Asthma is a complex disorder characterized by varying degree of airflow obstruction, airway hyperresponsiveness (AHR), airway inflammation, airway remodeling, mucus hypersecretion, and high serum immunoglobulin E (IgE).1,2 The etiology of asthma is complex and multifactorial. It is well known that T-helper type 2 (Th2) lymphocytes play a critical role in the initiation, progression and persistence of allergic diseases including asthma.3 However, the immunological basis of this disease is still controversial.
A subset of T lymphocytes, constitutively expressing CD25 (IL-2 receptor alpha chain) on their cell surface, has been recently identified in rodents and humans. This T cell subset, named CD4+CD25+ regulatory T cells (Tregs), is anergic to allogeneic or polyclonal activation, and actively possesses the suppressive capacity on responder (CD4+CD25-) and self-reactive T cells.4 Accounting for 2%-3% of total CD4+ T cells in humans, CD4+CD25high T cells exhibit the regulatory characteristics, which are identical to the Tregs.5 CD25 is not a unique marker for Tregs as it is also expressed by the activated/memory T cells. Several other markers have also been described for Tregs, such as surface-bound latency-associated peptide/transforming growth factor β1 (LAP/TGF-β1), glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), neuropilin-1, galectin-1, and lymphocyte activation gene-3. However, all these molecular markers are not unique for Tregs. Recent experiments have shown that forkhead winged-helix transcription factor forkhead box P3 (FOXP3) serves as the dedicated mediator of the genetic program governing Tregs development and function, therefore it is regarded as a reliable and specific marker for Tregs.6-8 Furthermore, toll like receptors (TLRs), mainly expressed on antigen-presenting cells (APC) as a bridge linking innate and adaptive immunity, also exist on Tregs, and directly modulate the quantity and quality of Tregs.9-13
Recent animal studies have demonstrated that Tregs can modify dendritic cells (DC) by down-regulating the expression of costimulatory molecules,14 and directly interfere with Th cell-dendritic cell interactions.15 Moreover, they could reverse airway eosinophilia and airway hyperresponsiveness by inhibiting Th2 response in allergic mice.16,17 Nevertheless, existing data regarding Tregs in patients with asthma are scarce and discrepant, and the mechanisms underlying Tregs-mediated immunosuppression remain to be elucidated.
The aim of the present study was to determine the expression of surface molecules CTLA4, TLR4, GITR, and membrane-bound LAP/TGF-β1 on human peripheral CD4+CD25high T cells, and to characterize their features between patients with atopic asthma and healthy subjects. We also investigated the effect of inhaled corticosteroid on these surface molecules, and analyzed the correlations between these surface markers and the clinical characteristics of asthma.
Sixty adult patients with a history of atopic asthma at least for one year and 23 healthy volunteers without atopy and asthma were enrolled. Asthma was diagnosed according to the GINA guidelines.18 The severity of asthma was classified as intermittent, mild persistent, moderate persistent and severe persistent based on the clinical features.18 Atopy was evaluated by one or more positive responses of the skin prick test (SPT) to a range of 25 common aeroallergens. All patients with asthma used short-acting inhaled β2-agonists as required, and received no inhaled or systemic glucocorticoids within a month. Of the 60 patients with asthma, 31 exhibited an acute exacerbation with an episode of rapidly progressive increase in shortness of breath, cough, wheezing, chest tightness, or some combination of these symptoms, and 29 were stable since they had a history of asthma but no asthmatic symptoms recently. Upon enrollment, all asthmatic patients underwent pulmonary function test, blood test for eosinophil count, chest radiography, and some necessary examinations for ruling out other pulmonary diseases. All subjects were nonsmokers, and those who suffered from heart disease, autoimmune disease and cancer were excluded. The subjects who suffered from airway infection within 4 weeks immediately after the examinations were also excluded. The characteristics of the subjects are detailed in Table. Written informed consent was obtained from each subject before the study, and the study protocol was approved by the Human Research Ethics Committee of Nanjing Medical University.
Proper instructions on how to perform the forced expiratory maneuver were illustrated to all subjects. Forced expiratory volume in one second percentage of the predicted value (%FEV1), forced vital capacity percentage of the predicted value (%FVC), and ratio of FEV1 to FVC (FEV1/FVC) were measured in triplicate using a spirometer (MicroLab, MicroMedical, Rochester, Kent, UK). The highest values were recorded.
Skin prick test
Skin prick test was conducted with a panel of common aeroallergen extracts in the presence of a positive histamine control and a negative vehicle control on the forearm. Twenty-five aeroallergens including Dermatophagoides pteronyssinus, Dermatophagoides farinae, Felis domesticus, Canis familiaris, cockroach, pollen, ragweed, mugwort, and moulds (ALK-Abelló A/S, Hørsholm, Denmark) were utilized. The test was considered positive if the wheal was greater than 3 mm in mean diameter.
Blood sampling and cell separation
Heparinized peripheral venous blood from each subject was collected in a sterile vacuum tube between 7:00 and 9:00 AM. Peripheral blood mononuclear cells (PBMCs) were enriched by Ficoll-Hypaque (TBD, Tianjin, China) gradient centrifugation within 4 hours. The cell viability determined by trypan blue exclusion assay was greater than 95%. Samples of peripheral venous blood were centrifuged for 10 minutes at 1300 × g at 4°C, and the sera were stored at -20°C.
Anti-CD3-APC (S4.1), anti-CD4-PE-Cy5.5 (S3.5), anti-CD25-FITC (CD25-3G10) biotin-conjugated anti-mouse IgG1, and PE-conjugated streptavidin were purchased from Caltag (Burlingame, CA, USA). Anti-CTLA4-PE (14D3), anti-TLR4-PE (HTA125) and human regulatory T cell staining kit were purchased from eBioscience (San Diego, CA, USA). Anti-LAP/TGF-β1 (27232) and anti-GITR-PE (110416) were purchased from R&D (Minneapolis, MN, USA). And corresponding isotype-matched controls were also obtained.
To detect surface markers on CD4+CD25high T cells, freshly isolated PBMC were stained with a combination of fluorochrome-labeled antibodies. In brief, 5×105 PBMCs were incubated directly with anti-CD4-PE-Cy5.5, anti-CD25-FITC, and each PE-conjugated antibody as the third color at the optimal concentrations recommended by the manufacturer. After incubation for 20 minutes at room temperature in the dark, the cells were washed twice with phosphate-buffered saline containing 0.1% (w/v) bovine serum albumin and 0.1% (w/v) NaN3 and finally fixed with 2% paraformaldehyde. For the staining of LAP, cells were incubated at room temperature for 20 minutes, each with 27232 antibody, biotin-conjugated anti-mouse IgG1, as well as PE-conjugated streptavidin, anti-CD4-PE- Cy5.5 and anti-CD25-FITC in the dark. FOXP3 staining was conducted using the human regulatory T cell staining kit following the manufacturer's protocols. Isotype-matched and fluorochrome-matched controls were used to set up the quadrants. Four-color flow cytometry was performed by FACSCalibur (Becton Dickinson, Mountain View, CA, USA) with CellQuest software. A total of 5000 CD4+ cells were acquired after gating the lymphocyte population by forward- and side-scattered properties. Gates were set so that the CD4+CD25- population was based on the isotype control while the CD25high population was determined relative to the low intensity of CD25 staining found on non-CD4+ T cells, as described previously.19 Cells expressing CD25 at levels above those of the isotype control and unstained cells but at lower expression levels than the CD25high cells were considered as CD25low (Figure 1).
PBMCs were cultured at a density of 1×106 cells/ml in 24-well flat-bottomed plates with AIM-V medium (GIBCO, Grand Island, NY, USA) containing 20 μmol/L mercaptoethanol (Sigma-Aldrich, Stockholm, Sweden) at 37°C in 5% CO2. Budesonide (AstraZeneca AB, Södertälje, Sweden) was added at various concentrations (10-8 mol/L, 10-7 mol/L and 10-6 mol/L). After incubation for 24 hours, cells were collected, washed twice with phosphate-buffered saline, and stained for flow cytometric analysis as described above.
Enzyme linked immunosorbent assay
The levels of total serum IgE were measured using a human IgE ELISA quantitation kit and a starter accessory package (Bethyl, Montgomery, TX, USA) according to the manufacturer's instructions. Serum high-sensitivity C-reactive protein (hs-CRP) concentrations were determined using the latex enhanced immunoturbidimetric assay (Dade Behring, Marburg, Germany). The minimum detection levels of serum IgE and hs-CRP were 6.51 IU/ml (15.625 ng/ml) and 0.15 μg/ml, respectively.
The data were expressed as mean ± standard deviation (SD). The Kruskal-Wallis test was used to calculate the differences among three groups. The data between two groups were compared using the Mann-Whitney U test. Correlations were analyzed using Spearman's rank-order correlation coefficient or Pearson's product-moment correlation coefficient as indicated. A P value of less than 0.05 was considered statistically significant. The statistical analysis was done using SPSS 11.0 software package (SPSS Inc., Chicago, IL, USA).
Preferential expression of CTLA4, TLR4, GITR, and membrane-bound LAP/TGF-β1 on human peripheral CD4+CD25high T cells
Expression of surface molecules CTLA4, TLR4, GITR, and LAP on CD4+CD25high T cells from healthy subjects was determined by flow cytometry. Human CD4+CD25high T cells constitutively and highly expressed membrane-bound LAP. The ratio of CD4+CD25highLAP+ T cells/CD4+CD25high T cells was (22.69±11.57)%, which was significantly higher than that of CD4+CD25low/-LAP+ T cells/ CD4+CD25low/- T cells ((3.03±2.52)%, P<0.01). Likewise, CD4+CD25high T cells expressed more CTLA4, TLR4, and GITR than did the CD4+CD25low/- T cells ((1.75±2.62)% vs (0.05±0.22)%, (3.36±2.68)% vs (1.17±1.39)%, (2.62±2.54)% vs (0.20±0.32)%, respectively, all P<0.01). Notably, expression of CTLA4, TLR4, and GITR was relatively lower than that of membrane-bound LAP (data not shown).
Equivalent numbers of CD4+CD25high T cells among healthy controls, stable asthmatics, and acute asthmatics
Next, we analyzed the difference of CD4+CD25high T cell counts in peripheral blood from healthy controls and patients with stable and acute asthma. The ratio of CD4+CD25high T cells/total CD4+ T cells was (2.91±1.30)% in healthy subjects, (3.43±1.22)% in stable asthmatics, and (3.32±1.51)% in acute asthmatics, respectively (Figure 2A). There was no statistical significance among them (P>0.05).
Expression of FOXP3 in the majority of CD4+CD25high T cells
FOXP3 is a reliable and specific marker for Tregs. As anticipated, most CD4+CD25high T cells expressed FOXP3, whereas CD4+CD25low/- T cells hardly expressed any FOXP3 (data not shown). Consistent with the above results, percentages of CD4+CD25highFOXP3+ T cells in CD4+CD25high T cells were not significantly different among healthy subjects, patients with stable asthma and acute asthma ((81.22±8.97)%, (77.91±10.15)%, and (84.34±5.10)%, respectively, P>0.05) (Figure 2B). Also, similar results were obtained from the median fluorescence intensity (MFI) of FOXP3 in CD4+CD25high T cells (41.98±8.28, 36.71±7.84, and 38.62±7.89, respectively, P>0.05).
Different expression of LAP, but not CTLA4, TLR4 and GITR, on the surface of CD4+CD25high T cells in healthy controls, stable asthmatics, and acute asthmatics
We examined whether numbers of CD4+CD25high T cells expressing membrane-bound LAP were different between healthy volunteers and asthmatics. Although both healthy volunteers and patients with stable asthma had a similar number of CD4+CD25highLAP+ T cells ((22.69±11.57)% vs (20.55±13.76)%, P>0.05), it was unexpectedly decreased in patients with acute asthma ((11.35±8.94)%, P<0.01) (Figure 3). As for CTLA4, TLR4 and GITR, there was no significant difference among healthy volunteers, patients with stable asthma and acute asthma ((1.75±2.62)% vs (2.35±3.91)% vs (1.89±2.37)%, (3.36±2.68)% vs (3.43±3.20)% vs (3.18±2.26)%, (2.62±2.54)% vs (3.23±3.34)% vs (3.05±2.26)%, respectively, all P>0.05).
Increased expression of LAP on the surface of CD4+CD25high T cells in patients with acute asthma after treatment with inhaled corticosteroid
All patients with asthma received a stepwise treatment after enrollment, including inhaled corticosteroid (budesonide), β2-agonists, and other medications, as recommended by GINA.18 Four weeks later, 6 asthmatic patients (3 with stable moderate asthma, 2 with acute moderate asthma, and 1 with acute severe asthma when enrollment) revisited. These patients were completely controlled with asthmatic symptoms after treatment according to the Asthma Control Test (www.asthmacontro.com). Peripheral venous blood was collected again. Changes of CD4+CD25high T cell counts and expression of surface markers, i.e., CTLA4, TLR4, GITR, and membrane-bound LAP, were compared before and after the treatment. CD4+CD25high T cell counts were slightly raised, but did not reach a statistically significant level ((4.55±2.89)% vs (2.59±1.32)%, P=0.337) after treatment with optimal inhaled corticosteroid (Figure 4A). CD4+CD25highLAP+ T cell percentages were evidently increased ((15.7±7.9)% vs (6.4±3.8)%, P=0.037) in parallel with clinical improvement (Figure 4B). Nevertheless, expression of CTLA4, TLR4, and GITR did not change significantly (data not shown).
Increased expression of LAP on the surface of CD4+ CD25high T cells after budesonide treatmentin vitro
We further studied the effect of glucocorticoids on membrane-bound LAP expression in vitro. In accordance with the result above, CD4+CD25highLAP+ T cells counts elevated in a dose-dependent manner, and reached a statistically significant level after the treatment with budesonide at a concentration of 1×10-7 mol/L (Figure 5).
Expression of LAP on the surface of CD4+CD25high T cells associated with total serum IgE, %FEV1, and severity degree in patients with asthma
Pearson's product-moment correlation coefficient showed that the percentage of CD4+CD25highLAP+ T cells in CD4+CD25high T cells was negatively correlated with the total serum IgE level in patients with asthma (r=-0.433, P=0.001, Figure 6A), but no significant correlation was found between the percentage of CD4+CD25highLAP+ T cells and hs-CRP (r=-0.182, P=0.164). In addition, Spearman's rank-order correlation coefficient depicted a moderate correlation between the percentage of CD4+CD25highLAP+ T cells and either %FEV1 (r = 0.255, P=0.049, Figure 6B) or severity degree of asthma (r=-0.396, P=0.002).
Tregs with suppressive properties on effector T cells have recently been focused on the homeostatic regulation of the immune system. Although accumulating evidence indicates an important role of Tregs in the development of experimental asthma, there are rare data available with respect to human subjects. Moreover, the results from independent studies are irreconcilable, and the exact mechanisms of Tregs modulating asthma remain poorly understood.
The present study demonstrates an equivalent number of CD4+CD25high T cells in peripheral blood from healthy subjects and patients with atopic asthma in different clinical status. The majority of CD4+CD25high T cells expressed FOXP3, a hallmark of Tregs.6 Patients with stable asthma had similar numbers of CD4+CD25highLAP+ T cells as compared with healthy subjects, whereas patients with acute asthma had decreased numbers of CD4+CD25highLAP+ T cells. After 4 weeks of treatment with inhaled corticosteroid, the expression of membrane-bound LAP/TGF-β1 on CD4+CD25high T cells was sharply increased. Furthermore, the expression of LAP on CD4+CD25high T cells was elevated in a dose- dependent manner after treatment with glucocorticoids in vitro. The percentages of CD4+CD25high T cells expressing membrane-bound LAP negatively were correlated with total serum IgE levels and severity grades of asthma, but positively correlated with %FEV1 in patients with asthma.
Shi et al20 reported an increased amount of CD4+CD25+ T cells in peripheral blood from asthmatics during acute exacerbation. Currently, we did not observe any significant differences of CD4+CD25high T cell counts between healthy controls and stable and acute asthmatics. This discrepancy possibly stems from distinction in the patients selected and cell subset aimed at. The aforementioned study recruited most patients who had been treated with inhaled or oral glucocorticoids, whereas only subjects with no glucocorticoids used within a month before sampling were selected in our study. It is well established that systemic treatment with glucocorticoids up-regulates the number of CD4+CD25+CD45RO+CD62L+ T cells in patients with asthma.21 Also, glucocorticoids can inhibit allergen- stimulated proliferation of CD4+CD25- T cells22 and induce a selective apoptosis in CD4+CD25- T cells,23 resulting in an indirectly increased frequency of CD4+CD25+ T cells. Furthermore, administration of glucocorticoids redistributes lymphocytes, which may leave Tregs in the periphery.21 Hartl et al24 demonstrated that percentages of CD4+CD25high T cells in peripheral blood from children with asthma who did not receive glucocorticoids were similar to those of controls, further supporting our findings. Notably, the numbers of CD4+CD25high T cells from controls in our study were slightly higher than those described by Hartl et al.24 This difference may be due to patients' age because the number of human peripheral CD4+CD25high Treg cells increases with age.25 To confirm the concept that treatment with glucocorticoids could increase the frequency of peripheral CD4+CD25high T cells, we compared the quantitative change of this cell subset in 6 patients with asthma before and after 4 weeks of inhaled budesonide. Although we discovered a slight increase after the treatment, there was no statistical significance between them. We speculate that dosage and course of the treatment may influence the final results. Furthermore, the form of medication should be also considered. Actually, a study of systemic lupus erythematosus26 indicated that only the patients (≥5 mg/day) other than those receiving continuous glucocorticoids (<5 mg/d) in 3 months before sampling had an increased percentage of CD4+CD25high T cells. Further investigations are therefore needed to clarify this issue in asthma.
Toll like receptors (TLRs) play a pivotal role in the immune response to pathogen-associated molecular patterns, and provide a critical link between the innate and adaptive immune systems.27 Caramalho et al9 highlighted that murine Tregs selectively expressed TLR4, 5, 7, and 8, and ligation of TLR4 on these cells enhanced their suppressive capacity in some situations. Afterwards, growing studies demonstrated the expression of various TLRs on murine or human Tregs with distinct roles.11-13 Our study provided evidence that human CD4+CD25high T cells preferentially expressed TLR4 protein with a higher level than did CD4+CD25low/- T cells. This finding was consistent with prior report that both ex vivo isolated Tregs and T cell clones expressed more TLR4 mRNA than their CD4+CD25- counterparts.10 Nevertheless, we found no significant difference in the number of CD4+CD25high T cells expressing TLR4 between atopic asthmatics and healthy controls. Our findings and the unique role of TLR4 on Tregs suggest that impaired quality, not quantity, of TLR4 as well as additional adaptor molecule in the TLR4 signaling pathway may be involved in the pathogenesis of asthma. Further investigations are intriguing to illuminate how TLR4 and other TLRs control Tregs function.
Although the mechanisms underlying the ability of Tregs to suppress immunity remain uncertain, most studies demonstrate a unique function of Tregs via cell-cell contact process and/or secretion of suppressive cytokines,28 involving multiple molecules such as GITR, CTLA-4, TGF-β, and IL-10. TGF-β is a multifunctional cytokine which displays both stimulatory and inhibitory effects. LAP is the amino-terminal domain of TGF-β precursor peptide. It remains non-covalently associated with the TGF-β peptide after cleavage and forms a latent TGF-β complex. Investigations have demonstrated that TGF-β is critical in the expansion and generation of Tregs.29,30 Nakamura et al31 for the first time reported that murine CD4+CD25+ regulatory T cells expressed high levels of membrane-bound LAP/TGF-β1, mediating cell contact-dependent immunosuppression. Furthermore, they confirmed this role of membrane-bound LAP/TGF-β1 in human CD4+CD25high T cells.32 TGF-β mediates its effects on cells through a heteromeric complex consisting of type I and II receptor components. In an adoptive transfer mouse model of colitis, CD4+CD45RBhigh T cells bearing a dominant negative form of TGF-β receptor II which can not respond to TGF-β, are resistant to the control by CD4+CD25+ regulatory T cells.33 A sudden decline of active membrane-bound TGF-β could impair the function of Tregs and their protection against autoimmune diabetes.34 Like membrane-bound TGF-β, CTLA-435 also contributes to the cell-contact suppressive sequelae, yet GITR stimulation acts as a counterbalance.36 As a synergistic effect, CTLA-4 signaling facilitates TGF-β mediated suppression by intensifying the TGF-β signal at the site of suppressor cell-target cell interaction.37 In the present study, we observed that human circulating CD4+CD25high T cells preferentially expressed surface molecules, namely CTLA-4, GITR, and membrane-bound LAP/TGF-β1, at higher levels than did CD4+CD25low/- T cells. Despite an equivalent expression of membrane- bound LAP existed between stable asthmatics and controls, patients with asthma who suffered from an acute exacerbation had less CD4+CD25high LAP+ T cells than those with stable asthma and healthy controls. After 4 weeks of treatment with inhaled corticosteroid, these patients had a significant increase of CD4+CD25high LAP+ T cells in parallel with amelioration of clinical symptoms. Although the interference of other medications administrated simultaneously can not be excluded, the in vitro experimental results did verify these findings. The correlation analysis confirmed that the percentages of CD4+CD25high LAP+ T cells inversely were correlated with total serum IgE levels and severity grades in patients with asthma, and positively correlated with %FEV1. We also took serum hs-CRP into consideration since it may serve as a surrogate marker of airway inflammation in asthma.38 Unexpectedly, no correlation was found between the percentages of CD4+CD25high LAP+ T cells and hs-CRP in patients with asthma. We have to point out that we did not investigate whether the suppressive capacities of CD4+CD25high T cells are regulated by the surface expression of TGF-β. Not withstanding its limitation, the present study does suggest that membrane-bound TGF-β on the CD4+CD25high T cells is a potential candidate for predicting the severity of asthma, and it may contribute to the sustained remission of asthma.
In summary, our study did not demonstrate quantitative defects in the numbers of peripheral CD4+CD25high T cells in patients with atopic asthma in distinct clinical status. The glucocorticoids-induced expression of membrane-bound TGF-β in asthmatics and its correlation with clinical characteristics suggest that CD4+CD25high T cells expressing membrane-bound TGF-β may be involved in the pathogenesis of atopic asthma. Strategies targeting Tregs, especially surface molecule TGF-β, are fascinating and promising for future asthma therapies.
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