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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Giant cell arteritis (GCA) is a large-vessel vasculitis of unknown origin. Recent findings indicate that at least 2 separate lineages of CD4+ T cells, Th1 and Th17 cells, participate in vascular inflammation. The pathways driving these T cell differentiations are incompletely understood, but may provide novel therapeutic targets. This study was undertaken to identify cytokines involved in the pathogenesis of GCA.

Methods

Thirty GCA patients fulfilling the American College of Rheumatology criteria, with active disease or disease in remission, and 30 age-matched controls were included. Levels of 27 cytokines were determined in culture supernatants, and flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and immunohistochemical analysis of temporal artery samples were performed.

Results

Multiparametric analysis of cytokines produced by PBMCs associated with GCA disease activity identified a signature involving interleukin-2 receptor (IL-2R), IL-12, interferon-γ (IFNγ), IL-17A, IL-21, and granulocyte–macrophage colony-stimulating factor (GM-CSF). An expansion of Th1 and Th17 cells and a decrease in Treg cells were observed in the peripheral blood of patients with active GCA. An expansion of IL-21–producing CD4+ T cells was also observed in patients with active GCA and correlated positively with Th17 and Th1 cell expansion. Immunohistochemical analysis revealed IFNγ, IL-17A, and IL-21 expression within inflammatory infiltrates. Stimulation of purified CD4+ T cells with IL-21 increased Th1 and Th17 cell frequencies and decreased FoxP3 expression. In contrast, blockade of IL-21 using IL-21R-Fc markedly decreased the production of IL-17A and IFNγ and increased FoxP3 expression.

Conclusion

Our findings indicate that IL-21 plays a critical role in modulating Th1 and Th17 responses and Treg cells in GCA, and might represent a potential target for novel therapy.

Giant cell arteritis (GCA) is a large-vessel vasculitis of unknown origin that affects persons older than 50 years, but the disease risk is highest among patients between 75 and 85 years (1). GCA causes aortitis and vasculitis of the extracranial branches of the aorta and spares intracranial vessels, and is associated with elevated levels of biologic parameters of inflammation (erythrocyte sedimentation rate and/or C-reactive protein) (1, 2). Typical manifestations of GCA include blindness, headache, scalp tenderness, and jaw claudication (1, 2). Ocular ischemic complications may occur in 25% of patients, leading to irreversible vision loss in 15% (2, 3). The diagnosis of GCA may be difficult in some cases, and temporal artery biopsy represents the gold standard for diagnosis, although the results of histologic analysis are normal in 20% of patients (4).

Recent studies have provided new insights into the pathogenesis of GCA. In animal models, mice deficient in interferon regulatory factor 4 (IRF-4) binding protein, a protein that inhibits interleukin-17A (IL-17A) production by controlling the activity of IRF-4 transcription factor, rapidly developed a large-vessel vasculitis due to inappropriate synthesis of IL-17A (5). In humans, IFNγ-producing Th1 cells and IL-17A–producing Th17 cells are implicated in GCA. Th1 and Th17 cells are markedly expanded in the peripheral blood of patients with active GCA compared to patients with inactive GCA, and Th1 and Th17 cells coexist within vasculitis inflammatory infiltrates. In addition, Th17 cell levels decrease, while Th1 responses persist, during steroid treatment (6). These findings suggest that a treatment strategy targeting Th1 and Th17 immune responses (7), in combination with corticosteroid treatment, should be explored in order to decrease the dose and duration of steroid exposure and the risk of relapse. Although the results described above have undoubtedly contributed to our understanding of the pathophysiology of GCA, data on cytokines that act upstream of these predominant Th1 and Th17 responses and represent potential targets for novel therapy are lacking.

Using a systematic and unsupervised large-scale assessment of 27 cytokines and chemokines produced by peripheral blood mononuclear cells (PBMCs) from GCA patients, we performed a multiparametric analysis in order to identify a signature that discriminates patients with active GCA from those with inactive GCA, and in particular to identify new cytokines involved in the pathogenesis of GCA. We confirmed that Th1- and Th17-related cytokines were associated with disease activity. We demonstrated an expansion of IL-21–producing CD4+ T cells in the peripheral blood of GCA patients that was correlated with disease activity, Th1 and Th17 expansion, and a decrease in resting and activated memory Treg cells. Stimulation of CD4+ T cells with IL-21 increased Th1 and Th17 differentiation and decreased Treg cell frequency. Conversely, IL-21 blockade with IL-21R-Fc restored Th1, Th17, and Treg cell homeostasis in GCA patients. Our findings suggest that IL-21 plays a critical role in the pathogenesis of GCA and might represent a potential target for novel therapy.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Patients.

The study population consisted of 30 consecutive adult patients (9 men and 21 women with a mean age of 72 years [range 60–88 years]) who fulfilled the American College of Rheumatology criteria for GCA (8). Patients were divided into 2 groups according to disease activity: patients with active and untreated GCA (n = 14) and patients with disease in remission (n = 16). Patients with active GCA were defined as patients with vasculitis manifestations and biologic inflammatory syndrome who were not receiving corticosteroids or immunosuppressants. Patients with GCA in remission were defined as patients who did not have clinical manifestations or elevated levels of inflammation parameters. Patients with disease in remission were receiving corticosteroids (median dosage 15 mg/day [range 5–30 mg/day]; n = 13) or were untreated (n = 3). Thirty age-matched control subjects (8 men and 22 women with a mean age of 73 years [range 56–86 years]) were included for the measurement of cytokines and chemokines in culture supernatants; 20 of these controls were included in the flow cytometric analysis of PBMCs. Controls did not have inflammatory or autoimmune disorders, cancer or a history of cancer, or bacterial, viral, or fungal infectious diseases and were not receiving any immunosuppressive or immunomodulating agents. The study was performed according to the principles of the Declaration of Helsinki, and informed consent was obtained from all subjects.

Analysis of cytokine production.

PBMCs from GCA patients and age-matched controls were stimulated for 4 hours with 50 ng/ml phorbol myristate acetate (PMA) and 1 mM ionomycin (Sigma-Aldrich) in the presence or absence of brefeldin A (BD PharMingen). Supernatants from cells cultured in the absence of brefeldin A were harvested and immediately frozen at −80°C. Cells cultured in the presence of brefeldin A were stained for cell surface markers and then permeabilized with Cytofix/Cytoperm buffer (BD PharMingen) and stained with fluorescein isothiocyanate (FITC)–conjugated IFNγ (BD PharMingen), Alexa Fluor 647–conjugated IL-17A (eBioscience), and Alexa Fluor 647–conjugated IL-21 (BioLegend). Data were acquired using a Navios flow cytometer and analyzed with Navios analysis software (Beckman Coulter).

Levels of the following 25 cytokines or chemokines in culture supernatant were measured using Human Cytokine 25-Plex according to the recommendations of the manufacturer (Invitrogen): granulocyte–macrophage colony-stimulating factor (GM-CSF), eotaxin, IFNα, IFNγ, IL-1 receptor antagonist (IL-1Ra), IL1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17A, CXCL10 (IFNγ-inducible protein 10 [IP-10]), CCL2 (monocyte chemotactic protein 1 [MCP-1]), CXCL9 (monokine induced by IFNγ [MIG]), CCL3 (macrophage inflammatory protein 1α [MIP-1α]), CCL4 (MIP-1β), CCL5 (RANTES), and tumor necrosis factor α (TNFα). Levels of IL-21 (BioLegend) and IL-23 (R&D Systems) were determined by enzyme-linked immunosorbent assay.

Analysis of cell surface markers and FoxP3 expression.

PBMCs were stained with the following conjugated monoclonal antibodies, at predetermined optimal dilutions, for 30 minutes at 4°C: energy-coupled dye (ECD)–conjugated CD3, PCy7-conjugated CD4, ECD-conjugated CD4, PCy7-conjugated CD8, allophycocyanin (APC)–conjugated CD8, FITC-conjugated CD16, ECD-conjugated CD19, phycoerythrin (PE)–conjugated CD27, FITC-conjugated CD28, FITC-conjugated CD45RO, APC-conjugated CD45RA, PE-conjugated CD56, PCy7-conjugated HLA–DR (all from Beckman Coulter), PE-conjugated CD25, PCy7-conjugated CD38, FITC-conjugated CD56, FITC-conjugated CD62L, FITC-conjugated IgD (all from BD PharMingen), PE-conjugated CCR7 (R&D Systems), and FITC-conjugated CD127 (eBioscience). Intracellular detection of FoxP3 was performed on fixed and permeabilized cells using appropriate buffer (eBioscience). Data were acquired using a Navios flow cytometer and analyzed with Navios analysis software.

Purification of CD4+ T lymphocytes from GCA patients.

Peripheral total CD4+ T cells were isolated from PBMCs using immunomagnetic depletion (Miltenyi Biotec) with a purity of each population of >95%. Purified CD4+ T cell populations were cultured in X-Vivo 20 medium (Lonza) supplemented with 2% penicillin–streptomycin (1 × 106 cells/ml) and stimulated in 48-well plates coated with anti-CD3/CD28 monoclonal antibodies under the following conditions: with anti-CD3/CD28 alone, with recombinant human IL-21 (rhIL-21) (50 ng/ml; BioVision), or with rhIL-21R-Fc chimera (100 μg/ml; R&D Systems). FoxP3 expression was analyzed using flow cytometry as described above, and intracellular cytokine production was analyzed after restimulation with PMA and ionomycin and flow cytometry.

Immunohistochemical analysis.

Detection of IL-17A+, IFNγ+, IL-6+, IL-21+, CCL20+, CXCL8+, and FoxP3+ cells was performed on fixed, paraffin-embedded temporal artery samples from 3 GCA patients and temporal artery samples without inflammatory infiltrates from 3 controls. Dewaxed slides were subjected to antigen retrieval by heating in citrate buffer (pH 6.0). Before incubation with primary antibodies, Fc receptor was blocked with 2% bovine serum albumin. Slides were incubated overnight with rabbit anti-human polyclonal IL-21 (dilution 1:20) (catalog no. 500-P191; PeproTech), goat anti-human polyclonal IL-17A (dilution 1:20; R&D Systems), rabbit anti-human polyclonal IFNγ (dilution 1:1,000; Abcam), rabbit anti-human polyclonal IL-6 (dilution 1:400; Abcam), rabbit anti-human polyclonal CCL20 (working dilution 1:30; Abcam), goat anti-human polyclonal CXCL8 (dilution 1:20, R&D Systems), and rat anti-human polyclonal FoxP3 (dilution 1:100; eBioscience). Antibody binding was visualized with diaminobenzidine tetrahydrochloride (Dako). Isotype-matched primary antibodies were used as a negative control.

Statistical analysis.

Data are presented as the mean ± SEM for continuous variables and as percentages for qualitative variables. Fisher's exact test was used to compare qualitative variables, and the nonparametric Mann-Whitney and Wilcoxon tests were used to compare continuous variables, as appropriate. P values less than 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism software, version 4.0 and InStat, version 3.0 for Windows.

Cytokine and chemokine levels were determined in all 30 GCA patients. Data were log10 transformed and standardized by subtracting the mean and dividing by the standard deviation. Student's t-test with Benjamini-Hochberg false discovery rate method (q ≤ 0.05) was used to identify cytokines that were differentially expressed between the 2 groups (patients with active disease versus patients with disease in remission). In order to reduce the statistically significant cytokine vector (i.e., signature) to the most informative classifiers, Pearson's correlation coefficients between these cytokines were calculated, and highly correlated cytokines (r > 0.9) were removed by a stepwise procedure. The selected data set was analyzed by hierarchical clustering using Euclidean distance and the complete linkage method. Based on the signature obtained as described above, we aimed to build a model predictive of the membership of a test sample in a pathophysiologic condition. For this purpose, a stepwise procedure against each response variable (clinical groups) was performed to assess the predictive power of the signature. Since our focus was to evaluate how well a linear combination of variables explains the difference between data classes, linear discriminant analysis models seemed best suited to our approach. Leave-one-out cross-validation was used to estimate the accuracy of the predictive model. This method allowed us to calculate the prediction error rate and sensitivity, specificity, and positive and negative predictive values of our model. All data-processing steps and statistical and multivariate analyses were performed using R (http://www.r-project.org/).

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Increased levels of Th1- and Th17-related cytokines in GCA patients.

Levels of cytokines and chemokines were measured in culture supernatants after PBMCs were stimulated for 4 hours with PMA and ionomycin. Compared to age-matched controls, patients with active GCA had increased mean levels of GM-CSF (53.3 pg/ml in patients versus 37.0 pg/ml in controls; P = 0.0005), IL-1β (387.5 pg/ml in patients versus 67.7 pg/ml in controls; P = 0.02), IL-6 (41.5 pg/ml in patients versus 19.7 pg/ml in controls; P = 0.001), IL-12 (28.6 pg/ml in patients versus 19.3 pg/ml in controls; P = 0.03), IFNγ (837.3 pg/ml in patients versus 274.0 pg/ml in controls; P < 0.0001), TNFα (610.0 pg/ml in patients versus 275.0 pg/ml in controls; P = 0.001), IL-17 (33.5 pg/ml in patients versus 10.7 pg/ml in controls; P = 0.0003), IL-21 (48.3 pg/ml in patients versus 16.2 pg/ml in controls; P < 0.0001), and IL-23 (75.8 pg/ml in patients versus 17.1 pg/ml in controls; P = 0.001).

Compared to patients with GCA in remission, patients with active GCA had increased mean levels of GM-CSF (53.3 pg/ml in patients with active GCA versus 35.3 pg/ml in patients with GCA in remission; P = 0.002), MIP-1α (965.5 pg/ml in patients with active GCA versus 442.4 pg/ml in patients with GCA in remission; P = 0.01), IL-2R (94.5 pg/ml in patients with active GCA versus 50.7 pg/ml in patients with GCA in remission; P = 0.007), IL-12 (28.6 pg/ml in patients with active GCA versus 13.8 pg/ml in patients with GCA in remission; P = 0.002), IFNγ (837.3 pg/ml in patients with active GCA versus 214.3 pg/ml in patients with GCA in remission; P = 0.0002), TNFα (610.0 pg/ml in patients with active GCA versus 269.5 pg/ml in patients with GCA in remission; P = 0.01), IL-17 (33.5 pg/ml in patients with active GCA versus 10.7 pg/ml in patients with GCA in remission; P = 0.0003), and IL-21 (48.3 pg/ml in patients with active GCA versus 15.1 pg/ml in patients with GCA in remission; P < 0.0001). (Details are available from the author upon request.) No difference was found between groups for the remaining cytokines measured (eotaxin, IFNα, IL-1Ra, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-13, IL-15, MIG, IP-10, MCP-1, MIP-1β, and RANTES) (data not shown).

Identification of a signature associated with GCA disease activity by multiparametric analysis of cytokines.

We next performed a multiparametric analysis of cytokines produced by PBMCs associated with GCA disease activity. Eight cytokines were significantly differentially expressed in culture supernatants from patients with active GCA and patients with GCA in remission. After Benjamini-Hochberg correction, 6 remained significantly differentially expressed (IL-2R, IL-12, IFNγ, IL-17A, IL-21, and GM-CSF). However, because we chose to withdraw highly correlated cytokines (r > 0.90) within the signature, IL-21, which was highly correlated with IL-17A and IFNγ, was excluded from the final signature. Finally, a signature involving IL-2R, IL-12, IFNγ, IL-17A, and GM-CSF provided the lowest prediction error of 13%. An average linkage hierarchical clustering analysis confirmed that GCA patients segregated into 2 main groups on the basis of the selected immunologic signature (Figure 1). Leave-one-out cross-validation revealed that this signature had a sensitivity of 79%, a specificity of 94%, and positive and negative predictive values of 92% and 83%, respectively, for discriminating patients with active disease from patients with inactive disease.

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Figure 1. Hierarchical clustering analysis discriminating patients with active giant cell arteritis (aGCA; n = 14) from patients with GCA in remission (rGCA; n = 16), using 5 cytokines identified by Student's t-test and Benjamini-Hochberg correction. Transformed expression levels are indicated by the color scale. IFNγ = interferon-γ; IL-2R = interleukin-2 receptor; GM-CSF = granulocyte–macrophage colony-stimulating factor.

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Th1 and Th17 cell levels are increased in the peripheral blood of GCA patients and correlate with disease activity.

We next examined the frequency of IL-17A– and IFNγ-producing T cells in PBMCs after 4 hours of stimulation with PMA and ionomycin. We observed an increase in the mean percentage of IFNγ-producing CD4+ Th1 cells in the peripheral blood of patients with active GCA (22.1%) compared to patients with GCA in remission (10.1%) and controls (11.8%) (P = 0.0005 versus patients with GCA in remission and P = 0.002 versus controls). The mean percentage of IFNγ-producing CD8+ T cells was also increased in patients with active GCA (59.8%) compared to patients with GCA in remission (36.6%) and controls (47.8%) (P = 0.004 and P = 0.05, respectively) (Figures 2A and B), as was the mean percentage of IL-17A–producing CD4+ Th17 cells (2.5% in patients with active GCA versus 0.7% in patients with GCA in remission and 0.5% in controls; P < 0.0001 for both) (Figures 2C and D).

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Figure 2. Th1 and Th17 effector responses in GCA. Peripheral blood mononuclear cells from 14 patients with active GCA, 16 patients with GCA in remission, and 20 age-matched controls were stimulated for 4 hours with phorbol myristate acetate and ionomycin. A, Representative dot plots of IFNγ-producing CD4+ (Th1) cells, after gating on CD3+ T cells. Numbers are the percentages of cells in each subpopulation. B, Frequencies of IFNγ-producing CD4+ (Th1) cells and CD8+ T cells. C, Representative dot plots of IL-17A–producing CD4+ (Th17) cells, after gating on CD3+ T cells. Numbers are the percentages of cells in each subpopulation. D, Frequencies of IL-17A–producing CD4+ (Th17) cells. IFNγ-producing CD8+ T cells and Th17 cells were markedly expanded in patients with active GCA. In B and D, circles indicate individual samples; horizontal lines indicate the mean. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. See Figure 1 for definitions.

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Decrease in resting and activated memory Treg cells and increase in cytokine-secreting T cells in GCA.

We explored a possible defect in T cell regulation associated with the Th1 and Th17 imbalance. We found that the mean percentage of CD4+FoxP3+CD25highCD127− Treg cells was decreased in peripheral blood from patients with active GCA (2.9%) and patients with GCA in remission (3.2%) compared to age-matched controls (4.4%) (P = 0.006 and P = 0.04, respectively), while no difference was noted between patients with active GCA and patients with inactive GCA. (Data are available from the author upon request.)

We next analyzed the expression of CD45RA and CD25 among the CD4+ T cells, in order to better delineate CD45RA+CD25++ resting Treg cells, CD45RA−CD25+++ activated memory Treg cells, and cytokine-secreting CD45RA−CD25++FoxP3low nonsuppressive T cells containing cells with Th17 cell potential. The mean percentages of resting Treg cells were decreased in patients with active GCA (0.9%) and patients with GCA in remission (1.0%) compared to controls (1.3%) (P = 0.02 and P = 0.14, respectively), as were the mean absolute numbers of resting Treg cells (6.2/mm3 in patients with active GCA and 7.3/mm3 in patients with GCA in remission versus 11.1/mm3 in controls; P = 0.02 and P = 0.11, respectively). The mean percentages and absolute numbers of activated memory Treg cells were also decreased in patients with active GCA and patients with GCA in remission versus controls (0.5% in patients with active GCA and 0.8% in patients with GCA in remission versus 1.0% in controls [P < 0.0001 and P = 0.04, respectively]; 4.1/mm3 in patients with active GCA and 5.1/mm3 in patients with GCA in remission versus 8.3/mm3 in controls [P = 0.003 and P = 0.03, respectively]). In contrast, the mean percentages and absolute numbers of CD45RA−CD25++FoxP3low nonsuppressive T cells were increased in patients with active GCA compared to patients with GCA in remission and controls (3.8% in patients with active GCA versus 3.0% in patients with GCA in remission and 2.8% in controls [P = 0.01 and P = 0.05, respectively]; 30.5/mm3 in patients with active GCA versus 19.9/mm3 in patients with GCA in remission and 20.5/mm3 in controls [P = 0.02 and P = 0.09, respectively]).

Increased production of IL-21 by CD4+ T cells in GCA and correlation of IL-21 levels with Th1 and Th17 responses.

Given the increased levels of IL-21 in culture supernatants, we analyzed intracellular production by T cells using flow cytometry. IL-21–producing CD4+ T cells were markedly expanded in the peripheral blood of patients with active GCA (mean 8.2%) compared to patients with GCA in remission (mean 3.3%) and controls (mean 2.4%) (P < 0.0001 for both) (Figures 3A and B). IL-21–producing CD4+ T cells displayed the phenotype of central memory T cells, as indicated by the expression of CD45RO and CD27. The expansion of IL-21–producing CD4+ T cells was positively correlated with the expansion of Th1 cells (r2 = 0.70, P < 0.0001) (Figure 3C), IFNγ-producing CD8+ T cells (r2 = 0.53, P < 0.0001), and Th17 cells (r2 = 0.69, P < 0.0001) (Figure 3D). No correlation with Treg cell frequency was found. The level of IL-21 measured in culture supernatants was also positively correlated with the levels of IFNγ (r2 = 0.40, P = 0.0002), IL-12 (r2 = 0.29, P = 0.002), TNFα (r2 = 0.38, P = 0.0003), and IL-17A (r2 = 0.77, P < 0.0001). (Data are available from the author upon request.) In contrast, IL-21 levels were not correlated with IL-6 levels in culture supernatants (r2 = 0.08, P = 0.13).

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Figure 3. IL-21 is produced by CD4+ T cells and correlates with Th1 and Th17 responses. Peripheral blood mononuclear cells from 14 patients with active GCA, 16 patients with GCA in remission, and 20 age-matched controls were stimulated for 4 hours with phorbol myristate acetate and ionomycin. A, Representative dot plots of IL-21–producing CD4+ T cells, after gating on CD3+ T cells. Numbers are the percentages of cells in each subpopulation. B, Frequencies of IL-21–producing CD4+ T cells. A marked enrichment in IL-21–producing CD4+ T cells was noted in patients with active GCA compared to patients with GCA in remission and age-matched controls. Circles indicate individual samples; horizontal lines indicate the mean. ∗∗∗ = P < 0.001. C and D, Correlations between IL-21–producing CD4+ T cells and Th1 cells (C) and between IL-21–producing CD4+ T cells and Th17 cells (D) in patients with GCA. IL-21–producing CD4+ T cells were positively correlated with Th1 and Th17 cells. See Figure 1 for definitions.

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Increased T cell activation in peripheral blood from GCA patients, determined by phenotypic analysis.

To identify perturbations in T cell homeostasis, frequencies of naive T cells (CD45RA+CD62L+), central memory T cells (CD45RA−CD62L+), effector memory T cells (CD45RA−CD62L−), and terminally differentiated effector memory T cells (CD45RA+CD62L−CD27−CD28−) were compared among CD4+ or CD8+ T cell subsets from patients with active GCA, patients with GCA in remission, and age-matched controls. Among CD4+ T cells, the mean percentage of central memory T cells was decreased in patients with active GCA (24.0%) and patients with GCA in remission (18.2%) compared to controls (42.0%) (P = 0.0005 and P < 0.0001, respectively), while the mean percentages of effector memory T cells and terminally differentiated effector memory T cells were increased in patients with active GCA and patients with GCA in remission compared to controls (effector memory T cells 29.7% in patients with active GCA and 42.7% in patients with GCA in remission versus 24.5% in controls [P = 0.30 and P < 0.0001, respectively]; terminally differentiated effector memory T cells 12.7% in patients with active GCA and 17.0% in patients with GCA in remission versus 2.6% in controls [P = 0.0004 and P < 0.0001, respectively]).

Among CD8+ T cells, the mean percentage of naive T cells was increased in patients with active GCA (26.3%) compared to patients with GCA in remission (10.9%) and controls (17.0%) (P = 0.005 and P = 0.06, respectively). The mean percentage of central memory T cells was decreased in patients with active GCA (12.2%) and patients with GCA in remission (4.6%) compared to controls (17.8%) (P = 0.14 and P = 0.0001, respectively), and the mean percentage of effector memory T cells was decreased in patients with active GCA (23.8%) compared to patients with GCA in remission (29.5%) and controls (33.3%) (P = 0.03 and P = 0.21, respectively). The mean percentage of terminally differentiated effector memory T cells was increased in patients with GCA in remission (56.9%) compared to patients with active GCA (37.7%) and controls (33.0%) (P = 0.004 and P = 0.0008, respectively). No difference was found in B cell or natural killer cell percentages between groups.

To further explore T cell activation, we compared the expression of HLA–DR on T cells. The mean percentage of HLA–DR+CD4+ T cells was increased in patients with active GCA (31.4%) compared to patients with GCA in remission (14.0%) and controls (8.8%) (P = 0.002 and P < 0.0001, respectively), and the mean percentage of HLA–DR+CD8+ T cells was increased in patients with active GCA (64.8%) compared to patients with GCA in remission (49.2%) and controls (15.3%) (P = 0.01 and P < 0.0001, respectively). The expansion of IL-21–producing CD4+ T cells was positively correlated with HLA–DR expression on CD4+ T cells (r2 = 0.35, P = 0.0006) and CD8+ T cells (r2 = 0.19, P = 0.02).

Expression of IFNγ, IL-17A, and IL-21 within inflammatory infiltrates in temporal artery samples from patients with vasculitis.

Immunohistochemical analysis of paraffin-embedded temporal artery specimens from 3 patients with GCA and 3 controls was used to investigate the pattern of expression of IFNγ, IL-17A, IL-21, IL-6, CCL20 (known to be a potent chemoattractant of Th17 cells), and FoxP3. Inflammatory infiltrates from GCA patients expressed IFNγ, IL-17A, IL-21, IL-6, and CCL20, as compared to noninflammatory temporal artery samples. In contrast, no FoxP3 or CXCL8 expression was detected in any of the specimens from the GCA patients or controls (Figure 4).

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Figure 4. Cytokine and chemokine expression in temporal artery specimens from GCA patients. Immunohistochemical analysis of IFNγ, IL-17A, IL-21, IL-6, CCL20, FoxP3, and CXCL8 expression in noninflammatory temporal artery specimens from age-matched controls and in inflammatory infiltrates from GCA patients was performed. IFNγ, IL-17A, IL-21, IL-6, and CCL20 were expressed within inflammatory infiltrates from GCA patients. Original magnification × 270; original magnification in insets × 540. See Figure 1 for definitions.

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Critical role of IL-21 in modulating Th17 differentiation and FoxP3 expression in GCA patients.

We stimulated purified CD4+ T cells for 5 days with anti-CD3/CD28 with or without rhIL-21. The addition of rhIL-21 increased the mean proportion of Th1 cell frequencies (from 8.0% to 11.7% in control samples [P = 0.003] and from 15.4% to 19.3% in GCA patient samples [P = 0.02]) and Th17 cell frequencies (from 0.9% to 2.0% in control samples [P = 0.04] and from 2.0% to 2.3% in GCA patient samples [P = 0.01]), and decreased FoxP3 expression (from 3.1% to 1.1% in control samples [P = 0.01] and from 2.9% to 1.7% in GCA patient samples [P = 0.05]) (Figure 5).

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Figure 5. IL-21 modulates Th1 and Th17 responses and FoxP3 expression in GCA. Purified CD4+ T cells from controls (open bars) and GCA patients (solid bars) were stimulated with anti-CD3/CD28 alone or with anti-CD3/CD28 and IL-21. A, Dot plots of FoxP3+CD25high Treg cells. Numbers are the percentage of FoxP3+CD25high Treg cells. B–D, Frequencies of FoxP3+CD25high Treg cells (B), IL-17A–producing cells (C), and IFNγ-producing cells (D) in purified CD4+ T cells from controls and GCA patients stimulated as indicated. Bars show the mean ± SEM. ∗ = P < 0.05; ∗∗ = P < 0.01. See Figure 1 for definitions.

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In order to more precisely determine whether the increase in the proportion of Th1 and Th17 cells and the decrease in the expression of FoxP3 after the addition of rhIL-21 was related to increased proliferation or to increased differentiation of naive CD4+CD25− T cells into effector Th1 and Th17 cells rather than into Treg cells, we monitored the proliferation of CD4+ T cells using a 5,6-carboxyfluorescein succinimidyl ester (CFSE) proliferation assay in association with IFNγ, IL-17A, and FoxP3 intracellular staining in samples from GCA patients (n = 5). We observed that the addition of rhIL-21 was not associated with either an increased proliferation of Th1 cells (mean 37.2% CFSElow Th1 cells in the absence of rhIL-21 versus 39.1% CFSElow Th1 cells in the presence of rhIL-21; P = 0.51) or a decreased proliferation of Treg cells (mean 73.6% CFSElow Treg cells in the absence of rhIL-21 versus 73.1% CFSElow Treg cells in the presence of rhIL-21; P = 0.51). In contrast, we found a slight increase in Th17 cell proliferation in GCA patient samples after the addition of rhIL-21 (mean 48.5% CFSElow Th17 cells in the absence of rhIL-21 versus 53.0% CFSElow Th17 cells in the presence of rhIL-21; P = 0.008).

Last, we analyzed the effect of IL-21 blockade using IL-21R-Fc chimera on Th1, Th17, and Treg cells in samples from GCA patients after 2 days of culture. The addition of IL-21R-Fc decreased the mean proportion of IFNγ-producing CD4+ T cells (Th1 cells) (17.0% with anti-CD3/CD28 alone; 21.2% with anti-CD3/CD28 plus rhIL-21; and 13.3% with anti-CD3/CD28 plus IL-21R-Fc) and IL-17A–producing CD4+ T cells (Th17 cells) (2.6% with anti-CD3/CD28 alone; 3.5% with anti-CD3/CD28 plus rhIL-21; and 1.7% with anti-CD3/CD28 plus IL-21R-Fc) and increased the mean proportion of FoxP3+ Treg cells (3.7% with anti-CD3/CD28 alone; 2.8% with anti-CD3/CD28 plus rhIL-21; and 5.8% with anti-CD3/CD28 plus IL-21R-Fc) (Figure 6).

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Figure 6. Blockade of IL-21 with IL-21R-Fc restores effector T cell and Treg cell homeostasis. Purified CD4+ T cells from GCA patients (n = 3) were stimulated with anti-CD3/CD28 alone, anti-CD3/CD28 and recombinant human IL-21 (rHuIL-21), or anti-CD3/CD28 and IL-21R-Fc. A and B, Dot plots of FoxP3 expression (A) and IL-17A and IFNγ production (B). Numbers are the percentages of cells in each subpopulation. C, IL-17A production, IFNγ production, and FoxP3 expression in CD4+ T cells from GCA patients stimulated as indicated. Bars show the mean ± SEM. See Figure 1 for other definitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

In the present study, we aimed to identify cytokines that could act upstream of and drive the predominant Th1 and Th17 responses recently observed in GCA patients (6), and that could represent a potential target for novel therapy. To address this question, we used a systematic and unsupervised multiparametric analysis in order to identify a cytokine signature associated with disease activity and analyzed peripheral blood and inflammatory infiltrates from GCA patients.

Our systematic large-scale assessment of cytokines produced by PBMCs was a novel approach to evaluate the association between a large set of 27 cytokines and chemokines, including Th1-, Th2-, and Th17-related cytokines and other proinflammatory cytokines and chemokines, and disease activity. We first identified an immunologic signature that discriminates patients with active GCA from patients with inactive GCA. This signature involved Th1 and Th17 cytokines, indicating the importance of both pathways in the pathogenesis of the disease and the possibility that targeting these effector cells may aid in the management of GCA. It also suggests that monitoring these cytokines could help in assessing disease activity in GCA patients. We confirmed our findings using fluorescence-activated cell sorting and immunohistochemical analyses, which demonstrated the expansion of Th1 and Th17 cells in peripheral blood and the expression of IFNγ and IL-17A within vasculitic inflammatory infiltrates from GCA patients. These results clearly confirm the recent findings of Deng et al, who demonstrated the role of these two pathogenic Th1 and Th17 pathways (6). However, despite the implication of both pathways, Deng et al showed that Th17 cells were sensitive to corticosteroid-mediated suppression, whereas Th1 responses persisted in treated patients (6), suggesting that either both of these pathways or the cytokines that may drive upstream Th1 and Th17 responses could be targeted for treatment.

We next assessed the frequency of Treg cells in GCA patients and analyzed the expression of CD45RA and CD25 among CD4+ T cells in order to better delineate CD4+ T cells expressing FoxP3 (9). We observed a decrease in natural Treg cells in GCA patients that included both resting and activated memory Treg cells, while cytokine-secreting nonsuppressive T cells were increased in patients with active GCA. These findings are in contrast with the findings of Deng et al, who did not observe a decrease in Treg cells (6). However, in their study, Deng et al defined Treg cells as CD4+FoxP3+ cells, whereas it is has now been clearly demonstrated that CD4+FoxP3+ cells are composed of 3 phenotypically and functionally distinct subpopulations: CD45RA+CD25++FoxP3low resting Treg cells and CD45RA−CD25+++FoxP3high activated memory Treg cells, both of which were suppressive in vitro, and cytokine-secreting CD45RA−CD25++FoxP3low nonsuppressive T cells containing cells with Th17 cell potential (9).

The systematic assessment of cytokines undertaken in the present study, combined with previously obtained data on cytokines involved in Th1 and/or Th17 differentiation and Treg cell homeostasis, allowed us to identify IL-21 as a candidate cytokine that could modulate both Th1 and Th17 responses and Treg cells. We showed that expansion of IL-21–producing CD4+ T cells correlated positively with Th1 and Th17 cell expansion and with T cell activation. We also observed that IL-21 was critical in modulating Th1 and Th17 differentiation and FoxP3 expression. The addition of IL-21 to stimulated purified CD4+ T cells slightly increased Th1, and more importantly Th17, cell frequencies and decreased FoxP3 expression. The blockade of the IL-21 pathway with IL-21R-Fc restored the balance between Th1 and Th17 cells and Treg cells, by suppressing IFNγ and IL-17A production and increasing FoxP3 expression by CD4+ T cells.

IL-21, the most recently identified member of the type 1 cytokine family (10), is produced by activated CD4+ T cells but targets a much broader range of cells (11). IL-21 was shown to be induced by IL-6 in activated T cells, and to potently induce Th17 differentiation and suppress FoxP3 expression. Thus, it was considered to be an autocrine cytokine that is sufficient and necessary for Th17 differentiation (12). IL-21 was also shown to suppress conversion of resting Treg cells into activated memory Treg cells (12–14), supporting the notion that it has a deleterious effect on Treg cell homeostasis. Besides its role in the balance between Treg cells and Th17 cells, the role of IL-21 in promoting Th1 responses was recently reported in celiac disease, an intestinal inflammatory disease characterized by marked infiltration of the intestinal mucosa with Th1 cells secreting IFNγ and expressing the Th1-associated transcription factor T-bet. In this model, enhanced IL-21 messenger RNA and protein expression was seen in duodenal samples from patients with celiac disease, and blockade of IL-21 activity in a culture of biopsy samples obtained from patients with celiac disease reduced the expression of the Th1-related transcription factor T-bet and IFNγ secretion (15).

In order to more precisely define the role of IL-21 in the increase in Th1 and Th17 cells and the decrease in Treg cells, we monitored the proliferation of CD4+ T cells using a CFSE proliferation assay. We observed that IL-21 did not affect the proliferation of Th1 cells and Treg cells, while it slightly increased the proliferation of Th17 cells. These findings suggest that naive T cells could preferentially differentiate into Th17 cells rather than into Treg cells through IL-21 stimulation, but also that IL-21 could increase the proliferation of Th17 cells. Both of these hypotheses could account for the increased proportion of Th17 cells observed in GCA. Consistent with the findings of the present study, it was previously hypothesized that in inflammatory conditions naive CD4+CD25− T cells could differentiate into effector Th17 cells rather than into Treg cells, and that IL-21 could accomplish this function (16).

Finally, a role of IL-21 in large-vessel vasculitis was previously suggested from studies of mice deficient in IRF-4 binding protein, which rapidly developed a vasculitis sharing similarities with GCA and showed inappropriate IL-21 synthesis in addition to increased IL-17A production (5). We have also recently demonstrated a role of IL-21 in Behçet's disease, another model of systemic vasculitis, in driving inflammatory lesions (17), suggesting that IL-21 could be a key cytokine in different models of large-vessel vasculitis. Taken together, these data support the notion that IL-21 is a key cytokine in the pathogenesis of GCA and the potential benefit of using IL-21 blockade as a therapy in patients who are receiving corticosteroids and/or experience relapses. However, our findings that GCA patients treated with corticosteroids did not show increased production of IL-21 by CD4+ T cells, while the central memory CD4+ T cell count was similar between patients with active disease and those with inactive disease, also suggest that corticosteroids might suppress the production of IL-21 by CD4+ T cells, possibly through a decrease in IL-6 production.

In conclusion, our study is the first to demonstrate a function of IL-21 in the pathogenesis of GCA. IL-21 modulates Th1 and Th17 differentiation and FoxP3 expression and is correlated with disease activity. Our findings suggest that IL-21 plays a critical role in GCA pathogenesis and might represent a promising target for novel therapy.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Cacoub had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Terrier, Geri, Saadoun, Cacoub.

Acquisition of data. Terrier, Geri, Chaara, Allenbach, Rosenzwajg, Costedoat-Chalumeau, Fouret, Musset, Benveniste, Six, Klatzmann, Saadoun, Cacoub.

Analysis and interpretation of data. Terrier, Geri, Chaara, Allenbach, Rosenzwajg, Costedoat-Chalumeau, Fouret, Musset, Benveniste, Six, Klatzmann, Saadoun, Cacoub.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Nathalie Ferry, Véronique Bon-Durand, and Cornélia Degbé for technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES