Published online before print June 27, 2008
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,1,2
* Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom;
UCB, Granta Park, Great Abington, Cambridge, United Kingdom; and
UCB, Slough, United Kingdom
3 Correspondence: Inflammatory Cell Biology Lab, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, UK. E-mail: S.G.Ward{at}bath.ac.uk
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: T cells • chemokines • cell trafficking • inflammation • signal transduction
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Th17 cells were first identified and detected in murine (m)EAE models, where expansion of encephalitogenic T cells in vitro in the presence of IL-23 or IL-12, followed by adoptive transfer to naïve, recipient mice demonstrated that IL-23 alone rendered the cells pathogenic [1
]. These pathogenic cells had the phenotype of IL-17+ IFN-
–, as demonstrated by intracellular cytokine detection. Subsequently, it was demonstrated that mTh17 cells develop from naïve T cells in the presence of TGF-β and IL-6 and proinflammatory cytokines such as IL-1β and TNF-
[6
], and IL-23 acts as a maintenance factor for the memory Th17 pool [7
]. In addition, it has been demonstrated that IL-21 is capable of autocrine regulation of mTh17 cells, and the Th1 and Th2 cytokines IFN- and IL-4, respectively, inhibit differentiation of Th17 cells [8
9
10
11
]. The identification of the retinoid-related orphan receptor t (RORt) and more recently, ROR, as Th17-specific transcription factors, has established the unique and distinct Th17 cell population [12
, 13
].
Recent publications have highlighted different requirements for the generation and maintenance of human (h) Th17 cells. Despite the critical function of TGF-β in the differentiation of mTh17 cells, this cytokine appears dispensable for the generation of hTh17 cells [14
, 15
]. In addition, although IL-1β is an effective inducer of IL-17 expression in activated hCD4+ T cells, only transient up-regulation of RORt occurs following IL-1β stimulation [15
]. IL-6 alone is a poor inducer of hTh17 cell differentiation but in combination with IL-1β, promotes sustained RORt expression [15
]. Finally, although IL-23 plays a role in maintenance of the mTh17 phenotype by acting on memory T cells, it appears to play a role in the differentiation of naïve human T cells toward a Th17 phenotype [16
, 17
].
The ability of specific T cell subsets to migrate toward chemoattractants is required for basic immune surveillance and adaptive immune responses, and expression profiles of chemokine receptors have been instrumental in the characterization of subsets of human memory T cells with distinct migratory capacity and effector functions. Hence, CCR7 expression discriminates between lymph node homing central memory T cells and tissue homing effector memory T cells [18
]. In addition, CXCR3, CXCR6, and CCR5 are preferentially expressed on Th1 cells [19
], and CCR3, CCR4, CCR8, and the PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes, are expressed on Th2 cells [20
, 21
]. Much evidence supports an evolutionarily conserved role for PI-3K and its D-3' phosphoinositide lipid products (e.g., phosphatidylinositol 3,4,5-trisphosphate) in cell motility and chemoattractant-induced migration [22
, 23
]. Activation of PI-3K (predominantly the Gβ-dependent p110 PI-3K isoform) is a biochemical response to most T cell-expressed chemokine receptors [24
], yet paradoxically, it is now clear that activation of PI-3K by chemokines can be a dispensable signal for directional T cell migration and is specifically determined by the chemoattractant, cell type, and differentiation state [25
, 26
].
Although the chemokine receptor expression profile of Th1 and Th2 cells has been explored, the Th17 chemokine receptor expression profile is less well understood. To date, identification of Th17 cells mainly relies on intracellular IL-17 detection using flow cytometry. Many researchers report high proportions of Th17 cells generated within specific culture conditions, however many non-IL-17-producing cells also exist within these systems. Therefore, the identification of Th17-specific surface marker(s) would enable phenotypic characterization and isolation of Th17 cells and significantly aid the understanding of Th17 biology. Studies of hTh17 cells have shown the existence of CCR2+ CCR5– [27 ] and CCR6+ CCR4+ [28 ] Th17 subsets, and more recently, the expression of CCR6 on IL-17-producing CD4+ and CD8+ T cells suggests a specific role for CCR6 in hTh17 cell biology [29 ]. However, although chemokine receptor expression has been characterized extensively in hTh17 cells, the situation in mTh17 cells is less clear, and the consequences for the migratory capacity of these cells has yet to be explored. In this study, we report that increased proportions of mCD4+ T cells cultured under Th17-polarizing conditions expressed surface CCR2, CCR6, and CCR9 compared with nonpolarized cells. Although agonists for CCR2, CCR6, and CCR9 stimulated biochemical signaling and migratory events in cells cultured under Th17-polarizing conditions, only the agonists for CCR2 and CCR6 elicited migratory responses of IL-17+ cells, which were shown to be PI-3K/Akt-dependent. These results therefore provide insight into the biochemical mechanisms by which Th17-polarized cells may be recruited during autoimmune inflammation and provide a rationale for the use of PI-3K inhibitors in treating these diseases. We reveal differences in phenotypic markers of mTh17 cells, most notably, that CCR6 expression on mIL-17+ cells does not fully define the mIL-17+ population.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
All chemicals and tissue-culture reagents were purchased from Sigma-Aldrich (Poole, UK) unless otherwise specified. The ECL kit was obtained from Amersham International (Little Chalfont, UK). Akt inhibitor (Akti)-1/2 was purchased from Calbiochem (Nottingham, UK). Recombinant (r)hTGF-β1 was purchased from Sigma-Aldrich. rmIL-1β, IL-2, IL-6, and TNF- were purchased from Peprotech (London, UK). rmIL-23 and anti-IFN- antibody (clone 37895) were purchased from R&D Systems (Abingdon, UK). Anti-IL-4 antibody (clone 11B11) was produced at UCB (Slough, UK). Cell culture medium comprised RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 12.5 mM HEPES, and 50 µM 2-ME.
Anti-CD4-Tri-Color (clone RM4-5), anti-IFN--FITC, and anti-IFN--AF610 (clone XMG1.2) were purchased from Invitrogen (Paisley, UK). Anti-CCR5-PE (clone C34-3448) and anti-IL-17-PE (clone TC11-18H10.1) were purchased from BD Biosciences (Oxford, UK). Anti-CCR3-FITC (clone 83101), anti-CCR6-FITC (clone 140706), and anti-CCR9-FITC (clone 242503) were purchased from R&D Systems. Anti-CCR7-PE (clone EBI-1) was purchased from eBioscience (San Diego, CA, USA). Anti-CCR2 (clone MC-21) was a kind gift from Professor Matthias Mack (University of Regensburg, Germany). Anti-rat IgG-FITC was purchased from Sigma-Aldrich. All isotype control antibodies were purchased from Invitrogen or BD Biosciences. All chemokines were purchased from Peprotech with the exception of CCL25 (R&D Systems). Phospho-specific polyclonal antibody recognizing Akt phosphoSer 473 was purchased from Cell Signaling Technologies (New England Biolabs, Hitchin, UK). Goat anti-protein kinase B was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies for immunoblotting were purchased from Dako (Glostrup, Denmark).
Cell preparation
Spleens from DO11.10 mice were removed and disaggregated through a 40-µm filter, and erythrocytes were lysed with RBC lysis buffer (Sigma-Aldrich). Cells were negatively selected using a CD4+ T cell isolation kit (Miltenyi Biotec, Woking, UK) according to the manufacturer’s instructions. The resulting cells were found to be >90% pure, as assessed by flow cytometry. Spleens from Balb/c mice were treated as outlined above. The resulting leukocytes were incubated with mitomycin C (50 µg/ml) for 30 min at 37°C followed by four washes in culture medium.
Generation of Th17 and Th0 cells
CD4+ T cells were cocultured with mitomycin C-treated Balb/c splenocytes (5x105/ml and 2.5x106/ml, respectively) in the presence of 200 ng/ml OVA323–339 peptide (University of Southampton, Southampton, UK) and appropriate polarizing conditions. Th17-polarizing conditions were 1 ng/ml TGF-β1, 20 ng/ml IL-6, 10 ng/ml IL-1β, TNF-, and IL-23, and 10 µg/ml anti-IFN- and anti-IL-4 antibodies. Nonpolarizing Th0 conditions were 10 ng/ml IL-2. All cell cultures were incubated at 37°C + 5% CO2 in a humidified environment and received IL-2 (10 ng/ml) on Day 3 and were used on Day 7. Prior to use, viable cells were isolated by density centrifugation over Nycoprep 1.077Å (Axis Shield, Kimbolton, UK) at 550 g for 20 min. Cells were removed from the interface, and viability was determined by trypan blue exclusion.
Flow cytometry
For detection of extracellular antigens (CD4 and chemokine receptor expression), cells were washed and stained with 10 µg/ml appropriate antibody in 100 µl staining buffer (PBS, 0.25% BSA) for 30 min on ice. Unbound antibody was then removed by washing the cells twice in staining buffer, and data were acquired with an EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA, USA) using EXPO32 ADC software or a FACSCanto II (Becton Dickinson, San Jose, CA, USA) using Becton Dickinson FACSDiVa software.
For detection of intracellular antigens, cells were stimulated with 50 ng/ml PMA, 500 ng/ml ionomycin, and GolgiStop (BD PharMingen, Oxford, UK) for 4 h at 37°C, followed by staining of extracellular antigens with antibody diluted in PBS + 1% FCS for 30 min on ice. Cells were then fixed and permeabilized using the Becton Dickinson Cytofix/Cytoperm Plus kit (BD Biosciences) following the manufacturer’s instructions and stained for intracellular cytokines (IL-17 and IFN-) with appropriate antibodies for 30 min in Perm/Wash buffer on ice (BD Biosciences). Data were acquired by flow cytometry as described above. Th17 cells were defined as IL-17+ IFN-–.
Secreted cytokine detection assay
Soluble IL-17 and IFN- protein levels were measured simultaneously within cell culture supernatants on Day 7 using a multiplex Luminex assay (Qiagen, Crawley, UK). Cell culture supernatant (100 µl) was incubated with a suspension of analyte capture antibody-conjugated microspheres, according to the manufacturer’s instructions (Qiagen). After further incubation with biotinylated detection antibodies (R&D Systems) and PE-conjugated streptavidin (Qiagen), the mean fluorescent intensity (MFI) of each distinct bead type was acquired using a Luminex100IS machine (Qiagen) and analyzed using Developer Workbench Software (Qiagen). A standard curve generated from proteins of known concentration was used to convert MFI to concentration values. The limits of detection were 12.3 pg/ml for IL-17 and 6.2 pg/ml for IFN-.
Gene expression analysis
Total cellular RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. RNA (1 µg) was used to synthesize cDNA using an Applied Biosystems (ABI; Foster City, CA, USA) high-capacity cDNA RT kit in a total volume of 100 µl following the manufacturer’s protocol. Taqman real-time quantitative PCR reactions were performed using the ABI 7900HT sequence detection system. cDNA (200 ng) was analyzed in triplicate. Data were analyzed using 
comparative threshold (CT) values obtained from the ABI software SDS 2.3 using automatic threshold and baseline and GAPDH as endogenous control. Expression fold-changes were calculated using the CT method of relative quantification (RQ) [30
]. The mean of the CT values of the Th0 samples was used as the calibrator value to calculate the CT. The RQ (or fold-change) was calculated using the formula 2–CT. GAPDH was used as an endogenous/housekeeper gene to normalize the CT values to the amount of cDNA present in each well (CT value). Statistical analyses were performed using the log value of RQ.
Cell migration assays
Cell migration assays were performed using 24-well Transwell® chemotaxis plates (Corning, Corning, NY, USA) with a 5-µm pore-size polycarbonate filter. Briefly, lower chambers contained 500 µl agonist, and 100 µl cells were added to the upper chamber of the Transwell (1x106/well), and the assay proceeded for 3 h at 37°C. For inhibitor studies, cells were incubated with LY294002 or Akti-1/2 for 30 min at 37°C before addition to the Transwell. Migrated cells were then collected from the lower chamber and stimulated with 50 ng/ml PMA, 500 ng/ml ionomycin, and GolgiStop for 3 h. After stimulation, cells were stained for extracellular antigens, fixed with 4% paraformaldehyde, and stored at 4°C overnight. Cells were permeabilized with Perm/Wash buffer and stained for intracellular antigens. Flow-count fluorospheres (Beckman Coulter) were added to each sample prior to acquisition to quantify the number of IL-17+ versus IFN-+ cells that had migrated into the lower chamber compared with input cells. Data were acquired using flow cytometry.
Immunoblotting
Cells were stimulated (8x106 cells/ml and 500 µl/sample) and incubated at 37°C in RPMI 1640. Reactions were terminated by the addition of ice-cold lysis buffer [50 mM Tris-HCl, pH 7.5, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 1 mM sodium molybdate, 10 mM sodium fluoride, 40 µg/ml PMSF, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 0.7 µg/ml pepstatin]. The samples were then centrifuged at 10,000 g 4°C for 10 min, and supernatants were transferred to new tubes. A volume of 5x sample buffer [5% (w/v) SDS, 50% (v/v) glycerol, 200 mM Tris-HCl, pH 6.8, bromophenol blue, and 5% (v/v) 2-ME] was added, and the samples were boiled for 5 min. The solubilized proteins were electrophoresed through 10% polyacrylamide/SDS gels and transferred by electroblotting onto nitrocellulose membranes, which were incubated for 1 h with 10 ml blocking solution [1% nonfat milk/0.05% sodium azide in TBS; 10 mM Tris (pH 7.5) and 100 mM NaCl], and then incubated overnight with 10 ml of the appropriate antibody in TBS + 0.1% Tween (1/1000 dilution). Membranes were incubated for 2 h with HRP-conjugated secondary antibody (1/10,000 dilution), and then proteins were visualized with ECL. For reprobing, membranes were stripped by incubation in stripping buffer [62.5 mM Tris (pH 6.8), 2% SDS, and 100 mM 2-ME] at 60°C for 30 min.
Measurement of Akt protein kinase activity
The Omnia® lysate assay (Invitrogen) was used to assess protein kinase activity of Akt. Polarized Th17 cells (5x106) per point were washed twice in RPMI 1640 and incubated at 37°C in serum-free RPMI for 60 min. If required, cells were incubated with inhibitors diluted in RMPI 1640 for the final 30 min of this period. Cells were left unstimulated or stimulated with chemokines at the required concentrations diluted in RPMI 1640. Stimulations were terminated by aspiration of the supernatant followed by the addition of ice-cold PBS and subsequent centrifugation at 1500 rpm for 5 min. The PBS was removed by aspiration, and the cell pellet was resuspended in 100 µl ice-cold cell extraction buffer provided with the assay kit. The cells were rotated at 4°C for 30 min before centrifugation at 13,000 rpm for 20 min and also at 4°C. The clarified cell extracts were then assayed for Akt activity according to the manufacturer’s specifications.
Statistics
An unpaired Student’s t-test or one-way ANOVA with Bonferroni correction were used for statistical analysis. P < 0.05 was considered as significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in vitro results in the development of a Th17 phenotype with IL-23 acting on memory CD4+ T cells to maintain the Th17 phenotype [6
, 7
]. Th17 cells have been identified using intracellular flow cytometry and predominantly express IL-17 with a few reports describing a small proportion of IL-17+ IFN-+ cells [31
]. We assessed the effect of Th17-polarizing and nonpolarizing conditions on total mCD4+ splenocytes from DO11.10 mice following activation with the OVA peptide323–339 using flow cytometry (Fig. 1 A and B
). The proportion of IL-17+ IFN-– cells was elevated significantly in Th17-polarized cultures (16.9%±0.9, n=12) compared with nonpolarized cultures (3.4%±0.3, n=12; P<0.001; Fig. 1C
). In addition, the proportion of IFN-+ IL-17– cells was decreased significantly in Th17-polarized cultures (5.6%±0.8, n=12) compared with nonpolarized cultures (8.5%±0.7, n=12; P<0.01; Fig. 1C
). Analysis of secreted cytokines within cell culture supernatants revealed a significantly higher concentration of IL-17 in Th17-polarized conditions (7401 pg/ml±1723 pg/ml, n=4) compared with nonpolarized conditions (230 pg/ml±36 pg/ml, n=5; P<0.001). Conversely, levels of IFN- were significantly lower under Th17-polarizing conditions (73 pg/ml±5 pg/ml, n=4) compared with nonpolarizing conditions (737 pg/ml±179 pg/ml, n=5; Fig. 1D
; P<0.001). In addition, significantly elevated IL-17 and RORt mRNA expression levels were detected in cells cultured under Th17-polarizing conditions compared with nonpolarizing conditions (Fig. 1 E and F)
.
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Figure 1. Phenotype of CD4+ T cells cultured under Th17- or Th0-polarizing conditions. CD4+ DO11.10 splenocytes were cultured under Th17-polarizing conditions or Th0-nonpolarizing conditions for 7 days. Intracellular IL-17 and IFN- detection was determined by flow cytometry. Representative flow cytometry plots from cells cultured under Th17-polarizing conditions (A) or nonpolarizing Th0 conditions (B) are shown. FL3 and FL2, Fluorescence 3 and 2, respectively. Numbers in each quadrant represent the proportion of stained cells within the total cell preparation. Mean intracellular flow cytometry data from different experiments are shown (n=8; C). Cell culture supernatant from cells cultured for 7 days was analyzed by Luminex to determine the concentrations of soluble IL-17 and IFN- (n=4; D). Quantitative analysis of IL-17 (E) and (F) RORt RNA expression of cells from Th17-polarizing conditions compared with nonpolarizing Th0 conditions. Results are expressed as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Figure 2. Chemokine receptor expression and functional responses of CD4+ T cells cultured under Th17- or Th0-polarizing conditions. CD4+ DO11.10 splenocytes were cultured under Th17-polarizing conditions or Th0-nonpolarizing conditions for 7 days and stained with fluorescently conjugated anti-CC chemokine receptor antibodies (A) and anti-CXC chemokine receptor antibodies (B; n=4). Migration of total Th17-polarized cells (C) or Th0-nonpolarized cells (D) was carried out in response to mCCL2, mCCL12, mCCL20, and mCCL25. Data are representative of three separate experiments. Results are expressed as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) Th17-polarized cell lysates (20 µl, equivalent to 1.6x106 cells) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with an antibody specific for the active Ser473-phosphorylated form of Akt, and protein was visualized with ECL. The blots were stripped and reprobed with anti-Akt antibody to verify equal loading and efficiency of protein transfer. Data are representative of three separate experiments. C, Control.
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Activation of the PI-3K-dependent signaling pathway is a well-documented biochemical event elicited by most chemokine receptors [24 , 25 ]. We therefore used this readout to explore whether CCR2, CCR6, and CCR9 detected on Th17-polarized cells were biochemically functional. Direct measurement of PI-3K products in primary cells is technically formidable, so phosphorylation of proteins downstream of PI-3K, such as Akt, is often used as a surrogate readout of PI-3K signaling output. The Th17-polarized cells were stimulated with agonists for CCR2 (CCL2), CCR6 (CCL20), and CCR9 (CCL25; 0.1, 1, or 10 nM), resulting in the concentration-dependent phosphorylation of residue Ser473 on Akt, with maximal phosphorylation occurring at 0.1 nM CCL2 and 1 nM CCL20 and CCL25 (Fig. 2E) . The phosphorylation of Akt in response to CCR2 and CCR6 agonists correlated with increased protein kinase activity, which was sensitive to inhibition of PI-3K with LY294002 (Fig. 3 ).
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Figure 3. CCR2 and CCR6 agonists stimulate LY294002-sensitive Akt protein kinase activity in Th17-polarized cells, which cultured under Th17-polarizing conditions, were incubated with LY294002 (A and C) or Akti-1/2 (B and D) at the concentrations indicated for 30 min before being stimulated with 10 nM CCL2 (A and B) or 10 nM CCL20 (C and D) for 5 min and lysed. Akt protein kinase activity was measured as described in Materials and Methods. Results are shown as the change in rate of fluorescence, which corresponds to change in rate of phosphorylation of target peptides by Akt. Results are expressed as mean ± SEM (n=4); **, P < 0.01. F, change in rate of fluoresence.
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Figure 4. Chemokine receptor expression and migration of Th17 cells, which cultured under Th17-polarizing conditions, were dual-stained for expression of intracellular IL-17 and surface expression of CCR2 (A), CCR6 (B), or CCR9 (C). The proportion of IL-17+ cells coexpressing CCR2, CCR6, or CCR9 is the value in the top right-hand quadrant (chemokine receptor+/IL-17+) calculated as a percentage of the sum of the two right-hand quadrant values (chemokine receptor+/IL-17+ and chemokine receptor–/IL-17+). Hence, 17% of cells are IL-17+ (15+2%) in A; therefore, 2% of 17% is  11% (see text for further values). Th17-polarized cells were placed in the upper chamber of a 24-well Transwell chemotaxis plate, and migration to increasing concentrations of CCL2 (D), CCL12 (E), CCL20 (F), or CCL25 (G) was determined. Postmigration cells were stimulated for 3 h with 50 ng/ml PMA, 500 ng/ml ionomycin, and GolgiStop and stained with antibodies to CD4, IL-17, and IFN-. Fluorescent beads were added to samples prior to data acquisition to quantify the number of migrated cells. Results are expressed as mean ± SEM (n 4); *, P < 0.05; **, P < 0.01.
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+ cells were detected postmigration using intracellular flow cytometry. Concentration-dependent migratory responses to CCL2, CCL12, and CCL20 were observed for IL-17+ cells (Fig. 4 D-F)
. In contrast, CCL25 elicited no significant migratory response effect for IL-17+ cells (Fig. 4G)
. The agonists examined did not significantly induce migration of IFN-+ cells (Fig. 4 D-G)
. Comparison of the proportions of IL-17+ cells pre- and postmigration demonstrated that the CCR2 agonists CCL2 and CCL12 and the CCR6 agonist CCL20 increased the proportion of IL-17+ cells in a concentration-dependent manner by a maximum of 10%. No significant enrichment of IL-17+ cells was seen after migration of Th0 cells to any of the chemokines, presumably as a result of the low frequency of these cells under nonpolarized culture conditions (data not shown).
CCR2- and CCR6-mediated migration is PI-3K/Akt-dependent
Although PI-3K has an evolutionary, conserved role in cell migration, its role in T cell migration remains controversial [26
]. We therefore explored whether PI-3K-dependent signals were required for the migration of Th17-polarized cells. The PI-3K inhibitor LY294002 significantly inhibited the migration of IL-17+ cells in response to 100 nM CCL2 and 30 nM CCL20 with pIC50 values of 5.73 ± 0.22 (n=3) and 5.68 ± 0.18 (n=4), respectively (Fig. 5 A and B
). These data are consistent with the affinity estimates reported for LY294002 in other cell systems [33
]. Although the inhibitory effects of LY294002 are well-documented, several off-target effects have also been identified, including affinity for mammalian target of rapamycin and casein kinase 2 [34
]. We therefore used another pharmacological tool to assess the contribution of PI-3K/Akt signaling to migration of IL-17+ cells, namely, Akti-1/2, which has shown strong selectivity for Akt 1 and Akt 2 with no reported off-target effects up to 50 µM concentrations [35
]. Akti-1/2 significantly inhibited the migration of IL-17+ cells in response to 100 nM CCL2 and 30 nM CCL20 with pIC50 values of 5.75 ± 0.08 (n=3) and 5.78 ± 0.13 (n=4), respectively (Fig. 5 A and B)
.
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Figure 5. Effect of PI-3K and Akti inhibition on CCR2- and CCR6-mediated Th17 cell migration. Cells cultured under Th17-polarizing conditions were incubated with increasing concentrations of LY294002 or Akti-1/2 (100 nM–100 µM) for 30 min and placed in the upper chamber of a 24-well Transwell chemotaxis plate, and migration to 100 nM CCL2 (A) or 30 nM CCL20 (B) was determined. Postmigration cells were stimulated for 3 h with 50 ng/ml PMA, 500 ng/ml ionomycin, and GolgiStop and stained with antibodies to CD4, IL-17, and IFN-. Fluorescent beads were added to samples prior to data acquisition to quantify the number of migrated cells. Results are expressed as mean ± SEM (n3).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, to polarize CD4+ splenocytes from DO11.10 mice toward a Th17 phenotype. Analysis of the surface expression profile of chemokine receptors on cells maintained under Th17-polarizing conditions revealed significant increases in the proportion of cells expressing the chemokine receptors CCR2, CCR6, and CCR9 when compared with nonpolarized cells. Agonists for CCR2, CCR6, and CCR9 stimulated migratory and biochemical responses of cells maintained under Th17-polarizing conditions. However, only agonists for CCR2 and CCR6 were able to elicit a modest enrichment of IL-17-producing cells within the migrated population. Finally, pan-isoform inhibitors of PI-3K/Akt signaling inhibited CCR2- and CCR6-stimulated cell migration in a concentration-dependent manner.
Th17 polarization resulted in significantly higher proportions of IL-17+ T cells as assessed by intracellular cytokine expression and a corresponding production of soluble IL-17. However, in our experiments, a small percentage of cells (approximately 6%) maintained under Th17-polarizing conditions did not express IL-17 but expressed IFN-. Although the differentiation of Th1 and Th17 was initially thought to be mutually exclusive, it is interesting to note the identification of a subset of cells sharing features of Th1 and Th17 cells including IFN-+ expression [36
, 37
]. Moreover, the possibility of differentiated Th17 cells showing flexibility in their cytokine production profile is not without precedent, as cells producing IL-4 and IFN- have also been reported [38
]. Overall, less than 20% of the population maintained under Th17-polarizing conditions exhibited intracellular expression of IL-17. The phenotype of the remaining >80% of cells within the population maintained under Th17-polarizing conditions remains unclear. The presence of anti-IL-4 antibodies in the culture medium makes it unlikely that Th2 cells contribute to this population. Similarly, IL-6 has been shown to inhibit TGF-β-induced forkhead box p3 expression [39
]; therefore, the presence of IL-6 in the Th17-polarizing medium makes it unlikely that a large number of regulatory T cells would be present. We were unable to detect CD62 ligand expression on Th17-polarized cells, suggesting that these cells had entered into a differentiation program (data not shown). The inability to differentiate greater proportions of IL-17+ cells within our culture system may be our use of total CD4+ T cells comprising naïve and memory populations. The isolation of naïve and memory populations prior to polarization is currently being explored.
We detected a significant decrease in the proportion of Th17-polarized cells expressing CXCR3 compared with nonpolarized cells. Based on the role of CXCR3 in Th1 cell biology, the decreased CXCR3 expression on Th17-polarized cells may in part reflect the decreased proportion of IFN-+ cells observed under these culture conditions. The increased expression of CCR2, CCR6, and CCR9 on CD4+ T cells under Th17-polarizing conditions has several implications for inflammatory diseases. For example, CCR6 is a mucosal homing receptor present on dendritic cells and effector memory T cells [40
, 41
] that has also been implicated in cutaneous, airway, and intestinal mucosal immunity [42
, 43
] and several inflammatory conditions including rheumatoid arthritis [5
, 44
] and psoriasis [45
, 46
], in which IL-17 has been shown to play a role. Recent evidence has revealed that dectin-1-mediated fungal immune recognition preferentially induced hTh17 cells characterized by expression of CCR6 and CCR4 [17
]. Singh et al. [29] have demonstrated that all IL-17-producing hCD4+ T cells expressed surface CCR6 and were enriched for RORt mRNA expression. Furthermore, IL-17 production from CCR6-expressing CD8+ T cells was readily detected. These data suggest a role for CCR6 in mediating the immune responses of IL-17-producing T cells [29
]. Our description of functional CCR6 expression on mTh17 cells is in agreement with recent observations from Hirota et al. [47
] that CCR6 is functionally expressed on mTh17 in vitro and in vivo. However, Hirota et al. [47
] reported that CD4+ T cells from mutant mice harboring a mutation of the gene encoding Zap70 spontaneously differentiate to arthritogenic Th17 cells, which predominantly express CCR6. The discrepancy between this and the relatively low level of CCR6 expression on IL-17+ cells observed in our study probably reflects differences in strains of mice and model systems used in each study. Nevertheless, it is interesting to note that this study also reported only a small percentage of IL-17+ cells (15–30%) following in vitro induction of Th17 cells from Balb/c mCD4+ T cells. This is a significantly higher percentage level of CCR6 expression on IL-17+ cells than we observed but serves to underline the fact that the level of chemokine receptor expression on Th17 cells will depend on the differentation strategy and model used [47
].
CCR2 is expressed on monocytes, activated memory T cells, B cells, and basophils in humans and in peritoneal macrophages in mice. Increased expression of CCR2 has been strongly associated with several inflammatory conditions including multiple sclerosis and rheumatoid arthritis and corresponding animal models of each disease, e.g., EAE and collagen-induced arthritis [48 49 50 51 ]. CCR2+ CCR5– hTh17 cells have been described previously [27 ], consistent with our findings using mTh17-polarized cells.
Interactions between CCL25 and CCR9 are involved in thymic development and the generation of gut-specific immunological memory [52 , 53 ]. In the gut, CCL25 is highly expressed by epithelial cells lining the small intestinal villi but is less-prominently expressed in the colon [52 , 53 ]. Evidence from CCR9–/– mice indicates that CCR9 is necessary for steady-state intestinal T cell development and/or migration [54 55 56 ]. Our demonstration of increased proportions of Th17-polarized cells expressing CCR9 is interesting, as IL-17 levels are elevated in inflammatory bowel disease (IBD) [57 ], and the IL-23R gene is a susceptibility factor for IBD [58 ]. Indeed, IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6 in experimental models [59 ]. Paradoxically, however, addition of the anti-IL-17 antibody worsened intestinal inflammation in a murine model of colitis [60 ].
We have shown, using pan-isoform PI-3K inhibitor and Akti-1/2, that the migration of mTh17 cells is dependent on PI-3K and its downstream effector Akt. Much interest has arisen recently in the role of PI-3K and inflammatory diseases, especially as two isoforms, p110
and p110, appear to be expressed primarily in leukocytes [61
]. Treatment of mice with inhibitors of p110 suppressed the progression of joint inflammation in mouse models of rheumatoid arthritis [62
] and prolonged survival in murine models of systemic lupus erythematosus [23
]. Both diseases have been shown to have elevated levels of IL-17 [63
, 64
]. Available evidence indicates that p110 is the predominant isoform required for cell migration in response to a chemokine agonist [24
, 25
, 65
]. Interestingly, although migration of leukemic cell lines and freshly isolated human T cells toward CXCL12 is PI-3K-dependent, directional migration of activated human T cells maintained ex vivo in response to the same chemokine is PI-3K-independent [24
25
26
, 65
]. In addition, PI-3K is a dispensable signal for migration of leukemic cell lines and hTh2 cells responding to CCL22 [25
, 26
]. Our new observations relating to the requirement of PI-3K signaling for migration of Th17-polarized cells strengthen and extend the notion that the contribution of PI-3K to chemotaxis depends on the individual chemoattractant agonist/chemokine receptor interaction, cell type, and/or its differentiation state [24
, 25
]. The different dependency of Th17 versus Th2 cell migration [26
] on PI-3K-dependent signaling offers exciting therapeutic opportunities whereby PI-3K isoform-selective inhibitors might be used to suppress Th17-driven inflammatory/autoimmune responses, while leaving other arms of the immune response unaffected. Moreover, analysis of migrated cells that responded to CCR2 and CCR6 agonists reveals a greater number of IL-17+ cells compared with the premigration population. Broad-spectrum inhibitors of PI-3K/Akt signaling demonstrated concentration-dependent inhibition of Th17 migration in response to CCR2 and CCR6 agonists. All four agonists tested (CCL2, CCL12, CCL20, and CCL25) were capable of eliciting migration and/or PI-3K phosphorylation of Th17-polarized cells, indicating functional and biochemical responsiveness of these receptors. However, in our study, despite the ability of CCL25 to stimulate biochemical responses, CCL25/CCR9 interactions had little effect on migratory responses of IL-17+ cells. The reasons for this are currently unclear but may reflect differences in functional efficacy of CCL25 versus CCL2 and CCL20. In this regard, CCL25 was much less potent than CCL2 or CCL20 in its ability to stimulate Akt phosphorylation.
In summary, this paper shows that CCR2, CCR6, and CCR9 receptors expressed on Th17 cells are biochemically functional. However, postmigration phenotypic analysis demonstrated that only the agonists for CCR2 and CCR6 elicited migratory responses of Th17-polarized cells via PI-3K-dependent mechanisms. The identification of unique cell surface Th17 markers may be useful in developing experimental tools to aid the phenotypic characterization and isolation of Th17 cells from mixed cell populations. Whether CCR2, CCR6, or CCR9 is coexpressed on the same cell population or represents distinct subpopulations of IL-17+ cells remains unclear. The unique chemokine receptor expression pattern of Th17 cells provides a basis for their recruitment to specialized inflammatory conditions in vivo and may also provide applications for CCR2, CCR6, and CCR9 antagonists and PI-3K isoform-selective inhibitors in defined inflammatory settings.
2 Current address: Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK.
Received April 9, 2008; revised May 14, 2008; accepted May 30, 2008.
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t directs the differentiation program of proinflammatory IL-17+ T helper cells Cell 126,1121-1133[CrossRef][Medline] and ROR Immunity 28,29-39[CrossRef][Medline] and p110, in lymphocyte chemotaxis and homing J. Immunol. 173,2236-2240 inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus Nat. Med. 11,933-935[Medline]-producing Th1 subset is the major source of IL-17 in experimental autoimmune encephalitis J. Neuroimmunol. 183,96-103[CrossRef][Medline] and is highly expressed in human dendritic cells J. Exp. Med. 186,837-844 and its ligand CCR6 in rheumatoid arthritis Lab. Invest. 83,579-588[Medline]/CCL20 and CC chemokine receptor 6 in psoriasis J. Immunol. 164,6621-6632(+) gut intraepithelial lymphocytes Blood 98,2626-2632 isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia Blood 106,1063-1066 suppresses joint inflammation and damage in mouse models of rheumatoid arthritis Nat. Med. 11,936-943[Medline] is the dominant isoform involved in migratory responses of human T lymphocytes: effects of ex vivo maintenance and limitations of non-viral delivery of siRNA Cell. Signal. 19,2528-2539[CrossRef][Medline]This article has been cited by other articles:
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