Abstract
An aberrant mitogen-induced polarization of peripheral blood T cells has been associated with type 1 diabetes (T1D). We studied, in T1D, type 1 and 2 cytokine-induced expression of the interleukin-12 receptor β2 chain (IL-12Rβ2 chain), which plays a critical role in regulating T-cell polarization. Peripheral blood lymphocytes from children with newly diagnosed T1D (n = 10; mean age 10 years), from children with longstanding T1D (n = 8; mean age 12·9 years) and from healthy children (n = 15; mean age 11·5 years) were stimulated with phytohaemagglutinin (PHA) in a type 1 (IL-12 and anti-IL-4) or a type 2 (IL-4 and anti-IL-12) cytokine environment. Secretion of interferon-γ (IFN-γ), IL-5 and IL-13, as detected by enzyme-linked immunosorbent assay (ELISA), and expression of the IL-12Rβ2 chain on CD4 and CD8 cells by flow cytometry, were analysed. Children with newly diagnosed and longstanding T1D had lower expression levels of the IL-12Rβ2 chain on IL-12Rβ2 chain-positive CD4 T cells (for a type 1 or a type 2 cytokine environment: P = 0·01 and P = 0·002 or P = 0·02 and P = 0·01, respectively) and on IL-12Rβ2 chain-positive CD8 T cells (for a type 1 or a type 2 cytokine environment: P = 0·007 and P = 0·0007 or P = 0·003 and P = 0·01, respectively) when compared to healthy children. A decreased percentage of IL-12Rβ2 chain-expressing CD4 T cells (P = 0·07 and P = 0·03) and CD8 T cells (P = 0·004 and P = 0·01) and increased secretion of IL-13 (P = 0·006 and P = 0·04) in a type 1 cytokine environment was seen in both groups of patients. Peripheral blood T cells from patients with both newly diagnosed and longstanding T1D showed poor polarization towards type 1 cells.
Keywords: IL-12R, IL-13, T cells, type 1 diabetes
Introduction
Type 1 diabetes (T1D) is an autoimmune disease caused by an immune-mediated destruction of the insulin-producing β cells in pancreas. The development of the destructive autoimmune response is believed to be facilitated by an aberrant polarization of the T cells. It has been reported that T helper 1 (Th1) cells and their cytokines, which support the function of cytotoxic T cells, are inductive for the autoimmune destruction of β cells in T1D. T helper 2 (Th2) cells and their cytokines have been considered to be protective.1
Antigen-specific CD4 T helper cells can be divided into two groups of cells expressing different cytokine profiles and effector functions, namely Th1 and Th2 cells. Th1 cells produce interferon-γ (IFN-γ) and interleukin (IL)-2, which are involved in cell-mediated immune responses. Th2 cells produce IL-4, IL-5 and IL-13, and support the humoral immune responses. Th1 and Th2 cells also reciprocally regulate the function of each other through their respective cytokines.2
In patients newly diagnosed with T1D, an enhanced production of type 1 cytokines – IFN-γ and tumour necrosis factor-α (TNF-α)3 – and a low production of type 2 cytokines – IL-44 – in mitogen-stimulated peripheral blood mononuclear cells (PBMC) have been reported. However, recent studies have questioned this paradigm of type 1 immune deviation in circulating lymphocytes. It has been shown that PBMC from patients with T1D have a decreased secretion of IFN-γ and IL-10 in response to stimulation with phytohaemagglutinin (PHA).5 It has also been shown that patients newly diagnosed with T1D have a low spontaneous and mitogen-stimulated secretion of IFN-γ by PBMC.6,7 Recently, Lohmann et al. reported a reduced expression of Th1-associated chemokine receptors combined with a decreased IFN-γ response on peripheral blood lymphocytes at the diagnosis of T1D, but this phenomenon was not observed in patients with a longer disease duration.8
The differentiation of naive T cells towards either Th1 or Th2 cells is influenced by the surrounding cytokine environment in which the T-cell activation occurs. The production of IL-12 by activated macrophages and dendritic cells plays a critical role in regulating the polarization of T cells by inducing Th1 responses.9 Differentiation of naive T cells into Th2 cells is induced by IL-4 and suppressed by IFN-γ.10 CD8 T-cytotoxic cells have also been reported to differentiate into T-cytotoxic type 1 (Tc1) and T-cytotoxic type 2 (Tc2) cells in a similar manner as for T helper cells.11
The activities of IL-12 are mediated through a high-affinity IL-12 receptor (IL-12R), which plays a critical role in regulating the T-cell polarization. This receptor is primarily expressed on activated T and natural killer (NK) cells and consists of two subunits: β1 and β2.12 The β1 chain is expressed by both Th1/Tc1 and Th2/Tc2 cells, whereas the β2 subunit, which has been reported to be the signal-transducing component, is selectively expressed on Th1 and Tc1 cells.13,14 The IL-12Rβ2 chain is not expressed on naïve/resting T cells.14
We stimulated peripheral blood T cells by mitogen in a type 1 or a type 2 cytokine environment and studied the T-cell polarization in children with T1D and in healthy children by comparing the expression of the IL-12Rβ2 chain and the amounts of cytokines secreted from the T cells.
Materials and methods
Subjects
PBMC were isolated from freshly collected, heparinized venous blood samples from children with newly diagnosed T1D (< 3 months after diagnosis) (n = 10, mean age 10 years; range: 6–14 years), from children with longstanding T1D (> 18 months after diagnosis) (n = 8, mean age 12·9 years; range: 6–18 years) and from healthy children (n = 15, mean age 11·5 years; range: 8–12 years). The diagnosis of T1D was based on World Health Organization criteria (1999).
Cell culture
The cells were separated by gradient centrifugation in Cell Preparation Tubes (Becton-Dickinson, Franklin Lakes, NJ) at 1500 g for 20 min. The cells were washed three times with RPMI-1640 (Invitrogen, Paisley, Scotland, UK) supplemented with 5% inactivated human AB+ serum (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) and were then diluted to 1 × 106 cells/ml in RPMI-1640 supplemented with inactivated human AB+ serum (5%), l-glutamine (2 mmol/l; Invitrogen) and gentamicin (25 µg/ml; Sigma-Aldrich Inc., St Louis, MO). The monocytes were depleted from the cell suspension by using plastic adherence (1 hr at 37° in 5% CO2). The non-adherent lymphocytes (2 × 106 cells/well) were cultured at 37° in 5% CO2. The lymphocytes were stimulated on day 0 with either PHA (5 µg/ml; Difco Laboratories, Detroit, MI), IL-12 (200 pg/ml) and anti-IL-4 (50 ng/ml) from BD Pharmingen (San Diego, CA) for generating a Th1/Tc1-line, or with PHA (5 µg/ml), IL-4 (0·4 ng/ml; Sigma-Aldrich Inc.) and anti-IL-12 (1 µg/ml; BD PharMingen) for generating a Th2/Tc2-line. On day 2, the T-lymphocyte lines were expanded by the addition of IL-2 (32 pg/ml; BD PharMingen). On day 4, half of the medium was replaced and the cytokines (IL-12 or IL-4), antibodies (anti-IL-4 or anti-IL-12) and IL-2 were added again. On day 4, supernatants from the T-lymphocyte cultures were collected for further analysis of cytokine profiles.
Flow cytometry
After 6 days of culture, the lymphocytes were collected for fluoresence-activated cell sorter (FACS) analysis. The lymphocytes were stained with 5 µl/300 000 cells of anti-fluorescein isothiocyanate (FITC)-CD4, anti-FITC-CD8, anti-phycoerythrin (PE)/IL-12Rβ2, as well as isotype-matched controls (BD PharMingen), for 30 min. After washing with phosphate-buffered saline (PBS) (Medicago AB, Uppsala, Sweden) supplemented with 0·5% bovine serum albumin (BSA) (Difco Laboratories, Detroit, MI), the cells were resuspended in 160 µl of PBS supplemented with 0·5% BSA. The labelled cells were analysed with three-colour flow cytometry using a FACSCalibur and cellquest software (Becton-Dickinson, San José, CA).
The lymphocytes were gated on forward- and side-scatter. A total of 15 000 cells were acquired, and two-parameter dot-plots were created. For isotype controls, 5000 cells were acquired. Compensation was performed to adjust for spectrally adjacent dye pairs. The quadrants in the dot-plots were placed according to the staining of the unstained cells and isotype controls for each sample. The number of CD3+ cells was > 95% within the gate.
Enzyme-linked immunosorbent assay (ELISA) for IFN-γ
Maxisorb plates (Nunc, Roskilde, Denmark) were coated with monoclonal anti-human IFN-γ (M-700 A; Endogen, Woburn, MA) at a concentration of 2 µg/ml (50 µl/well). After overnight incubation at +4°, plates were washed with PBS-Tween and then blocked in PBS containing 1% BSA for 30 min. A standard curve was prepared by using recombinant human IFN-γ (19751 N; Pharmingen, San Diego, CA). Supernatant samples (100 µl/well) and standards were incubated for 2 hr. After washing with PBS-Tween, biotinylated monoclonal anti-human IFN-γ (M-701; Endogen) was added at a concentration of 0·5 µg/ml (50 µl/well), and the plates were incubated for 90 min. Streptavidin–alkaline phosphatase complex (Zymed, San Francisco, CA) was added and p-nitrophenyl phosphate (Medix, Kauniainen, Finland) was used to develop the colour for reading at 405 nm. The detection level of the assay was 50 pg/ml.
ELISA for IL-13
The Pelikine Compact human IL-13 ELISA kit (CLB, Amsterdam, the Netherlands) was used for IL-13 analysis, according to the manufacturer's instructions.
ELISA for IL-5
ELISA for IL-5 (Pharmingen) was performed according to the protocol used in the IFN-γ ELISA. The antibodies (and concentrations) used in this assay were purified rat monoclonal anti-human IL-5 (1 µg/ml; 18051D) and biotinylated rat monoclonal anti-human IL-5 (0·5 µg/ml; 18522D). Dilutions of recombinant human IL-5 (19651V) were used to create a standard curve. The detection level of the assay was 30 pg/ml.
Statistical analysis
Differences between the groups were analysed by the Mann–Whitney U-test and the Kruskal–Wallis test using graphpad software (GraphPad Software Inc., San Diego, CA). P-values below 0·05 were considered significant, and P-values below 0·1 were considered a trend. No correction has been made for the comparisons.
Ethical considerations
Informed consent was obtained from all participants, and the study was approved by the Regional Ethics Committee for Human Research at the Faculty of Health Sciences, Linköping University, Sweden.
Results
The generation of type 1 and type 2 T cells in healthy children
The number of IL-12Rβ2 chain-expressing CD4 and CD8 T cells was higher when the cells were cultured in a type 1 cytokine environment than in a type 2 cytokine environment. In healthy children the median number of IL-12Rβ2 chain-expressing CD4 or CD8 T cells was 50·25% and 55·74%, respectively, after stimulation in a type 1 cytokine environment. After stimulation in a type 2 cytokine environment, the median number of IL-12Rβ2 chain-expressing CD4 or CD8 T cells was 3·9% and 2·33%, respectively.
The median fluorescence intensity of IL-12Rβ2 chain expression on IL-12Rβ2 chain-positive T cells was also significantly higher in CD4 and CD8 T cells stimulated in a type 1 cytokine environment compared to a type 2 cytokine environment in healthy children (for CD4 T cells medians 28·64 versus 19·28, respectively), and for CD8 T cells (medians 25·71 versus 18·27, respectively).
Figure 1 illustrates examples of IL-12Rβ2 expression in CD4 T cells, when cultured in a type 1 or a type 2 cytokine environment, in a representative case of healthy children.
Figure 1.
Representative illustration of interleukin-12 receptor β2 (IL-12Rβ2) expression on CD4 T cells. A representative case is shown of the expression of IL-12Rβ2 on CD4 T cells in a type 1 and a type 2 cytokine environment (a and b, respectively) for a healthy child. The percentage values refer to IL-12Rβ2-expressing CD4 T cells.
In healthy children, a higher level of IFN-γ was secreted after PHA stimulation in a type 1 cytokine environment than in a type 2 cytokine environment, whereas secretion of IL-5 and IL-13 were higher after stimulation with PHA in a type 2 cytokine environment than in a type 1 cytokine environment (Table 1).
Table 1. Secretion of interferon-γ (IFN-γ), interleukin (IL)-13 and IL-5 from T cells cultured for 4 days in a type 1 or a type 2 cytokine environment in healthy children.
| Cytokine environment | ||
|---|---|---|
| Cytokine | Type 1 | Type 2 |
| IFN-γ | 143 078***1(6485–1 205 000) | 4403 (365–135 044) |
| IL-13 | 71***1 (16·9–281·7) | 525·2 (112–4573) |
| IL-5 | 132·1**1 (20·5–1762) | 1560 (18·2–8112) |
Results are expressed in pg/ml of cytokine as median value (range).
The P-value represents significant differences in cytokine secretion in healthy children.
P < 0·0001
P < 0·001.
Percentage of CD4 and CD8 T cells expressing the IL-12Rβ2 chain in children with newly diagnosed and longstanding T1D, and in healthy children
The number of CD4 T cells expressing the IL-12Rβ2 chain after stimulation in a type 1 cytokine environment was lower in children with newly diagnosed and longstanding T1D than in healthy children (medians 26·54, 11·25 and 50·25; P-values 0·07 and 0·03, respectively; Fig. 2a).
Figure 2.
Percentage of CD4+ and CD8+ T cells, respectively, expressing the interleukin-12 receptor β2 (IL-12Rβ2) chain, when cultured in a type 1 (a, b) or a type 2 (c, d) cytokine environment. The P-value represents significant differences in the percentage of CD4+ and CD8+ T cells expressing the IL-12Rβ2 chain in children with newly diagnosed or longstanding type 1 diabetes (T1D) and healthy children. **P < 0·001, *P < 0·05 and ns, not significant. Horizontal lines indicate median values.
The number of IL-12Rβ2 chain-expressing CD8 T cells after stimulation in a type 1 cytokine environment was lower in children with newly diagnosed T1D (P = 0·004) and longstanding T1D (P = 0·01) than in healthy children (medians 23·55, 23·45 and 55·74%, respectively, Fig. 2b).
The number of CD4 or CD8 T cells expressing the IL-12Rβ2 chain after stimulation in a type 2 cytokine environment did not differ significantly between the groups (Fig. 2c,2d).
Expression of the IL-12Rβ2 chain on IL-12Rβ2 chain-positive CD4 and CD8 T cells in children with newly diagnosed and longstanding T1D, and in healthy children
The median fluorescence intensity for the IL-12Rβ2 chain on IL-12Rβ2 chain-positive CD4 cells stimulated in a type 1 cytokine environment was significantly lower in children with newly diagnosed T1D (P = 0·01) and longstanding T1D (P = 0·002) than in healthy children (medians 18·9, 15·54 and 28·64, respectively, Fig. 3a). The IL-12Rβ2 chain-expressing CD4 T cells stimulated in a type 2 cytokine environment also expressed less IL-12Rβ2 chain in children with newly diagnosed T1D (P = 0·02) and longstanding T1D (P = 0·01) than in healthy children (medians 15·63, 15·79 and 19·28, respectively, Fig. 3c).
Figure 3.
Expression of the interleukin-12 receptor β2 (IL-12Rβ2) chain on IL-12Rβ2 chain-positive CD4+ and CD8+ T cells. Expression of the IL-12Rβ2 chain on IL-12Rβ2 chain-positive CD4+ and CD8+ T cells, respectively, cultured in a type 1 (a, b) or a type 2 (c, d) cytokine environment. The P-value represents significant differences in expression of the IL-12Rβ2 chain in CD4+ and CD8+ T cells in children with newly diagnosed or longstanding type 1 diabetes (T1D) and healthy children. ***P < 0·0001, **P < 0·001, *P < 0·05 and ns, not significant. Horizontal lines indicate median values.
The expression of the IL-12Rβ2 chain on IL-12Rβ2 chain-positive CD8 T cells was also lower after stimulation in a type 1 cytokine environment in children with newly diagnosed T1D (P = 0·007) and longstanding T1D (P = 0·0007) than in healthy children (median intensity 18·94, 16·01 and 25·71, respectively, Fig. 3b). In a type 2 cytokine environment, the IL-12Rβ2 chain-expressing CD8 T cells also had a lower expression of IL-12Rβ2 chain in children with newly diagnosed T1D (P = 0·003) and longstanding T1D (P = 0·01) than in healthy children (median intensity 14·96, 15·2 and 18·27, respectively, Fig. 3d).
IFN-γ, IL-13 and IL-5 secretion of T cells in children with newly diagnosed and longstanding T1D, and in healthy children
IL-13 secretion of T cells cultured in a type 1 cytokine environment was higher in children with newly diagnosed T1D (P = 0·006) and longstanding T1D (P = 0·04) than in healthy children (medians 194·5 pg/ml, 183·5 pg/ml and 71 pg/ml, respectively, Fig. 4a). No significant difference between the groups was seen in the IL-13 secretion of type 2 cells (Fig. 4b).
Figure 4.
Secretion of interleukin-13 (IL-13) from cultured T cells. Secretion of IL-13 from T cells cultured in a type 1 (a) or a type 2 (b) cytokine environment for 4 days. The P-value represents significant differences in IL-13 secretion of T cells in children with newly diagnosed or longstanding type 1 diabetes (T1D) and healthy children. **P < 0·001, *P < 0·05 and ns, not significant. Horizontal lines indicate median values.
After 4 days of culture there were no differences between the groups regarding secretion of IFN-γ or IL-5, in either cytokine environment.
Discussion
In comparison to healthy children, we found that children with newly diagnosed and longstanding T1D had a lower percentage of CD4 and CD8 T cells expressing the IL-12Rβ2 chain when cultured in a type 1 cytokine environment. Moreover, in comparison to healthy children, children with newly diagnosed and longstanding T1D also had a decreased expression of the IL-12Rβ2 chain on IL-12Rβ2 chain-positive CD4 and CD8 T cells stimulated with mitogen in a type 1 or type 2 cytokine environment. In addition, we observed enhanced PHA-induced secretion of IL-13 of T cells cultured in a type 1 cytokine environment in children with newly diagnosed and longstanding T1D.
Decreased expression of the IL-12Rβ2 chain, together with elevated production of IL-13 from T cells cultured in a type 1 cytokine environment, suggests an impaired polarization of T cells towards a type 1 immune response in T1D. As the secretion of IL-13, a marker of activation of type 2 cytokine response, was increased in T1D when measured on day 4 preceding the analyses of the IL-12Rβ2 chain, enhanced secretion of IL-13 in a type 1 cytokine environment could be responsible for down-regulation of the IL-12Rβ2 chain.15,16 Alternatively, our results may indicate a primary defect in the up-regulation of IL-12Rβ2 chain in T1D. Our results further showed that a low intensity of the IL-12Rβ2 chain on IL-12Rβ2 chain-expressing CD4 and CD8 T cells was even found in type 2 cytokine stimulation, which supports the view that aberrant regulation of the IL-12Rβ2 chain is connected to T1D, and may not be restricted to type 1 cytokine stimulation, but is a general defect.
Several other studies have reported differences between children with T1D and healthy children in T-cell deviation, supporting a poor type 1 response in T1D. Recently, Lohmann et al. showed a reduced expression of the Th1-associated chemokine receptors CCR5 and CXCR3, and a reduced secretion of the Th1 cytokines IFN-γ and TNF-α, in freshly isolated PBMC from children with newly diagnosed T1D, but this was reversed in most patients 6 months after diagnosis.8 The level of the Th2-associated chemokine receptor, CCR4, was reduced in children with both newly diagnosed and longstanding T1D. The secretion of IL-4 from PBMC stimulated by PHA was shown to be reduced in both newly diagnosed individuals and in patients with longstanding T1D. Reduced production of IFN-γ and IL-10 from PBMC stimulated with PHA has been reported by Mayer et al. at the diagnosis of T1D in children and young adults.5 Kukreja et al. reported that PMA and ionomycin stimulation of PBMC showed that the CD4 T cells were defective in intracellular IFN-γ in patients with both newly diagnosed and longstanding T1D.7 There were no differences in IL-4 secretion between patients with newly diagnosed or longstanding T1D and healthy controls. Also, IFN-γ-, IL-4- and transforming growth factor-β (TGF-β)-specific mRNA levels had been reported to be decreased in unstimulated PBMC from children with newly diagnosed T1D.6 Karlsson et al. reported increased numbers of PBMC that spontaneously produced IFN-γ in high-risk relatives of patients with T1D. However, those high-risk relatives who developed T1D during the follow-up, as well as the children with T1D, had a lower ratio of IFN-γ/IL-4 than the high-risk relatives who did not develop T1D.17
On the other hand, some previous studies have indicated contrasting findings of reduced IL-44 and increased IFN-γ3 in circulating lymphocytes from patients with T1D. Rapoport et al. showed that PHA stimulation of PBMC from children with longstanding T1D produced an overall higher level of IFN-γ than PBMC from healthy children.18 The IFN-γ secretion was also prolonged and associated with decreased secretion of IL-4 and IL-10 in children with longstanding T1D.
The duration of the stimulation of PBMC, choice of mitogen and the age of patients and controls studied may, at least partly, explain the discrepancies seen in cytokine secretion between the different studies. We studied here the effect of in vitro cytokine environment on deviation of circulating lymphocytes. In both healthy children and in children with T1D, the up-regulation of IL-12Rβ2 chain and secretion of IFN-γ was strongly associated with stimulation of T cells in a type 1 cytokine environment, whereas IL-5 and IL-13 secretion was more pronounced in T cells stimulated in a type 2 cytokine environment. As the mean age of patients and controls is considerably lower than in the other studies published, it is possible that poor differentiation to type 1 cells is associated with T1D diagnosed in young children. The polarization of T cells towards a type 1 response, including IL-12 and IFN-γ secretion, emerges with age.19
Our findings suggest that type 1 deviation is disturbed in patients with T1D. These results were unexpected in the light of the present view of pathogenesis of T1D and polarization of islet-infiltrating T cells. Our findings may reflect the polarization aberrancy associated with metabolic disturbances in T1D. Lohmann et al. found that reduced expression of CCR5 and IFN-γ was normalized after diagnosis.8 However, we did not see any differences between newly diagnosed patients and patients with a longer duration of T1D when the metabolic balance is usually better. Furthermore, the expression of the IL-12Rβ2 chain was not associated with the actual HbA1c levels of the patients (data not shown). In accordance with our results, Kukreja et al. also reported decreased IFN-γ secretion in both newly diagnosed patients and in patients with longstanding T1D.7
Our results are limited to the functional deviation of peripheral blood lymphocytes and do not exclude the presence of type 1 immune response locally in pancreatic islets. A regulatory defect characterized by a decreased IFN-γ response has been suggested to be associated with T1D,7 although the mechanisms of the defect have not been identified. In rheumatoid arthritis and spondyloarthropathy, a decreased IFN-γ response is seen in peripheral blood and in synovial-derived T cells, which is recovered after treatment with anti-TNF-α associated with clinical remission.20–22 Accordingly, the possibility that a decreased type 1 response of peripheral blood T cells is associated with the regulatory defect leading to clinical manifestation of autoimmune diseases should be considered in future studies.
Acknowledgments
This study was supported by grants from Swedish Child Diabetes Foundation (Barndiabetesfonden), Swedish Research Council (K2002-72X-11242-08A and K2003-72X-14690-01A), Swedish Diabetes Association and the Foundation for Strategic Research. We thank Gosia Smolinska for technical assistance and Ulla Ludvigsson for recruitment of healthy children.
References
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