The introduction of Global Initiative for Asthma (GINA) guidelines has enabled more patients with allergic asthma to control their asthma. However, many patients using standard treatments still progress to moderate or severe stages of asthma. Moreover, the pathogenesis of allergic asthma remains unknown.
T helper cells can generate an antigen specific, T cell response to an allergen, which plays a key role in the earliest stages of the establishment of allergic sensitization. In allergic asthma, activated T helper type 2 (Th2) cells and their secreted cytokines promote the development of eosinophilia and airway hyperresponsiveness.1 Experimental and clinical data indicate that Th2 cells and an imbalance in Th1/Th2 expression are central to the pathogenesis of asthma.2-6
However, in recent years, it was recognized that Th1/Th2 imbalance does not fully explain the aetiology of asthma. For example, reversing the Th1/Th2 imbalance does not fully control asthmatic symptoms in humans. Some studies have suggested that other CD4+ T cell subsets may play a role in asthma, including Th1 cells, Th17 cells and regulatory T cells (Treg).7 The expression profile of all CD4+ T cell subsets in allergic asthma has not been fully investigated. Therefore, to understand further the role of CD4+ T cells in asthma, we determined the relative abundance and activities of Th1, Th2, Th17 and CD4+CD25+ Treg cells in patients with mild or moderate to severe asthma.
Thirty-nine patients with chronic persistent asthma, based on positive results of allergy tests to house dust mites (UniCap, Pharmacia, Stockholm, Sweden), were selected for this study. The severity of asthma was assessed based on the GINA guidelines.8 Lung function tests including forced expiratory volume in the first second (FEV1) (% predicted) were performed. Participants also completed the Asthma Control Questionnaire (ACQ). The concentrations of Dermatophagoides pteronyssinus (D. pteronyssinus） specific immunoglobulin E (DP.sIgE) and total IgE were measured. None of the patients had been treated with systemic glucocorticoids within 1 month before the study and had never been treated with other immunosuppressive agents or desensitization therapy. Twenty healthy donors, nonsmokers with normal pulmonary function and negative allergy tests, were selected as a normal control group. Total IgE levels were also measured. This study was approved by the Research Ethics Board of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Written informed consent was obtained from all individuals.
Ten millilitres of heparinized peripheral venous blood were collected from each participant. Plasma was isolated from peripheral blood and stored at –80°C until used to measure the concentrations of cytokines. Peripheral blood mononuclear cells (PBMCs) were obtained from peripheral blood by Ficoll-Hypaque density centrifugation (2500 r/min for 20 minutes at room temperature). PBMCs were suspended at a density of 2×106 cells/ml in Roswell Park Memorial Institute (RPMI) media 1640 with GlutaMAX supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 10% heat inactivated, fetal calf serum (Gibco, USA).
Flow cytometric analysis
For analysis of Th1, Th2 and Th17 cells, the cell suspension was stimulated with 20 ng/ml phorbol 12-myristate-13-acetate and 1 μg/ml ionomycin in the presence of 2 mmol/ml monensin (Sigma-Aldrich, USA) in 24-well plates. After 4 hours of culturing (37°C; 5% CO2), the cells were transferred to tubes and washed once in phosphate buffered saline (PBS). The cells were then incubated with phycoerythrin-cy5 (PE-cy5) antihuman CD4 (BD Pharmigen, USA) at 4°C for 30 minutes. After surface staining, the cells were fixed and permeabilized according to the manufacturer's instruction, and then stained with fluorescein isothiocyanate (FITC) antihuman interferon (IFN)-γ (BD Pharmigen) plus phycoerythrin antihuman interleukin (IL)-4 (BD Pharmigen) or PE antihuman IL-17A (eBioscience, USA).
For analysis of Treg cells, the cell suspension was transferred into tubes and washed once in PBS. The cells were stained with FITC antihuman CD4 and allophycocyanin antihuman CD25 at 4°C for 30 minutes. The cells were then incubated with PE antihuman Foxp3 after fixation and permeabilized according to the manufacturer's instruction. All of antibodies and reagents were from eBioscience.
Isotype controls were used to correct nonspecific binding. All stained cells were analysed by flow cytometry.
PBMCs from 18 patients with mild asthma, 15 patients with moderate to severe asthma and 16 healthy donors were cultured in 24-well plates at 2×106 cells/ml per well in the presence of RPMI 1640 medium and 5 μg/ml recombinant D. pteronyssinus antigen (rDerP1, Prospec-Tany, Israel). The cells were incubated for 48 hours (37°C; 5% CO2). The cell culture supernatants were then collected and stored at 80°C for analysis of cytokine.
Enzyme linked immunosorbent assays (ELISA)
The concentrations of IFN-γ, IL-4, IL-17, IL-10 and transforming growth factor (TGF)-β1 in plasma and PBMC culture supernatant were measured by ELISA in accordance with the manufacturer's instructions (R&D Systems, USA). The concentrations of total IgE and specific IgE were also measured by ELISA. All samples were measured in duplicate.
SPSS 11.0 was used for statistical analysis. Homogeneity of variance in three groups was tested first. If each group showed homogeneity, analysis using one way analysis of variance (ANOVA) followed by Student Newman Keuls test. When heteroscedasticity was present in each group, the data were analysed using Mann-Whitney test and were expressed as medians (interquartile range). Pearson's correlation analysis was used to analyse the relationship between the expression of CD4+ T cells and the severity of asthma. Statistical significance was defined as P <0.05.
General characteristics of subjects
According to the GINA guidelines, the 39 patients with allergic asthma could be divided into two subgroups: 22 had mild asthma and 17 had moderate to severe persistent asthma. There were no significant differences in terms of age or gender among the two subgroups of patients with asthma and the normal control group. Total IgE levels were significantly higher in all patients with asthma than in the normal control group. However, there were no differences between the mild and moderate to severe asthma subgroups in terms of total IgE and DP.sIgE levels. FEV1 (% predicted) were significantly lower in patients with moderate to severe asthma than in those with mild asthma, but the mean scores for ACQ were higher in patients with moderate to severe asthma than in those with mild asthma. The characteristics of the study subjects are summarized in Table 1.
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Table 1. Patient characteristics
Th1/Th2 imbalance in allergic asthma
The frequency of Th2 cells (CD4+IL-4+ cells) was markedly higher in patients with mild (3.59% (2.76%–5.72%)), and moderate to severe asthma (4.49% (2.52%–6.49%)) than in the control group (1.71% (1.19%–2.63%)). By contrast, there were no differences in the expression of Th1 cells (CD4+IFN-γ+ cells) among the three groups (Figure 1). An imbalance in Th1/Th2 cells was present in both subgroups of patients with asthma. The Th1/Th2 ratio was significantly lower in patients with mild (4.22 (1.97–5.38)) and moderate to severe asthma (2.42 (1.86–3.91)) than in the control group (5.27 (3.61–8.65)). However, there were no differences in the frequency of Th2 cells or the Th1/Th2 ratio between the two subgroups of patients with asthma (Figure 1).
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Figure 1. Frequencies of Th1 and Th2 cells in CD4+ T cells among patients with allergic asthma and normal subjects. *P <0.01.
High levels of Th17 cells in peripheral blood of patients with moderate to severe allergic asthma
The percentage of Th17 cells (CD4+IL-17+ cells) in PBMCs was significantly higher in patients with moderate to severe asthma ((4.32±2.31)%) than in those with mild asthma ((2.03±1.02)%) and the normal control group ((1.55±0.87)%) (Figure 2). However, there was no difference in proportion of Th17 cells between patients with mild asthma and the control group. This observation indicated that the severity of asthma was associated with an increased proportion of Th17 cells.
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Figure 2. The frequencies of Th17 cells relative to blood CD4+ cells were examined by flow cytometry. A: Representative plots are from a single patient in each group. B: Frequencies of Th17 cells in each group. *P <0.01.
Low levels of Treg cells in peripheral blood of patients with moderate to severe allergic asthma
The frequency of Treg cells (CD4+CD25+Foxp3+ cells) was significantly lower in peripheral blood of patients with moderate to severe allergic asthma ((2.73±1.64)%) than in patients with mild asthma ((4.59±1.90)%) or the control group ((4.10±2.38)%) (Figure 3). This indicates that the severity of asthma is negatively correlated with the frequency of Treg cells.
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Figure 3. The frequencies of natural Treg cells relative to blood CD4+ cells were examined by flow cytometry. A: Representative plots are from a single patient in each group. B: Frequencies of Treg cells in each group. *P <0.05.
Plasma IFN-γ, IL-4, IL-17, IL-10 and TGF-β1 levels
Plasma IL-4 levels were significantly higher in patients with mild and moderate to severe asthma than in the control group. However, the plasma IL-17 levels were markedly higher in patients with moderate to severe asthma than in patients with mild asthma or normal control subjects. By contrast, the plasma IL-10 level was significantly lower in patients with moderate to severe asthma than in patients with mild asthma and the normal control group. In patients with moderate to severe asthma, the levels of TGF-β1 were lower and those of IFN-γ were higher than those in the control group, but were not significantly different to those in patients with mild asthma (Table 2).
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Table 2. Plasma IFNγ, IL-4, IL-17, IL-10 and TGF-β1 levels
Cytokine production in PBMCs cultured with rDerp1
PBMCs from patients with mild allergic asthma (n=17) or moderate to severe asthma (n=15) and normal control subjects (n=16) were cultured with rDerp1, an allergen of house mites. There was no difference in age or gender among the three groups. After 48 hours, the concentrations of IFN-γ, IL-4, IL-17, IL-10 and TGF-β1 in the culture supernatant were measured by ELISA. The levels of IL-4 were higher in patients with mild and moderate to severe asthma, than that in the normal control group. The levels of IL-17 were significantly higher in patients with moderate to severe asthma than in patients with mild asthma or the control subjects. By contrast, there were no differences in IFN-γ, IL-10 and TGF-β1 levels among the three groups (Table 3).
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Table 3. Cell culture supernatant levels of IFN-γ, IL-4, IL-17, IL-10 and TGF-β1
Correlation analysis in patients with allergic asthma
In patients with allergic asthma, the frequency of peripheral blood Th17 cells was negatively correlated to the percentage of Treg cells (r=–0.553, P <0.01). Total IgE levels were positively correlated to the frequency of Th2 cells (r=0.527, P <0.01). DP.sIgE levels were positively correlated to the frequency of Th2 cells (r=0.407, P <0.05) and plasma IL-4 levels (r=0.651, P <0.01), and negatively correlated to the Th1/Th2 ratio (r=–0.343, P <0.05). FEV1 (% predicted) in patients with asthma was negatively correlated to the frequency of Th17 cells (r=–0.658, P <0.01) and plasma IL-17 levels (r=–0.675, P <0.01), and positively correlated to the frequency of Treg cells (r=0.483, P <0.01). The mean ACQ score in patients with asthma was positively correlated to the frequency of Th17 cells (r=0.624, P <0.01) and serum IL-17 levels (r=0.663, P <0.01), and negatively correlated to the frequency of Treg cells (r=–0.507, P <0.01). Total IgE levels, DP.sIgE, FEV1 (% predicted), and mean ACQ scores were not correlated with the other CD4+ T cells or cytokine levels.
In this study, we assessed the CD4+T cell profiles in patients with allergic asthma, providing corroborative evidence for differences in CD4+ T cell phenotypes between the mild and moderate to severe stages of asthma. The increased Th2 cell response and the imbalance in Th1/Th2 cells in patients with allergic asthma, as found in our study, have been described elsewhere.6 We also found that the extent of the Th2 response and the Th1/Th2 imbalance were closely associated with the IgE response. Therefore, we have confirmed that the Th2 cell response plays a key role in the aetiology of allergic asthma. As in other allergic diseases, the Th2 cell response, which is activated by allergens, can initiate the IgE mediated allergic response and promote the development of asthma.
The role of Th1 cells in allergic asthma remains unclear. Results of many studies indicate that Th1 cells inhibit the progress of allergic asthma, but other studies indicate that Th1 cells can promote asthmatic inflammation.7,9 In our study, Th1 expression and the IFN-γ level in PBMCs exposed to rDerP1 did not increase in asthmatics. However, the plasma IFN-γ levels were increased in patients with moderate to severe asthma. IFN-γ can be secreted by cells other than PBMCs, including CD8+ T cells, suggesting that the elevated level of IFN-γ does not necessarily correspond to an enhanced Th1 response. Other studies have confirmed enhanced IFN-γ production in asthmatic patients, particularly in uncontrolled patients.10,11
Th17 cells, defined by their production of IL-17A/F and IL-22, play important roles in autoimmune and inflammatory diseases.12 In asthmatic patients, IL-17 mRNA expression was increased in sputum and was correlated with an increased frequency of neutrophils.13 The effect of Th17 cells and IL-17 on promoting eosinophil and neutrophil recruitment into airways is made use of in models of asthma.14,15 We found that the frequency of Th17 cells was markedly increased in patients with moderate to severe asthma, in addition to IL-17 levels in plasma and the culture supernatant of PBMCs stimulated by Der p1. These results indicate that the Th17 response was highly correlated with the severity of asthma.
CD4+CD25+ Treg cells, which specifically express the transcription factor Foxp3, are major regulators of autoimmunity and are critical for the control of antigen specific inflammation.16,17 Treg cells exert their roles through direct cell to cell contact or via cytokines such as IL-10 and TGF-β. A recent study in paediatric patients with asthma found a lower percentage of CD4+CD25+ T cells in bronchoalveolar lavage fluid than in healthy children or children treated with corticosteroids.18 Our study showed that the frequency of Treg cells and plasma IL-10 and TGF-β1 levels were decreased in moderate to severe asthma. These data suggest that deficiencies in Treg cells are correlated with the severity of asthma.
It was recently reported that there is a close connection between the differentiation into Th17 cells and Treg cells. Naïve CD+ T cells can differentiate into Treg cells in the presence of TGF-β with or without the presence of IL-6.19 Therefore, the Th17/Treg balance maintains normal immune responses besides the Th1/Th2 balance. It has been confirmed that the Th17/Treg imbalance contributes to the development of autoimmune diseases, such as systemic lupus erythematosus20 and primary nephrotic syndrome.21 According to our results, moderate to severe asthma is associated with increased Th17 and decreased Treg levels. Furthermore, the presence of Th17 cells was negatively correlated with Treg cells in patients with allergic asthma. We also found that the increased Th17/IL-17 level and Treg deficiency were correlated with a decline in FEV1 (% predicted) and higher mean ACQ scores. FEV1 (% predicted) are important markers for the severity of asthma. The ACQ scores could also be used to assess the level of control of asthma. A decline in FEV1 (% predicted) and higher mean ACQ scores are associated with worsening of asthma. Therefore, we believe that imbalances in Th17/Treg cells and Th1/Th2 cells, play an important role in asthma. An imbalance in Th1/Th2 cells may initiate allergic asthma, while an imbalance in Th17/Treg cells is associated with worsening of allergic asthma.
In conclusion, we found marked imbalances in the Th1/Th2 and Th17/Treg ratios in patients with allergic asthma compared with nonasthmatic controls. An increase in the Th2 cell response and an imbalance in Th1/Th2 cells were found in patients with allergic asthma across a range of disease severities. Furthermore, we found an increased Th17 cell response and a deficiency in Treg cells in patients with moderate to severe allergic asthma and conclude that the severity and control of asthma may be affected by the imbalance in Th17/Treg cells. These results provide valuable insight on how to treat asthma according to the CD4+ T cell imbalance found at different stages of asthma. A limitation of our study is that the local CD4+ T cell phenotype and its association with airway inflammation were not determined. Further studies are needed to clarify the relationship between Th1/Th2 and Th17/Treg, and assess which of these cells plays a more important role in allergic asthma.
1. Holgate ST. Pathogenesis of asthma. Clin Exp Allergy 2008; 38: 872-897.
2. Gruning G, Warnock M, Wakil AE, Venkayya R, Brombacher F, Rennick DM, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998; 282: 2661-2663.
3. Foster PS, Hogan SP, Ramsay AJ, Matthaei KI, Young IG. Interleukin 5 deficiency abolishes eosinophilia, airway hyperreactivity, and lung damage in a mouse asthma model. J Exp Med 1996; 183: 195-201.
4. Brusselle G, Kips J, Joos G, Bluethmann H, Pauwels R. Allergen-induced airway inflammation and bronchial responsiveness in wild-type and interleukin-4-dificient mice. Am J Respir Cell Med Biol 1995; 12: 254-259.
5. Del Prete GF, De Cari M, D'Elios MM, Maestrelli P, Ricci M, Fabbri L, et al. Allergen exposure induces the activation of allergen-specific Th2 cells in the airway mucosa of patients with allergic respiratory disorders. Eur J Immunol 1993; 23: 1445-1449.
6. Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, Bentley AM, et al. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992; 326: 298-304.
7. Afshar R, Medoff BD, Luster AD. Allergic asthma: a tale of many T cells. Clin Exp Allergy 2008; 38: 1847-1857.
8. Bateman ED, Hurd SS, Barnes PJ, Bousquet J, Drazen JM, FitzGerald M, et al. Global strategy for asthma management and prevention: GINA executive summary. Eur Respir J 2008; 31: 143-178.
9. Randolph DA, Stephens R, Carruthers CJ, Chaplin DD. Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation. J Clin Invest 1999; 104: 1021-1029.
10. Boniface S, Koscher V, Mamessier E, EI Biaze M, Dupuy P, Lorec AM, et al. Assessment of T lymphocyte cytokine production in induced sputum from asthmatic: a flow cytometry study. Clin Exp Allergy 2003; 33: 1238-1243.
11. Magnan A, Mély L, Camilla CA, Badier MM, Montero-Julian FA, Guillot CM, et al. Assessment of the TH1/TH2 Paradigm in whole blood in atopy and asthma: increased IFN-gamma-producing CD8+ T cells in asthma. Am J Respir Crit Care Med 2000; 161: 1790-1796.
12. Tesmer LA, Lundy SK, Sarkar S, Fox DA. Th17 cells in human disease. Immunol Rev 2008; 223: 87-113.
13. Bullens DM, Truyen E, Coteur L, Dilissen E, Hellings PW, Dupont LJ, et al. IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx? Respir Res 2006; 7: 135.
14. Wakashin H, Hirose K, Maezawa Y, Kagami S, Suto A, Watanabe N, et al. IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice. Am J Respir Crit Care Med 2008; 178: 1023-1032.
15. Nakae S, Komiyama Y, Nambu A, Sudo K, Iwase M, Homma I, et al. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity 2002; 17: 375-387.
16. Kim JM, Rasmussen JP, Rudensky AY. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol 2007; 8: 191-197.
17. Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, et al. Selective depletion of Fox3+ regulatory T cells induces a scurfy-like disease. J Exp Med 2007; 204: 57-63.
18. Hartl D, Koller B, Mehlhorn AT, Reinhardt D, Nicolai T, Schendel DJ, et al. Quantitative and functional impairment of pulmonary CD4+CD25hi regulatory T cells in pediatric asthma. J Allergy Clin Immunol 2007; 119: 1258-1266.
19. Bettelli E, Carrier Y, Gao W, Korn T, Storm TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effecter Th17 and regulatory T cells. Nature 2006; 411: 235-238.
20. Yang J, Chu Y, Yang X, Gao D, Zhu L, Yang X, et al. Th17 and natural Treg cell population dynamics in systemic lupus erythematosus. Arthritis Rheum 2009; 60: 1472-1483.
21. Shao XS, Yang XQ, Zhao XD, Li Q, Xie YY, Wang XG, et al. The prevalence of Th17 cells and FOXP3 regulate T cells (Treg) in children with primary nephrotic syndrome. Pediatr Nephrol 2009; 24: 1683-1690.