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Proc Natl Acad Sci U S A. Apr 14, 2009; 106(15): 6232–6237.
Published online Mar 26, 2009. doi:  10.1073/pnas.0808144106
PMCID: PMC2669354
Immunology

In vivo activated monocytes from the site of inflammation in humans specifically promote Th17 responses

Abstract

Th17 cells are a recently defined subset of proinflammatory T cells that contribute to pathogen clearance and tissue inflammation by means of the production of their signature cytokine IL-17A (henceforth termed IL-17). Although the in vitro requirements for human Th17 development are reasonably well established, it is less clear what their in vivo requirements are. Here, we show that the production of IL-17 by human Th17 cells critically depends on both the activation status and the anatomical location of accessory cells. In vivo activated CD14+ monocytes were derived from the inflamed joints of patients with active rheumatoid arthritis (RA). These cells were found to spontaneously and specifically promote Th17, but not Th1 or Th2 responses, compared with resting CD14+ monocytes from the blood. Surprisingly, unlike Th17 stimulation by monocytes that were in vitro activated with lipopolysaccharide, intracellular IL-17 expression was induced by in vivo activated monocytes in a TNF-α- and IL-1β-independent fashion. No role for IL-6 or IL-23 production by either in vitro or in vivo activated monocytes was found. Instead, in vivo activated monocytes promoted Th17 responses in a cell-contact dependent manner. We propose that, in humans, newly recruited memory CD4+ T cells can be induced to produce IL-17 in nonlymphoid inflamed tissue after cell–cell interactions with activated monocytes. Our data also suggest that different pathways may be utilized for the generation of Th17 responses in situ depending on the site or route of accessory cell activation.

Keywords: antigen-presenting cell, IL-17, myeloid cell, rheumatoid arthritis, T cells

Th17 cells are a recently defined proinflammatory CD4+ T helper cell subset that is characterized by the production of IL-17. In mice, in vitro Th17 differentiation from naive CD4+ T cells requires CD3 and CD28-mediated stimulation in the presence of IL-6 or IL-21 and TGF-β. Th17 development is further increased when Th1 and Th2 skewing cytokines are blocked, and by the presence of IL-23, which promotes expansion and/or maintenance of the population. These Th17 differentiation conditions result in up-regulation of a specific transcription factor, RORγt, and the production of the Th17-related cytokines IL-17A, IL-17F, IL-21, and IL-22 (1, 2). In humans, stimulation of naive CD4+ T cells through CD3 and CD28 in the presence of TGF-β and IL-6 alone appears insufficient to induce Th17 differentiation (38), due to the bimodal action of TGF-β. TGF-β is essential for initial up-regulation of RORγt expression in human naive T cells, but, simultaneously, inhibits IL-17 expression, and additional proinflammatory cytokines such as IL-1β, IL-23, or IL-21 are required to overcome this inhibitory effect (9, 10).

Although it is clear that a Th17 population may be generated in vitro when human naive CD4+ T cells are cultured with anti-CD3/CD28-coated microbeads and exogenously added recombinant cytokines, it is less clear what the in vivo requirements for Th17 development are. Th17 differentiation is likely to occur when naive CD4+ T cells encounter antigen-presenting dendritic cells (DC) that produce the appropriate polarizing cytokines in the lymph nodes. Interestingly, there is accumulating evidence both in mice and humans that Th17 cells can be generated not only from naive CD4+ T cells, but also from effector memory CD4+ T cells (4, 5, 11), and even from highly differentiated CD4+CD25+ regulatory T cells (Tregs) (12, 13). The induction of Th17 responses from these differentiated CD4+ T cells seems to occur particularly in the presence of activated antigen-presenting cells (APCs) such as monocytes (4, 13) or DC (5). These findings suggest that Th17 cells may not only be generated in lymph nodes, but may also be induced in situ when memory or regulatory T cells enter a site of inflammation and encounter activated monocytes or DC. This plasticity may be essential to generate an early Th17 response in the tissue during infection, but could also result in aberrant (auto) immune activity. Indeed, Th17 cells have been linked to a number of chronic inflammatory and autoimmune disorders, including rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, and inflammatory bowel disease (1416).

To test the hypothesis that Th17 responses may be generated at nonlymphoid sites of inflammation in humans, we devised an experimental system, in which bulk CD4+ T cells from the blood of patients with active RA were cocultured with either resting CD14+ monocytes from peripheral blood (PB), or in vivo activated CD14+ monocytes from synovial fluid (SF) or synovial membrane (SM) from an inflamed joint. We found that in vivo activated monocytes spontaneously and specifically promoted Th17 responses in blood-derived CD4+ T cells. Intriguingly, in contrast to monocytes that were in vitro activated with LPS, in vivo activated monocytes induced IL-17 expression in an IL-1β/TNF-α-independent manner. Instead, cell-contact between monocytes and T cells was required, indicating that different pathways may be used for the generation of Th17 responses, which may in part depend on the site or route of monocyte activation.

Results

In Vitro Activated Monocytes Promote Th17 Responses in an IL-1β and TNF-α-Dependent Manner.

We recently demonstrated that the percentage of Th17 cells and their IL-17 production can be significantly increased when memory, but not naïve, CD4+ T cells are stimulated in the presence of LPS-activated monocytes (4). To investigate the underlying mechanism, purified CD4+ T cells and autologous CD14+ monocytes from healthy controls (HC) were cocultured with anti-CD3 mAb in the absence or presence of LPS, and neutralizing antibodies against TNF-α, IL-1β, IL-6, or IL-23. These proinflammatory cytokines are all produced by activated monocytes, and are proposed to have a role in Th17 induction in mice and humans. After 3 days, the percentage of IL-17positive IFN-γ negative T cells (henceforth called IL-17+ T cells) and the production of IL-17 were measured by intracellular cytokine (ICC) staining and ELISA. Effectiveness of the neutralizing mAbs was predetermined by appropriate dose titration and isotype control experiments (Fig. S1). In agreement with our previous data, addition of LPS to monocytes resulted in a significant increase in IL-17+ T cells and IL-17 production (Fig. 1), associated with an increase in TNF-α, IL-1β, and IL-6, but not IL-23 (Fig. S1). The increase in IL-17+ T cells was due to a direct effect of LPS on monocyte activation, and not by means of the effect of LPS on T cells, as shown by monocyte/LPS preculture experiments (Fig. S2). Blocking either IL-1β or TNF-α at the start of the culture did not prevent the increase in the percentage of IL-17+ T cells, but did significantly reduce IL-17 production by 45 ± 9 and 35 ± 9%, respectively (P < 0.05; Fig. 1). Blocking both IL-1β and TNF-α led to a synergistic effect in reducing both the percentage of IL-17+ T cells (50 ± 9%, n = 6) and IL-17 secretion (78 ± 7%, n = 6) relative to control conditions (Fig. 1 A–C). Blockade of IL-6 and IL-23 had no effect on either the percentage of IL-17+ T cells or IL-17 production. These data suggest that to inhibit induction of Th17 cells by LPS-activated monocytes, it is necessary to block both IL-1β and TNF-α, whereas blocking either cytokine separately is sufficient to impair IL-17 secretion. In all conditions, TCR ligation was required for both IL-17 expression and secretion, because in the absence of anti-CD3 mAb, IL-17 expression and secretion were significantly reduced by 68 ± 11 and 98 ± 1%, respectively (Fig. 1D). Based on these data, we assessed whether the role of the activated monocyte could be replaced by culturing purified CD4+ T cells with high levels of TCR ligation and costimulation using anti-CD3/CD28 microbeads, together with exogenously added IL-1β and TNF-α. The presence of anti-CD3 mAb alone did not trigger an IL-17 response, whereas the addition of anti-CD3/CD28 microbeads resulted in a similar level of IL-17 production, as seen with unstimulated blood monocytes (Fig. 1E). The addition of hrIL-1β and TNF-α without costimulation did not trigger IL-17 production, whereas the addition of these cytokines, along with costimulation and TCR ligation, triggered a profound Th17 response similar to that seen with LPS-activated monocytes. These data indicate that TCR ligation in the context of costimulation, IL-1β and TNF-α is sufficient to mount a sizeable Th17 response in human CD4+ T cells. To further investigate the role of costimulation or adhesion, we added blocking Abs to CD80/CD86, CD54, or CD40 to cocultures of CD4+ T cells, monocytes, anti-CD3 mAb, and LPS. However, blocking these separate costimulatory/adhesion pathways did not consistently reduce IL-17 expression or production (Fig. S3), suggesting that a certain level of redundancy exists in the cell membrane-derived signals required for Th17 responses.

Fig. 1.
LPS-activated monocytes drive Th17 responses in a TNF-α and IL-1β, but not IL-6 or IL-23-dependent manner. (A–C) CD4+ T cells from HC were cocultured with monocytes and anti-CD3 mAb in the absence (Control, white bars) or presence ...

In Vitro Activated Monocytes from RA Patients also Promote Th17 Responses in an IL-1β and TNF-α-Dependent Manner.

Because Th17 cells and IL-17A have been proposed to contribute to RA pathogenesis (15), we examined the induction of monocyte-driven Th17 responses in RA patients (n = 9) vs. HC (n = 16). The magnitude of the Th17 response observed in RA and HC cocultures was similar, indicating that CD4+ T cells from RA patients are not intrinsically different in terms of their ability to adopt a Th17 profile in the presence of resting or LPS-stimulated monocytes (Fig. 2A). Fig. 2B shows that in RA patients, like HC, the increase in IL-17+ T cells and IL-17 production caused by the addition of in vitro LPS-activated monocytes could be blocked by adding neutralizing Abs to IL-1β and TNF-α. To better characterize these IL-17+ cells, we investigated the coexpression of Th17-associated cytokines. IL-17A+ cells from both HC and RA patients predominantly coexpressed TNF-α (HC 53 ± 14 and RA 48 ± 12%), whereas coexpression of IFN-γ (HC 23 ± 5 and RA 19 ± 4%), IL-21 (HC 19 ± 5 and RA 20 ± 8%), and IL-22 (HC 9 ± 3 and 13 ± 4%) was also observed, albeit at lower levels. Addition of LPS to these cultures led to a global increase in Th17-associated cytokine expression, but did not change the proportion of IL-17+ cells coexpressing TNF-α, IFN-γ, IL-21, or IL-22 (Fig. 2 C and D).

Fig. 2.
LPS-activated monocytes from RA patients drive Th17 responses in a TNF-α and IL-1β-dependent fashion leading to an up-regulation of IL-17 and associated cytokines. (A) CD4+ T cells and autologous monocytes from PB of either HC (n = 16) ...

In Vivo Activated Monocytes from the Site of Inflammation Specifically Promote Th17 Responses.

Both the above data and previous work (35) indicate that the activation status of the APC is a critical determining factor in the induction of human Th17 cells in vitro. To place these findings in a pathophysiological context, we investigated the Th17-inducing capacity of in vivo activated monocytes by isolating CD14+ monocytes from SF of patients with active RA (for patient data, see Fig. S4). Fig. 3A shows that CD14+ monocytes are present at significantly increased numbers in the RA joints, compared with PB. Also, these SF-derived monocytes were highly activated with significantly increased expression of CD40, CD86, HLA-DR, and CD54, compared with their PB counterparts (n = 8; Fig. 3B). Then, we cocultured RA PB-derived CD4+ T cells with autologous RA PB or SF-derived monocytes in the presence of anti-CD3 mAb. Strikingly, a significantly higher percentage of IL-17+ T cells was detected in the presence of in vivo activated SF-derived monocytes, compared with resting PB-derived monocytes (Fig. 3C). This increase in the percentage of IL-17+ T cells was observed in all patients tested (n = 9), and was mirrored by a significant increase in IL-17 levels in the supernatants of these cultures. Interestingly, the increase in IL-17 was not due to a general increase in T cell activation, because neither the Th1 response, characterized by IFN-γ, nor the Th2 response, characterized by IL-4 were significantly altered (Fig. 3D). These data indicate that the increase in IL-17+ T cells and the corresponding increase in IL-17 production is a specific response induced by in vivo activated SF-derived monocytes.

Fig. 3.
In vivo activated monocytes drive Th17, but not Th1 or Th2 responses. (A and B) Percentage (A) and phenotype (B) of CD14+ cells in the mononuclear cell fraction of paired samples of PB (white bars) and SF (black bars) from patients with active RA (n = ...

In the above experiments, we used SF-derived CD14+ monocytes as a model system for in vivo activated monocytes. However, in RA, the main site of inflammation is the SM tissue. Using CD14+ monocytes isolated from the SM from a patient with RA undergoing joint surgery, we found that also SM-derived CD14+ monocytes induced an increased percentage of Th17 cells and IL-17 secretion when cocultured with autologous PB CD4+ T cells, compared with PB as well as SF-derived monocytes (Fig. 3E). Further evidence for the in vivo relevance of the Th17-inducing capacity of SM-derived monocytes was provided by the demonstration that CD4+IL-17+ cells were indeed present in the SM of this patient, as shown by flow cytometry of digested tissue (Fig. 3F) and confocal imaging (Fig. 3G).

SF-Derived Monocytes Drive IL-17 Expression in a TNF-α and IL-1β-Independent Manner.

We noted that the increase in Th17 cells and IL-17 production triggered by coculture with RA SF-derived monocytes was very similar to the increase seen when PB-derived monocytes from the same RA patients were stimulated with LPS (Fig. 4A). SF monocytes also induced coexpression of the Th17-associated cytokines IL-21 (n = 2), TNF-α (n = 5), and IFN-γ (n = 9). These data suggest that in vivo and in vitro activated monocytes share pathways that lead to enhancement of Th17 responses in humans. To investigate whether the Th17 response triggered by in vivo activated SF monocytes was mediated via IL-1β and TNF-α, we cultured CD4+ T cells from RA PB in the presence of anti-CD3 mAb and autologous SF-derived monocytes, in the absence or presence of anti-IL-1β and anti-TNF-α neutralizing antibodies. In striking contrast to LPS-triggered RA PB-derived monocytes (Fig. 2B), IL-1β and TNF-α blockade did not lead to a decrease in IL-17-expressing CD4+ T cells when in vivo activated SF monocytes were used (Fig. 4B). The addition of these blocking antibodies had some effect on IL-17 secretion, but this effect was not consistent in all patients (overall decrease 32 ± 21%). ELISA results on the supernatants taken from these experiments confirmed that the addition of anti-IL-1β and anti-TNF-α mAbs lead to a complete blockade of these cytokines, indicating that the lack of effect was not due to inefficient neutralizing capacity. Also, we found that SF-derived monocytes did not consistently express enhanced levels of TNF-α or IL-1β relative to PB monocytes either at the protein or mRNA level, irrespective of whether they were cultured with T cells or LPS (Fig. S5). This result underscores our observation that SF-derived monocytes promote IL-17 expression in a TNF-α and IL-1β-independent manner. We also assessed the presence of IL-23 and IL-6. No IL-23 was found above the detection level (15 pg/mL) in any of the supernatants containing either PB or SF monocytes, and we did not find consistently increased levels of IL-6 by SF-derived monocytes relative to PB-derived monocytes (Fig. S5). Also, IL-6 blockade either in combination with anti-TNF-α or anti-IL-1β mAb did not lead to significant changes in the percentage of IL-17+ T cells or IL-17 production (n = 3).

Fig. 4.
In vivo activated monocytes drive Th17 responses via a cell-contact dependent mechanisms. (A) The percentage of IL-17+CD4+ T cells (ICC, Left) and IL-17 production (ELISA, Right) induced by coculture of RA PB CD4+ T cells with autologous RA PB or SF-derived ...

To assess whether the increased Th17 response seen in the presence of SF-derived monocytes was mediated via a soluble factor other than TNF-α, IL-1β, Il-6, or IL-23, we conducted transwell experiments. CD14+ monocytes from RA PB, SF, or SM were added to a semipermeable membrane-containing insert, which was placed above wells containing CD4+ T cells and autologous monocytes from HC, in the presence of anti-CD3 mAb. Fig. 4C shows that the presence of in vivo activated monocytes from RA SF or SM in the transwell did not lead to an increase in IL-17+ T cells or IL-17 production in the bottom compartments in any of the donors. In contrast, when these synovial monocytes were added to autologous RA PB CD4+ T cells in coculture, increased Th17/IL-17 levels were observed, as compared with PB-derived monocytes (Fig. 3E). Together, these data indicate that Th17 induction in the presence of in vivo activated monocytes appears to rely on cell-contact, rather than soluble factors. Given the high expression of CD86, CD40, and CD54 on SF monocytes, we blocked these pathways. However, this approach did not lead to a decrease in the percentage of Th17 cells (n = 2), indicating redundancy in the system or the involvement of additional factors.

Discussion

Th17 cells are thought to contribute to inflammatory diseases by up-regulating a range of proinflammatory mediators, including TNF-α, IL-1, IL-6, IL-8, and matrix metalloproteinases (14, 15). IL-17 may also contribute directly to joint damage, because it was shown to act synergistically with TNF-α and/or IL-1β to induce cartilage destruction in vitro and in experimental arthritis in vivo (1719). In RA, SM IL-17 expression in synergy with TNF-α is predictive of joint damage progression (20), and, in juvenile idiopathic arthritis (JIA), the number of IL-17-expressing T cells is higher in patients with the more severe form of disease (21). Despite these data, very little is known about what drives human Th17 responses in vivo, particularly at sites of inflammation. In this study, the use of paired PB/SF samples from patients with active RA provided a unique opportunity to test our hypothesis that in vivo activated accessory cells, such as monocytes, can drive Th17 responses in humans. We show that CD14+ monocytes, taken from the site of inflammation, spontaneously and specifically induce a Th17 response, but not a Th1 or Th2 response in blood-derived CD4+ T cells. These data suggest that CD4+ T cells migrating into inflamed joints could be driven by direct interactions with activated monocytes toward a Th17 phenotype, and, thus, participate in the inflammatory response. Although it has previously been shown that Th17 cells and IL-17 are present at sites of human autoimmune inflammation, including RA (2224), MS (25), and psoriasis (24, 26), to our knowledge, it has not been demonstrated that APC taken from a site of inflammation can promote Th17 responses. As such, our data provide an insight into the formation of these highly pathogenic cells in vivo, and suggest that targeting of inflammatory monocytes could lead not only to a decrease in the production of proinflammatory cytokines, but also in a reduction in Th17 cells.

Recent literature indicates that, in addition to multiple cytokine signals, cell-derived factors may be required for the generation or expansion of Th17 cells (2, 27, 28). We show that in vitro activated monocytes from both HC and RA patients induce Th17 responses in an IL-1β/TNF-α-dependent fashion. Interestingly, blockade of either cytokine in isolation decreased IL-17 secretion, but not the percentage of IL-17+ T cells, suggesting that IL-17 secretion was impaired on a per cell basis. However, when both TNF-α and IL-1β were neutralized, also the percentage of Th17 cells was significantly reduced. These data suggest that IL-17 expression and secretion are controlled at different levels, with IL-17 secretion more easily perturbed than the percentage of IL-17-producing cells. Surprisingly, in the context of in vivo activated monocytes, the combined blockade of TNF-α and IL-1β did not affect the generation of Th17 cells, and only a moderate suppression of IL-17 secretion was observed. The continued generation of Th17 cells, despite TNF-α/IL-1β blockade, may explain why disease relapse is common when these treatments are withdrawn. In contrast to the in vitro activated PB monocytes, in vivo activated monocytes appear to rely on cell-contact with CD4+ T cells to promote Th17 responses, suggesting a more important role for cell-to-cell signals like costimulation or adhesion. Indeed, using an APC-free culture system, we found that the addition of a costimulatory signal, such as anti-CD28 mAb, was essential to generate a Th17 response. However, blockade of a single costimulatory or adhesion pathway (CD80/CD86, CD54, or CD40) in cocultures with either in vitro or in vivo activated monocytes did not decrease the percentage of Th17 cells. This result indicates a certain level of redundancy in the system or the involvement of additional factors that remain to be identified.

Together, our data indicate that Th17 responses may be induced via different pathways, depending on the route or site of activation of accessory cells. Accessory cells such as monocytes can be activated in situ by several mechanisms, including signaling through Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors by pathogens or danger signals, via cytokine-activation, or by T cell-mediated CD40-ligation (2932). The specific signaling cascades induced by each of these stimuli are likely to affect the way in which monocytes and T cells interact, and, thus, the ensuing T cell response. Also, in both mice and humans, different monocyte subsets with distinct phenotypic and functional characteristics exist, and these different subsets may further influence T cell outcome (30, 33, 34). In our experiments, the difference in the underlying mechanism of Th17 induction by LPS-stimulated vs. joint-derived monocytes may reflect differences between in vitro and in vivo activating conditions, or between signaling pathways linked to infectious vs. autoimmune processes. A better understanding of the molecular basis for this phenomenon is clearly important when considering treatment strategies in inflammatory disease. Importantly, although it is becoming increasingly clear that monocytes have a critical role in orchestrating T cell responses in vivo by affecting the polarization and expansion of both naive and memory T cell responses (30), we do not exclude that other APC such as DC may contribute in a similar fashion to the generation of Th17 responses at sites of inflammation.

In conclusion, our data show that in vitro activated monocytes induce Th17 responses in cocultured CD4+ T cells in an IL-1β/TNF-α-dependent fashion. We demonstrate that in vivo activated monocytes from the site of inflammation in RA also induce increased Th17 responses in a distinct manner, which is cell-contact dependent. Our data highlight the importance of monocyte/T cell interactions in the shaping of inflammatory T cell responses, and suggest that synovial monocytes may be a critical target in RA to control both excess cytokine production and stimulation of Th17 cells.

Materials and Methods

Cell Isolation from HC and RA Patients.

Ethical approval for the use of PB from healthy donors and PB, SF, and SM from RA patients was obtained from the Bromley and the Bexley and Greenwich Research Ethics Committees. RA patient samples were obtained from the Rheumatology outpatient clinic at Guy's Hospital with patients' informed consent. Disease activity was assessed by using the DAS28 on the day of sample collection. PB and SF mononuclear cells were isolated by density gradient centrifugation (Lymphocyte separation media; PAA) and used freshly. SF samples were obtained from inflamed knees. SM was removed from the elbow during a synovectomy procedure. The tissue was homogenized and then digested by using 10 μg/mL collagenase (Sigma) at 37 °C for 1 h, followed by cell isolation using density gradient centrifugation. CD4+ T cells (>99% purity as confirmed by flow cytometry) from PB and CD14+ monocytes (>90% purity) from PB, SF, and SM were isolated by magnetic cell separation by using a negative depletion kit for CD4+ T cells or anti-CD14 microbeads (Miltenyi Biotec), respectively.

Cell Culture.

Bulk CD4+ T cells (0.5 × 106/mL) were cultured in 24 well plates in the absence or presence of monocytes (0.5 × 106/mL) in RPMI-1640 medium, supplemented with 1% penicillin/streptomycin, 1% glutamine, and 10% heat-inactivated FCS (Cambrex). Cultures were stimulated with 100 ng/mL anti-CD3 (OKT3, Janssen-Cilag), and where indicated 100 ng/mL LPS (Escherichia Coli strain O111:B4; Sigma). Neutralizing/blocking mAbs to IL-1β (clone 8516, mIgG1; RnD Systems), IL-6 (clone 1936, mIgG2b; RnD Systems), TNF-α (Infliximab, chimeric h/mIgG1; Schering), CD80 (clone 2D10, mIgG1; BioLegend), CD86 (clone IT2.2, mIgG2b; BioLegend), CD40 (clone 82102, mIgG2b; RnD Systems), CD54 (clone HCD54, mIgG1; BioLegend), or isotype controls (mIgG1, clone 11711 and mIgG2b, clone 20116; RnD Systems) were used at 10 μg/mL, and to IL-23 (polyclonal goat IgG; RnD Systems) at 1 μg/mL. In the APC free set up, cells were cultured at 1 × 106/mL and stimulated with anti-CD3/CD28 microbeads (0.5 beads/cell; Invitrogen); hrTNF-α and IL-1β (RnD Systems) were added at 10 ng/mL.

Flow Cytometry.

Cells were stained for 30 min at 4 °C with anti-CD3-FITC, anti-CD4-PE-Cy5, anti-CD8-PE-Cy5, anti-CD14-PE-Cy5, anti-CD19-PE, anti-HLA-DR-perCPcy5.5 (Beckman-Coulter), anti-CD80-FITC, anti-CD86-PE (Immunotech), anti-CD40-PE (AbD Serotec), or anti-CD54-FITC (BD). For ICC, cells were restimulated at day 3 for 4–6 h with PMA (50 ng/mL; Sigma-Aldrich) and ionomycin (750 ng/mL; Sigma-Aldrich) with GolgiStop (according to manufacturer's instructions; Becton Dickinson) added during the last 3 h. Cells were stained for cell surface markers, fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin, then labeled with isotype control Abs (IgG1-FITC, IgG1-PE, Beckman-Coulter), anti-IFN-γ-FITC, anti-IL-17-PE, anti-IL-4-APC, anti-IL-21-APC (eBiosciences), anti-TNF-α-APC (AbD Serotec) or anti-IL-22-PE (RnD Systems). Cells were fixed in 2% paraformaldehyde, acquired by a FACsCalibur, and analyzed by using Cellquest software (Becton Dickinson) or FlowJo software (TreeStar). When stating IL-17+ or IFN-γ+ T cells, we refer to single positive cells.

ELISA.

Supernatants were collected after stimulation with PMA/ionomycin, and stored at −80 °C until detection of IL-17, IFN-γ, IL-1β, TNF-α, IL-6 (Invitrogen), or IL-23 (eBiosciences) by ELISA, according to manufacturer's instructions. Detection limit was 15 pg/mL.

Real-Time PCR.

RNA was extracted with the RNeasy mini kit (Qiagen) and cDNA transcribed by using a Reverse Transcription kit (PrimerDesign), according to manufacturers' instructions. The real-time PCR reacting was carried out by using the Precision qPCR Mastermix and specific primers for human IL-1β, TNF-α and IL-6 (PrimerDesign). Reactions were normalized to cyclophilin in a multiplex reaction performed on an ABI Prism 7900HT sequence detection system (Applied Biosystems).

Confocal Microscopy.

SM sections were embedded in OCT and frozen at −80 °C until use; 7-μm sections were cut and fixed with acetone for 10 min at −20 °C. Sections were then stained for CD4-Biotin overnight at +4 °C, followed by 1 h at room temperature with strep-Cy3 (both a kind gift from Arne Akbar, University College London, United Kingdom). DAPI and IL-17-Alexa 488 (eBiosciences) were added at +4 °C overnight. Confocal imaging was performed on a Leica SP2 (Leica Microsystems).

Statistical Analysis.

Significant differences were calculated with GraphPad 4.03 using the appropriate statistical tests.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Mr. Mark Evans for his assistance and advice in the confocal imaging. This work was supported by the Department of Health via the National Institute for Health Research Comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. H.G.E. is supported by a PhD studentship from the Medical Research Council.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808144106/DCSupplemental.

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