Tim-3 mRNA expression in peripheral blood lymphocytes from asthmatic patients

Abstract

The study aimed to detect the expression of the Th1-specific cell surface protein T cell Ig and mucin domain-containing molecule-3 (Tim-3) mRNA in peripheral blood lymphocytes isolated from asthmatic patients and to examine the correlation among Tim-3 mRNA, interleukin-4 (IL-4), interferon-γ (IFN-γ) level, and FEV1/FVC (force expiratory volume in one second/forced vital capacity) to explore the role of Tim-3 in the development and progression of asthmatic inflammation. Tim-3 mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). The IL-4 and IFN-γ levels were determined by using enzyme-linked immunosorbent assay (ELISA). The correlation among Tim-3 mRNA, IL-4, IFN-γ level, and pulmonary ventilatory capacity was analyzed. The expression of Tim-3 mRNA in patients with acute asthma exacerbation was 0.39±0.06, significantly higher than that in patients at the remission stage and controls (0.18±0.05 and 0.07±0.03, P<0.05). The level of IL-4 in patients with acute asthma exacerbation was 68.42±10.54, significantly higher than that in the patients at the remission stage and controls (41.83±9.37 and 32.75±8.16, P<0.05). The level of IL-4 in patients in remission was significantly higher than that in controls (P<0.05). The level of IFN-γ in patients with acute asthma exacerbation was 65.74±7.85, significantly lower than that in patients in remission and the control group (120.84±11.62 and 139.65±13.47, P<0.05). The level of IFN-γ in patients in asthma remission was significantly lower than that in controls (P<0.05). Tim-3 mRNA expression was positively correlated with the level of IL-4 (r=0.68, P<0.05) and negatively with the level of IFN-γ and pulmonary ventilatory capacity (r=-0.85, r=-0.76, both P<0.01). The increased expression of Tim-3 mRNA in peripheral blood lymphocytes might be involved in the development and progression of asthmatic inflammation.

Keywords

asthma / airway inflammation / peripheral blood lymphocyte / Tim-3

Introduction

Asthma is generally believed to be a consequence of the functional disorder of T lymphocytes, which is characterized as the imbalance of Th1 and Th2 cells; i.e., the predomination of Th2 cells in the T cell population during the asthmatic airway inflammation. This may be the underlying mechanism that triggers and maintains the pathological process of asthma [1]. Because the imbalance of Th1/Th2 plays an important role in the airway inflammation, a novel strategy for the treatment of asthma that either enhances Th1 response or inhibits Th2 response has been proposed [2]. The T cell Ig and mucin domain-containing molecule-3 (Tim-3) was reported to be a transmembrane protein preferentially expressed on Th1 cells and might down-regulate Th1 responses [3]. Tim-3, however, is also involved in Th2 immune responses. Accumulating evidence suggests that treatment with anti-Tim-3 Ab in the experimental murine allergic conjunctivitis (EC) can alleviate EC by up-regulating systemic interferon-γ (IFN-γ) production [4]. In order to study the role of Tim-3 in the pathogenesis of asthma, we employed reverse transcription polymerase chain reaction (RT-PCR) to detect the expression of Tim-3 mRNA in the peripheral blood lymphocytes of patients with asthma and examine the relationship between Tim-3 expression and the concentrations of IL-4 and IFN-γ in the supernatants and pulmonary function to explore the role of the Tim-3 gene in the development and progression of asthmatic inflammation.

Data and methods

Clinical data

From September in 2007 to March in 2008, 46 asthmatic patients were enrolled from the out-patients or hospitalized patients of the Department of Respiratory Diseases, Tongji Hospital (Wuhan, China). They included 21 cases (11 males and 10 females) in asthma remission (remission group), aged 33.2±13.5 years, ranging from 18 years to 59 years old; and 23 cases of mild to moderate asthma at the acute attack phase (12 males and 11 females) (acute attack group), aged 31.7±15.4 years, with ages ranging from 17 years to 55 years old. Asthma diagnosis was established on the basis of the Guidelines to the Global Asthma Prevention and Treatment [5]. Seventeen healthy subjects acted as normal controls (control group); they were 30.7±16.5 years old, with ages ranging from 21 years to 56 years old (9 males and 8 females). All of the subjects had no smoking history.

Methods

Isolation of peripheral blood T lymphocytes

Peripheral blood mononuclear cells (PBMC) were isolated from blood by Ficoll (Dakewei Biotech Co., Ltd., China) density gradient centrifugation, which was followed by passage through a sterile nylon wool column to collect non-adherent T cells [6].

RNA extraction and RT-PCR

The total RNA was extracted from peripheral blood T cells using Trizol (Promega, USA), in accordance with the manufacturer’s instructions, and cDNA was synthesized by using Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) (Promega, USA). The PCR reaction was carried out in a 20-μL system containing 1 μL of cDNA template from the RT reaction, 0.5 μL of each primer, and 10-μL 2×PCR mix. The primer sequences for Tim-3 were 5'-CCAAATCCCAGGCATAAT-3' (sense) and 5'-AAGCGACAACCCAAAGGT-3' (antisense), generating a fragment of 558 bp. Used as a control for RNA input, the primer sequences for β-actin were 5'-CCTGGACTTCGAGCAAGA-3' (sense) and 5'-GGGCAGTGATCTCCTTCT-3' (antisense), producing a fragment of 299 bp. Amplification was performed in the linear range under the following conditions: initial denaturation at 95°C, 5 min; denaturation at 95°C, 1 min; annealing at 56°C for Tim-3 and β-actin, 40 s; extension at 72°C, 1 min; and final extension at 72°C, 10 min. The RT-PCR products were analyzed by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light.

Cytokine measurement

The IFN-γ and IL-4 concentrations in the peripheral blood lymphocyte culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions (Dakewei Biotech Co., Ltd., China).

Pulmonary function testing

Routine spirometry, including the measurement of forced vital capacity (FVC) and force expiratory volume in one second (FEV1)/FVC%, was performed using pulmonary function meter.

Statistical analysis

The data were presented as \overline{x}±s. Comparisons were made using One-Way ANOVA. The correlations between variables were evaluated using Pearson’s correlation test for data with a normal distribution. Analyses were performed by employing the SPSS11.0 software package.

Results

Pulmonary ventilation function

The FEV1 and FEV1/FVC% in the acute attack group were lower than those in the remission group and control group (P<0.05), and those in the remission group were also lower than in the control group (P<0.05) (Table 1).

The Tim-3 mRNA expression in peripheral blood T lymphocytes

The Tim-3 mRNA expression in peripheral blood T lymphocytes in the acute attack group was higher than that in the remission group and control group (P<0.05), and the Tim-3 mRNA in the remission group was also higher than in the control group (P<0.05) (Table 1, Fig. 1).

IL-4 and IFN-γ concentrations in the supernatant of peripheral blood T lymphocytes

The IL-4 concentration in the supernatant of peripheral blood T lymphocytes in the acute attack group was higher than that in the remission group and control group (P<0.05), and those in the remission group were also higher than those in the control group (P<0.05) (Table 2).

The IFN-γ concentration in the supernatant of peripheral blood T lymphocytes in the acute attack group was lower than that in the remission group and control group (P<0.05), and that in the remission group were also lower than that in the control group (P<0.05) (Table 2).

Correlation analysis

There existed a positive correlation between the Tim-3 mRNA expression and the IL-4 concentration in the acute attack group (r=0.68, P<0.05). Significant negative correlations were found between the Tim-3 mRNA expression and the IFN-γ concentrations as well as the level of FEV1 and FEV1/FVC% in the acute attack group (r=-0.85, r=-0.76, P<0.01 for both).

There was a positive correlation between the Tim-3 mRNA expression and the IL-4 concentration in the remission group (r=0.63, P<0.05). Significant negative correlations were observed between the Tim-3 mRNA expression and the IFN-γ concentrations as well as the level of FEV1 and FEV1/FVC% in the remission group (r=-0.82, r=-0.71, P<0.01 for both).

Discussion

Naive T helper cells could be activated to differentiate into Th1 or Th2 cells by many factors, including cytokines, antigens of different kinds or of different delivery routes, and transcription factors. Th1 cells could mediate immune response to intracellular pathogens such as viruses and produce cytokines such as IFN-γ and interleukin-2 (IL-2). Moreover, Th2 cells could mediate immune responses to extracellular pathogens such as helmets and produce the cytokines such as IL-4, IL-5, and IL-13. Furthermore, Th1 and Th2 cells could also cross-regulate one other [7]. It was shown that if T helper cells differentiated into Th2 cells, they could block the pathological process of autoimmune diseases. However, if T helper cells differentiated into Th1 cells, they could hinder the pathological process of asthma and other allergic diseases [8, 9]. Therefore, the cell surface molecules that specifically expressed on Th1 or Th2 may be used as the molecular targets of immuno-regulation treatment. Tim-3, a recently discovered surface molecule, was shown to be a transmembrane protein preferentially expressed on Th1 cells. In a Th1-mediated model of multiple sclerosis (MS), Koguchi et al[10] used small interfering (si)RNA to reduce Tim-3 expression on CD4+T cells from MS cerebrospinal fluid (CSF) clones ex vivoand found that the siRNA inhibited Tim-3 expression on CD4+T cells and substantially enhanced cell proliferation and IFN-γ secretion, suggesting that Tim-3 expression on human T cells regulates cell proliferation and IFN-γ secretion. Fukushima et al[4] demonstrated that the treatment of EC with anti-Tim-3 Ab can ameliorate EC by inhibiting conjunctival eosinophil infiltration and elevating splenocyte IFN-γ production, which suggested that anti-Tim-3 Ab can be used for the treatment of Th2-mediated diseases since it enhances Th1 response along with a shift toward Th1-mediated immunity.

The underlying mechanisms of immune function disorder in asthma are involved. The imbalance of Th1/Th2 cells and cytokines were also implicated in the pathogenesis of asthma. The role of Tim-3 in the imbalance of Th1/Th2 in asthma has not yet been fully elucidated, and recent researches have been restricted to gene polymorphism [11]. In order to explore the role of Tim-3 in the pathogenesis of asthma, we used RT-PCR to detect Tim-3 mRNA expression in peripheral blood T lymphocytes in asthmatic patients.

It was shown that the level of Tim-3 mRNA in the acute attack group and remission group was increased significantly in peripheral blood T lymphocytes compared with the control group, suggesting that Tim-3 may partake in the inflammatory process of asthma. Moreover, the level of Tim-3 in the acute attack group was higher than in the remission group in peripheral blood T lymphocytes, which suggested that Tim-3 might also play a certain role in the persistent attack of asthma. Consistent with previous findings in asthmatic patients, the IFN-γ concentrations were markedly lower and the IL-4 concentrations were notably higher in the supernatant of peripheral blood T lymphocytes. Furthermore, the Tim-3 mRNA expression bore a significant negative correlation with the IFN-γ concentrations and a significant positive correlation with the IL-4 concentrations, indicating that the IFN-γ hyposecretion was associated with the high expression of Tim-3 mRNA in asthmatic patients. However, how Tim-3 regulates IFN-γ secretion is unknown so far. Mariat et al[12] found that Tim-3 could suppress Th1 response by regulating CD4+CD25+ regulatory T cells. Sui [13] also demonstrated that the level of Tim-3 mRNA expression was gradually decreased with the differentiation and maturation of DC, suggesting that Tim-3 may work through CD4+CD25+ regulatory T cells and dendritic cells.

The FEV1/FVC% is a single and optimal quantitative index that reflects the severity of airway obstruction in asthma. The results showed that there was a negative correlation between Tim-3 mRNA expression and the level of FEV1 and FEV1/FVC% in both the acute attack group and the remission group, suggesting that Tim-3 could be used as a reliable index to indirectly mirror the severity of asthma.

To sum up, this study showed that the expression of the Tim-3 gene in peripheral blood T lymphocytes in asthmatic patients was increased significantly, which can be used for reflecting the severity of airway inflammation in asthma and, when combined with pulmonary function, the measure can provide an objective indicator of anti-inflammatory treatment.

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