell population is promoted through the actions of IL-6 and transforming growth factor ␤ (TGF␤) on naive murine CD4⫹ T cells, while IL-23 is important in the maintenance and expansion of Th17 cells (14,15). In contrast, IFN␥, IL-2, IL-4, IL-25, and IL-27 inhibit the development of murine Th17 cells (12,16,17). Until very recently, there was a paucity of data on Th17 cells in humans. It is now apparent that there are significant differences between mice and humans with respect to Th17 cell differentiation. While TGF␤ is critical in the mouse, this cytokine is not needed for development of IL-17A–producing cells in humans and, indeed, may inhibit IL-17A production (18,19). IL-6 alone has been reported to be a poor inducer, and IL-1 to be a potent inducer, of IL-17A production by activated CD4⫹ T cells (18,19). However, AcostaRodriguez et al reported that IL-1 in combination with IL-6 promotes production of IL-17A and IFN␥ in the cells (19). IL-23 remains an important inducer of Th17 cells and of IL-17A production (18,20,21). In human T cells ex vivo, IL-12 inhibits IL-17A production (21). IL-2 has also been reported to up-regulate IL-17A expression in human peripheral blood mononuclear cells ex vivo, whereas IFN␥ inhibits IL-17A expression (22). The aim of the present study was to determine whether the genes for IL-17A–F are expressed in rheumatoid nodules in a pattern similar to that documented in the synovial lesions of patients with RA. There was particular interest in the expression of the IL-17A gene and those cytokines important in the regulation of Th17 cell differentiation. PATIENTS AND METHODS Sample collection. Nineteen nodules were obtained from 16 different patients with RA whose diagnosis fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for RA (23). Synovium was obtained from 4 patients with RA. Details on all of the patients are shown in Table 1. Ethics approval was obtained from the University of Otago Ethics Committee. IL-17 SUBSETS IN RHEUMATOID NODULES AND SYNOVIAL MEMBRANE 1603 Table 2. Primers and annealing temperatures for polymerase chain reaction (PCR) screening assays and for quantitative real-time PCR* Assay, gene, primer Screening IL-17A 5⬘ 3⬘ IL-17B 5⬘ 3⬘ IL-17C 5⬘ 3⬘ IL-17D 5⬘ 3⬘ IL-17E 5⬘ 3⬘ IL-17F 5⬘ 3⬘ IL-6 5⬘ 3⬘ IL-12p35 5⬘ 3⬘ IL-23p19 5⬘ 3⬘ TGF␤ 5⬘ 3⬘ ␤-actin 5⬘ 3⬘ Quantitative real-time PCR TGF␤ 5⬘ 3⬘ GAPDH 5⬘ 3⬘ Oligonucleotide sequence Annealing temperature, °C ATGACTCCTGGGAAGACCTCATTG TTAGGCCACATGGTGGACAATCGG 55 CTGGGGCTACAGCATCAACC GTGCAGCCCACAGCGATGGT 45 CCGTTCAGTGTGACCGCCGA GTTGGGAAGAGGCAGCCTGC 50 GCCAAAGAGATAGGGACGCA TTCATCAGTCAGCCATCGGT 45 TGAAGTGCTGTCTGGAGCAG TCCTCAGAATCATCCATGTC 42 GAAGACATCTCCATGAATT ACATACACACATACATTGTG 40 GTACATCCTCGACGGCATCTCAGC GGTTGGGTCAGGGGTGGTTATTGC 55 CCTGGACCACCTCAGTTTGG CTAAGGCACAGGGCCATCAT 50 CTGCTTGCAAAGGATCCACC TTGAAGCGGAGAAGGAGACG 62 GCGTGCTAATGGTGGAAAC ACTCCGGTGACATCAAAAGATAA 47 CGCCCTGGACTTCGAGCAAG GCCAGGGTACATGGTGGTGC 54 CAACAATTCCTGGCGATACCT GCTAAGGCGAAAGCCCTCAAT 60 TGCACCACCAACTGCTTAGC GGCATGGACTGTGGTCATGAG 60 * IL-17 ⫽ interleukin-17; TGF␤ ⫽ transforming growth factor ␤. Analysis of cytokine gene expression. Total RNA was extracted from 50–100 mg of nodule or synovial tissue using Qiagen RNeasy mini kits (Qiagen, Hilden, Germany). RNA (0.5 g or 1 g) was reverse transcribed at 42°C for 50 minutes using Superscript II (Life Technologies, Carlsbad, CA) and oligo(dT)12-18 primers. For screening assays, complementary DNA (cDNA) was amplified by polymerase chain reaction (PCR) under nonsaturating conditions with the respective gene-specific primer pairs and under various annealing temperatures, as detailed in Table 2. Each PCR amplification cycle consisted of a 15-minute denaturation at 95°C at the start of the reaction, followed by 35 cycles of denaturation at 94°C for 30–60 seconds of annealing and then an extension at 72°C for 30–60 seconds, with a final 10-minute extension at 72°C. Amplified products were analyzed by 1.3% agarose gel electro- phoresis with ethidium bromide staining. Controls for the PCR included reactions with known positive cDNA (pooled nodule reference [n ⫽ 5] or tonsil tissue) or in which no cDNA was added. Quantitative real-time PCR was undertaken using TaqMan gene expression assays for IL-6, IL-12, IL-23, and IFN␥ (Applied Biosystems, Foster City, CA) or SYBR Green assays for TGF␤ on 10 nodule samples from different patients. Primers used in the quantitative real-time PCR SYBR Green assays for TGF␤ (and for GAPDH as control) are detailed in Table 2. Statistical analysis. Analysis of the data was undertaken in triplicate samples. Results are expressed as the mean ⫾ SD ng RNA for each gene of interest, relative to the expression of GAPDH RNA. 1604 STAMP ET AL RESULTS Expression of IL-17A–F in rheumatoid nodules. IL-17A gene expression was present in 1 (5%) of 19 nodules (Figure 1A). Irrespective of this single positive nodule, 2 other nodules from the same patient were subsequently found to be negative for IL-17A. The positive nodule was from a tendon sheath, and the histologic characteristics of this nodule were similar to those of the other nodules. Another nodule obtained from a tendon sheath in a different patient was subsequently found to be negative for IL-17A. Despite the absence of IL-17A in nodule samples, IL-17A was readily detected in rheumatoid synovial tissue by PCR screening assays (Figure 1B). IL-17B, IL-17C, and IL-17D were found in the majority of nodule samples (Table 3). Only a minority of samples expressed IL-17E. In contrast, no expression of IL-17F was observed in any of the nodule samples (Table 3). Expression of genes for cytokines that regulate IL-17A production. In an attempt to explain the absence of IL-17A in rheumatoid nodules, cytokines known to be involved in the regulation of Th17 cell differentiation were examined. The results are summarized in Table 3. IL-6 and IL-23, which promote Th17 cell differentiation and IL-17A production, as well as IL-12, TGF␤, and IFN␥, which inhibit Th17 cell differentiation, were present in the nodules. We previously reported the absence of IL-4 (a potent inhibitor of Th17 cell differentiation) and IL-2 (a promoter of Th17) in rheumatoid nodules (5). Quantitative real-time PCR was undertaken to Figure 1. Representative polymerase chain reaction showing expression of interleukin-17A (IL-17A) in A, rheumatoid nodules and B, rheumatoid synovium. The amplicon is the expected size for IL-17A, at 468 bp. Table 3. Expression of IL-17 subtypes A–F and cytokines regulating IL-17A expression in rheumatoid nodules* Cytokine Nodules (n ⫽ 19) IL-17A IL-17B IL-17C IL-17D IL-17E IL-17F IL-6 TGF␤ IL-12p35 IL-23p19 1 (5) 17 (89) 18 (95) 16 (84) 3 (16) 0 (0) 17 (89) 19 (100) 9 (47) 13 (68) * Values are the no. (%) of nodules positive for each cytokine. See Table 2 for definitions. examine the balance between the expression of cytokines thought to promote or inhibit Th17 cell differentiation and the production of IL-17A. Overall, the pattern of cytokine gene expression was similar for the individual cytokines between each nodule sample (Figure 2). With regard to the cytokines that are known to stimulate IL-17A, the mean ⫾ SD gene expression for IL-6 was 2.28 ⫾ 2.2 ng, while that for IL-23 was lower, at 0.05 ⫾ 0.05 ng. With regard to the IL-17A–inhibitory cytokines IFN␥, TGF␤ and IL-12, all 3 were clearly present in the rheumatoid nodules (IFN␥ gene expression 0.67 ⫾ 0.68 ng, IL-12 0.48 ⫾ 0.23 ng, and TGF␤ 2.96 ⫾ 1.14 ng) (Figure 3). Figure 2. Quantitative RNA expression analysis of cytokines involved in Th17 cell differentiation and in interleukin-17 (IL-17) production in 10 different rheumatoid nodules. Each color and symbol represent data from an individual nodule. IFN␥ ⫽ interferon-␥; TGF␤ ⫽ transforming growth factor ␤. IL-17 SUBSETS IN RHEUMATOID NODULES AND SYNOVIAL MEMBRANE Figure 3. Quantitative real-time polymerase chain reaction analysis of cytokines involved in stimulation and inhibition of Th17 cells and of interleukin-17A (IL-17A) production in 10 different rheumatoid nodules. RNA expression for A, positive regulators of IL-17A (IL-6, IL-23) and B, inhibitors of IL-17A (IL-12, interferon-␥ [IFN␥], transforming growth factor ␤ [TGF␤]) is shown relative to that for GAPDH. Each circle represents data from an individual nodule; bars show the mean for each group. DISCUSSION The IL-17 family members IL-17A–F are variably expressed in rheumatoid nodules. The presence of IL17C, IL-17E, and IL-17F, but not that of IL-17B and IL-17D, in synovial fluid and peripheral blood mononuclear cells from patients with RA has previously been reported (10). Whereas the actions of IL-17A in RA have received significant attention, the role of the other IL-17 members has been less well investigated. In humans, the cellular sources of IL-17B and IL-17C have not been identified, and the actions of these 2 cytokines are not well defined. IL-17B has been identified in chondrocytes in the middle and deep zones of normal bovine cartilage (24). More recently, in a murine collagen-induced arthritis (CIA) model, levels of IL-17B and IL-17C were found to be elevated compared with those in controls. IL-17B was exclusively expressed in the inflamed cartilage, whereas IL-17C was expressed by a variety of cells, including CD4⫹ T cells, macrophages, and dendritic cells (25). Furthermore, in experiments using a murine fibroblast cell line, IL-17B and IL-17C induced expression of IL-1␤. In murine peritoneal exudate cells, IL-17B induced expression of IL-1␤, IL-6, IL-23, and TNF␣, and IL-17C induced expression of IL-1␤, IL-23, and TNF␣ (25). Adoptive transfer of IL-17B– and IL-17C–transduced CD4⫹ T cells exacerbated CIA in these mice. The results from these studies suggest that IL-17B and IL-17C have important proinflammatory effects in CIA. Although IL-17C has been reported in human synovial fluid mononuclear cells, IL-17B has not been found (10). Thus, confirmation of the presence of IL17B and IL-17C in rheumatoid nodules, as reported herein, gives further weight to the suggestions derived 1605 from animal models that IL-17 family members have a role in human disease. The nature and significance of their involvement is worthy of further investigation. Among peripheral blood cells, IL-17D is produced by resting CD4⫹ T cells and CD19⫹ B cells. Stimulation of human umbilical vein endothelial cells with IL-17D induces secretion of IL-6 and IL-8, but not IL-1␤, IFN␥, or TNF␣ (26). Although IL-17D has not been found in human synovial fluid or peripheral blood mononuclear cells (10), we were able to detect this cytokine in rheumatoid nodules. These data highlight another difference between the nodule, in which B cells are absent, and the synovium and suggest that particularly the T cells within the 2 lesions have differences. IL-17E (also known as IL-25) is produced by mast cells and induces Th2-type responses (27). Its role in RA has not been defined. The actions of IL-17F are similar to those of IL-17A. To date, IL-17A is the only T cell–derived cytokine found in significant quantities in rheumatoid synovium. IL-17A has a number of proinflammatory actions, both directly and through synergy with IL-1␤ and TNF␣, including stimulation of the production of IL-6, IL-8, IL-1␤, TNF␣, and prostaglandin E2 from monocyte/ macrophages and synoviocytes (28,29). In addition, IL17A may mediate the bone destruction observed in RA through induction of matrix metalloproteinases (30), stimulation of osteoclast precursors (31), inhibition of proteoglycan synthesis (32), and increased expression of RANK and RANKL (33). In animal models, inhibition of murine IL-17A with neutralizing antibodies has been shown to suppress the onset of experimentally induced arthritis, reduce the severity of the arthritis, and reduce synovial RANKL messenger RNA (mRNA) expression and bone erosion (34,35). In patients with RA, the levels of IL-17A mRNA in synovium along with the levels of IL-1␤, TNF␣, and IL-10 mRNA have been reported to be predictive of damage progression (36). Thus, IL-17A links both inflammation and bone destruction in RA. Whether IL-17A has a role in the extraarticular tissue– damaging lesions of RA, such as the rheumatoid nodule, has not been determined previously. We therefore assessed the presence of IL-17A in rheumatoid nodules. Despite the presence of IL-17A mRNA in rheumatoid synovial tissue, 18 of the 19 nodule samples did not express IL-17A mRNA. There are several explanations for only one nodule being found positive for IL-17A. First, this nodule was the only one obtained from a tendon sheath in this study. and although we found no histologic evidence in replicate 1606 tissue pieces, the sample analyzed for IL-17A may have been contaminated with synovial membrane tissue from the sheath. Alternatively, the morphologic features of nodules may differ depending on their location in the tissue. For example, RA-associated pulmonary nodules contain B lymphocytes, but these are not present in subcutaneous nodules (37). This latter possibility seems less likely, given that the only other nodule available for analysis from a tendon sheath was negative for IL-17A. In mice, it has been shown that IL-17A is produced by a distinct subset of CD4⫹ T cells known as Th17 cells. Differentiation of Th17 cells from naive CD4⫹ Th cells is driven by TGF␤ and IL-6. IL-23, which consists of the IL-12p40 subunit and a unique IL-23p19 subunit, has a critical role in maintenance and expansion of Th17 cells (14,15). The importance of IL-23 has been highlighted in a murine model of arthritis in which specific absence of the IL-23 gene conferred complete resistance to the development of CIA in mice. Furthermore, the resistance to CIA in IL-23–deficient mice correlated with the absence of IL-17A–producing T cells (38). In patients with RA, serum and synovial fluid concentrations of IL-23 have been reported to be higher compared with those in patients with osteoarthritis or healthy controls (39). Furthermore, IL-23p19 is upregulated in RA synovial fibroblasts, an effect that is mediated, at least in part, by IL-17A (39). Thus, there appears to be an important positive feedback loop between IL-23 and IL-17A, which may be important in driving synovial inflammation. In contrast, it has been demonstrated that the sensitivity of IFN␥-knockout mice to CIA is associated with increased IL-17A production, and administration of anti–IL-17A antibodies markedly reduces the incidence and severity of CIA (40). The importance of IL-23 in autoimmune diseases is also highlighted by the relationship between the IL-23 receptor (IL-23R) polymorphism and the prevalence of inflammatory bowel disease (41), whereas the data with respect to the role of IL-23R in RA are conflicting (42,43). In an attempt to explain the lack of IL-17A in rheumatoid nodules, we examined the expression of those cytokines suggested, in murine and human studies, to be involved in the regulation of Th17 cell differentiation and in the production of IL-17A. Although IL-6 was clearly present, there was only minimal expression of IL-23. In contrast, those cytokines suggested to inhibit IL-17A production, namely IFN␥, TGF␤, and IL-12, were all present. Thus, while T cells are present in STAMP ET AL rheumatoid nodules, our data suggest that these are not IL-17A–producing Th17 cells. Although the story of Th17 cell differentiation and IL-17A production in human T cells is far from complete, given the available data in human systems, the relative lack of IL-23 may explain, at least in part, the absence of IL-17A in rheumatoid nodules. However, other cytokines, as yet unidentified, may be important in regulating Th17 cell differentiation and IL-17A production. This is highlighted by recent evidence suggesting that IL-23R–negative T cells are still capable of producing IL-17A (19). The rheumatoid nodule and synovial lesion share a number of similarities. Monocyte/macrophages, T cells, and putative dendritic cells are found in both lesions (4). Expression of adhesion molecules is similar between the nodule and the synovium, with the exception of E-selectin, which has higher expression in the nodule (44). The cytokine profile of the 2 lesions is also similar and resembles that of a Th1 granuloma (5). Nevertheless, there are also significant differences between the 2 lesions. B cells and lymphoid follicles, which are present in the synovium, are typically absent from subcutaneous nodules. In comparison, the necrosis found in the nodule is usually absent in the synovium. Further evidence of differences between the 2 lesions comes from the response to therapy. For example, whereas infliximab and methotrexate can effectively suppress synovial inflammation, infliximab has no effect on subcutaneous nodules (45), and methotrexate can exacerbate nodules. The relative absence of IL-17A in the nodule as compared with its presence in the synovium highlights another important difference between the 2 lesions, particularly with regard to the T cell populations present. Recirculation of T cells between articular and extraarticular locations in RA has been hypothesized as a possible explanation for this difference (46). This is based, at least in part, on the similarities observed between the T cells in both lesions, including similar patterns of adhesion molecule expression and T cell receptor rearrangements (47). However, the absence of IL-17A in rheumatoid nodules suggests that there may be important differences between the T cell populations present within these 2 lesions. Recent evidence suggests that there is a distinct window early in the inflammatory phase during which the IL-17A response can be modulated (48). IL-17A production by murine T cells in vitro is suppressed by IL-27 when CD4⫹ T cells are in the early stages of T cell activation, but not when they are fully activated (49). IL-17 SUBSETS IN RHEUMATOID NODULES AND SYNOVIAL MEMBRANE Similarly, in murine models, suppression of Th17 cell development by IFN␥ and IL-4 appears to be limited to an early stage of Th17 cell differentiation, with mature Th17 cells being resistant to inhibition by IFN␥ and IL-4 (12). In a murine model of experimental autoimmune encephalomyelitis, IL-23 was reported to be critical in the induction phase, but not the effector phase, of the disease. Of note, fully differentiated T cells that induced encephalomyelitis could continue to produce IL-17A in the absence of IL-23 (50). Given these data, it is possible that the local environment during the initiation phase differs between synovial lesions and nodule lesions, thereby allowing expression of Th17 cells and IL-17A production in the synovium but not the nodule. In summary, IL-17 subtypes A–F are varyingly expressed in rheumatoid nodules. IL-17A is absent from the majority of nodules, despite its presence in rheumatoid synovium. Low levels of IL-23 and high levels of TGF␤ in the nodule may be the explanation for the absence of IL-17A. While IL-17A may have a role in the inflammatory process in the synovium, the nodule appears to be IL-17A independent. 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