A polymorphism in the interleukin-4 receptor affects the ability of interleukin-4 to regulate Th17 cells: a possible immunoregulatory mechanism for genetic control of the severity of rheumatoid arthritis

Twenty patients with established RA and 26 healthy individuals were enrolled in the study. The average age of the healthy individuals was 40.6 years (range, 21 to 62 years), and this group included 12 females and 14 males. The characteristics of the RA patients are summarized in Table S1 (Additional file 1). Health assessment questionnaires were completed by each patient, and disease activity scores were calculated on the basis of a 28-joint count and a visual analogue scale. Thirty milliliters of blood were collected from each subject. Twenty milliliters were saved for cell culture, 5 ml were saved for DNA isolation and genotyping and 5 ml were saved for serum. All study participants provided written informed consent. The research protocol was approved by the University of Michigan Institutional Review Board.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral whole blood of RA patients and healthy controls by gradient centrifugation over Histopaque-1077 (Sigma, St. Louis, MO, USA). Cell cultures were performed in RPMI 1066 medium (Lonza, Basel, Switzerland) with 10% fetal bovine serum, 1% penicillin G/1% streptomycin and 2% L-glutamine. PBMCs were activated with Orthoclone OKT3 (anti-CD3, produced in the University of Michigan Hybridoma Core) 1 μg/ml and either Th17-stimulating conditions alone (IL-23, 10 ng/ml; IL-1β, 5 ng/ml; IL-6, 10 ng/ml) or Th17-stimulating conditions with the addition of IL-4 (50 ng/ml). Cells were left in culture for 96 hours. Supernatants were collected from each culture condition and stored at -80°C for analysis by ELISA.

On day 5 of culture, the cells were restimulated with phorbol myristate acetate (5 ng/ml) and ionomycin (500 ng/ml) for 1 hour prior to addition of brefeldin A (10 μg/ml) for 5 more hours. The cells were washed and 1 × 10⁶ cells per sample were used for staining. Cells were first blocked with 20 μl of 10% human serum/10% mouse serum in PBS at 4°C for 15 minutes. The cells were surface-stained with antigen-presenting cell (APC)-labeled mouse anti-human CD4 (BD Bioscience (Palo Alto, CA, USA) or APC-conjugated mouse immunoglobulin G₁ (mIgG₁) isotype control (Ebioscience, San Diego, CA, USA), at 4°C for 30 minutes, washed twice with cold 2% newborn calf serum/phosphate-buffered saline (NCS/PBS) buffer and fixed overnight in 4% paraformaldehyde. The cells were then permeabilized with 0.5% saponin in 2% NCS/PBS. Intracellular cytokine staining was performed using fluorescein isothiocyanate (FITC)-labeled anti-human IFN-γ (BD Bioscience) and phycoerythrin (PE)-labeled anti-human IL-17A (Ebioscience), or FITC-conjugated mIgG₁ isotype control (Ebioscience) and PE-conjugated mouse IgG₁ isotype control (Ancell, Bayport, MN, USA). Samples were run on a BD Biosciences FACS Calibur flow cytometer and analyzed by CellQuest Pro (BD Bioscience).

Both culture supernatants and fresh sera were analyzed by ELISA for IL-17A levels. Flat-bottomed, high binding, 96-well plates (Corning Costar, Lowell, MA, USA) were coated overnight at 4°C with anti-human IL-17-purified antibody (Ebioscience) diluted to 1:500 with 0.1 M carbonate buffer, pH 9.4. On day 2, the plates were washed three times with 1 × PBS/0.05% Tween at 200 μl per well and blocked using 200 μl of PBS with 10% fetal calf serum per well for 2 hours. The plates were then washed three times with 200 μl of 1 × PBS/0.05% Tween per well. The standard curve was created in duplicate starting with a concentration of 2,000 pg/ml and serial twofold dilutions to 7.8 pg/ml. Supernatants and sera were assayed in triplicate at 100 μl per well, both undiluted and at a 1:5 dilution. The samples were then refrigerated at 4°C overnight, after which they were washed five times with 200 μl of 1 × PBS/0.05% Tween per well. A secondary biotinylated anti-IL-17 antibody and the detection reagent streptavidin horseradish peroxidase were added to each well and incubated at room temperature for 2 hours. The plates were washed seven times with 1 × PBS/0.05% Tween with 1-minute soaks between washes. Tetramethylbenzidine 100 μl were added to each well, and plates were kept in the dark at room temperature for 10 to 30 minutes. Stop solution, 2 N H₂SO₄, was added to each well. ELISA plates were read by a Synergy HT plate reader (Biotek, Winsooki, VT, USA) and analyzed by KC4 software (Biotek).

The data were analyzed with GraphPad Prism version 4.02 software (GraphPad Software Inc., San Diego, CA, USA). Paired comparisons were performed using a two-tailed t-test. Values of P ≤ 0.05 were considered significant. Dot plots were generated in CellQuest Pro.

We measured IL-17 secretion by ELISA of lymphocyte culture supernatants. In the healthy individuals there was a significant increase in the IL-17 level after the addition of Th17-stimulatory cytokines over baseline T cell stimulation with anti-CD3 (P < 0.01), and there was a significant decrease in the measured IL-17 level with the addition of IL-4 to the Th17-stimulatory conditions (Figure 1).

We then further examined these groups by specific genotype. In the I/I genotype group, addition of IL-4 led to a significant reduction in IL-17 production by cells that had been stimulated under Th17 conditions (P < 0.01). There was also a significant reduction in IL-17 production after the addition of IL-4 to cells from the I/V genotype group (P < 0.05). However, IL-4 was unable to significantly reduce IL-17 production in cell cultures from the V/V genotype group when the data were analyzed using paired comparisons (Figure 2A and 2B).

Of the 20 RA patients (85% women and 15% men), 4 were homozygous for isoleucine, 6 were heterozygous and 10 were homozygous for valine at amino acid 50 of the IL-4R (Table S1 in Additional file 1). The mean disease activity score (DAS) for the patients with an I/I genotype was 3.1, representing low disease activity. The mean DAS for the patients with the I/V genotype was 3.9, or moderate disease activity, and for the patients with the V/V genotype the mean DAS was 4.2, or high to moderate disease activity. The differences between these groups were not statistically significant, but suggest a trend toward association of the V allele with more active disease, notwithstanding the aggressive treatment that these patients were receiving.

There was not a significant increase in IL-17 production in RA patients in Th17-skewing conditions versus culture with anti-CD3 alone (P = 0.13) (Figure S1 in Additional file 1). IL-4 did suppress IL-17 production in vitro, albeit not to the extent seen in healthy controls. Comparing the RA groups, the extent of suppression of IL-17 production by IL-4 was intermediate and appeared to be similar among all genotype groups (Figure S2 in Additional file 1).

We also performed intracellular staining of cultured cells for both IL-17 and IFN-γ and examined the samples by flow cytometry. A set of representative flow cytometry histograms is shown in Figure 3 for each of the healthy control group genotypes. There was a more pronounced suppression of the percentage of IL-17⁺ cells in the I/I genotype culture, as shown in the top row of Figure 3A, compared to the suppression of IL-17⁺ cells in the V/V genotype culture, shown in the bottom row. In these cultures, the majority of IL-17⁺ cells were CD4⁺, but some CD4-IL-17⁺ cells were also observed. IL-4 likewise affected the expression of IL-17 by these CD4^- cells. Flow cytometry of cultured PBMCs activated under Th17 conditions showed that RA patients generated a higher percentage of IL-17⁺ and IL-17⁺/IFNγ⁺ (Th1/Th17) cells compared to controls (Figure 3B). A large proportion of the Th17 cells in both healthy individuals and patients with RA are of dual Th17/Th1 lineage. IL-4 generally reduced the number of IL-17⁺/IFNγ⁺ cells in parallel with reductions in the number of IL-17⁺/IFNγ^- cells (data not shown).

Consistent with in vitro generation of higher numbers of Th17 cells from RA mononuclear cells, we also observed higher serum IL-17 levels in the RA patients compared to the healthy individuals (P = 0.05) (Figure 4). These results, as well as the flow cytometry data summarized in Figure 3, are consistent with a recent report that documents expansion of the Th17 subset in RA patients compared to healthy individuals [24].