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Background: During subcutaneous immunotherapy (SCIT), there is a local mucosal shift from Th2 to Th1 type cytokine predominance and downregulation of interleukin (IL)-5 and eosinophilia. According to recent studies IL-10- and transforming growth factor (TGF)-β-induced tolerance is another key phenomenon in SCIT. Few data to date is available on mechanisms and roles of these cytokines in sublingual immunotherapy (SLIT).
Scope: This study was undertaken to analyse the allergen-induced in vitro mRNA expression of IL-4, IL-5, IL-10, TGF-β and interferon (IFN)-γ during SLIT in peripheral blood mononuclear cells (PBMC) of children with allergic rhinitis (AR).
Methods: Ten patients with AR undergoing pollen SLIT with a weekly dose of 200 000 SQ-U, 10 with a weekly dose of 24 000 SQ-U of glycerinated mixture of Betula verrucosa, Corylus avellana and Alnus glutinosa and 10 with placebo were included in the study. Peripheral blood mononuclear cell samples were collected and stimulated with pollen allergen extract prior to the treatment, after 1 and 2 years of the treatment. The cytokine mRNA expression was assessed using kinetic real time reverse transcription polymerase chain reaction (RT-PCR; TaqMan®).
Results: The in vitro allergen-induced mRNA expression of IL-5 by PBMC in the placebo group at 1 (P = 0.0065) and 2 (P = 0.013) years of therapy were increased in comparison with the highest dose. The expression of IL-10 mRNA was increased in the highest dose group (P = 0.0016) and the lower dose group (P = 0.034) at 2 years of therapy when compared with placebo. The change in the expression of allergen-induced TGF-β had an inversed correlation with the change of IL-5 (r = −0.38, P = 0.036) and positive correlation with the change of IL-10 (r = 0.58, P = 0.0019).
Conclusions: Sublingual immunotherapy induced a dose-dependent systemic allergen-specific immunological response in children with AR. During high-dose SLIT, there was activation of regulatory cytokine IL-10 and an inhibitory effect on IL-5 expression increase that was associated with TGF-β.
Allergic rhinitis (AR) is a common disorder with usually seasonal mucosal inflammation characterized by mast cell and eosinophil activation. The inflammation displays a predominant interleukin (IL)-4 expression (Th2 cells) in CD4+ T cells over interferon (IFN)-γ expression (Th1 cells). The clinical effect of sublingual immunotherapy (SLIT) has been shown in several studies. Of the 22 double blind placebo controlled (DBPC) SLIT trials published in peer-reviewed journals, all but three have confirmed statistically the clinical efficacy of SLIT with the most common allergens: grass, mites, birch and parietaria (1, 2).
Interleukin-10 is an anti-inflammatory cytokine, produced by T cells and dendritic cells that downregulates both Th1 and Th2 type responses (3–5). In addition, IL-10 has direct effects on T cells, including induction of non-responsive anergic state (6). Furthermore, IL-10 has inhibitory effects on mast cells and basophils (7, 8). Transforming growth factor (TGF)-β is another regulatory, pleiotropic cytokine that affects the growth, differentiation and function of a variety of cells, including T cells (9). Transforming growth factor-β antagonizes both Th1 and Th2 type inflammatory responses and is an essential cytokine in the immunosuppression induced by oral tolerance (9–11). CD4+ T cells secreting IL-10 and TGF-β have been designated regulatory T cells (Treg) and further divided into two subsets: Tr1 cells with predominance of IL-10 production and Th3 cells with predominance of TGF-β production (11–13). Interleukin-5 is a crucial cytokine in maturation, growth and activation of eosinophils (14).
In patients with AR treated with subcutaneous immunotherapy (SCIT) there is a local mucosal shift from Th2 to Th1 type cytokine predominance (15). Expression of IL-5 mRNA in the nasal mucosa is increased in AR and decreased after SCIT (16, 17). It has been firmly established that IL-10-induced tolerance is a key phenomenon in venom immunotherapy (VIT) and recently it has been shown that IL-10, TGF-β and CD4+CD25+ Treg are involved also in SCIT (18–22). Although their important role in SCIT is well established, there are to date no reports on the expression of these cytokines during SLIT.
This study was undertaken to investigate the allergen-induced in vitro mRNA expression of Th1 type effector cytokine IFN-γ, Th2 type cytokines IL-4 and IL-5 and regulatory cytokines IL-10 and TGF-β during SLIT in allergic children suffering from rhinoconjunctivitis caused by allergy to tree pollen.
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- Material and methods
The in vitro allergen-induced mRNA expression of IL-5 and ratio of IL-4/IFN-γ mRNA expression by PBMC at three time points in the three study groups are illustrated in Fig. 1. There is a significant difference between the placebo group and the dose group 2 at 1 (P = 0.0065) and 2 (P = 0.013) years in IL-5 mRNA expression. Low levels of IL-5 are maintained in the dose group 2 throughout the study. There are no significant differences in IL-4/IFN-γ ratio between the treatment groups at any time point. No significant differences are either seen when IL-4 and IFN-γ are studied separately.
Figure 1. In vitroallergen-induced interleukin (IL)-5 mRNA expression and the ratio of in vitro allergen-induced mRNA expression of IL-4 and interferon (IFN)-γ by peripheral blood mononuclear cell (PBMC) cultures of study patients assessed with real time kinetic reverse transcription polymerase chain reaction (RT-PCR; TaqMan®). The expression is shown as TaqMan index (IL-5), indicating fold increase in comparison with no stimulus, or ratio of TaqMan indices (IL-4/IFN-γ). The mean and SEM are shown separately for patients treated in dose group 2 (black square), dose group 1 (grey triangle) and placebo (white circle) before the treatment and after 1 and 2 years of therapy. The P-values refer to non-parametric unpaired comparison of dose group 2 to placebo group at each time point (Mann–Whitney U-test).
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The in vitro allergen-induced mRNA expression of regulatory cytokines IL-10 and TGF-β by PBMC are illustrated in Fig. 2. There is a significant difference between the placebo group and the dose group 2 (P = 0.016) and between the placebo group and the dose group 1 (P = 0.034) at 2 years in IL-5 mRNA expression. The expression of TGF-β mRNA remains low. However, when changes in the expression of allergen-induced TGF-β from commencement to 1 year is compared with the changes in mRNA expression of IL-5 and IL-10, it appears that TGF-β has an inversed correlation with IL-5 (r = −0.38, P = 0.036) and positive correlation with IL-10 (r = 0.66, P < 0.0001; Fig. 3). The best correlation between changes in TGF-β and IL-10 is seen in the highest dose group (dose group 2; r = 0.79, P = 0.0045). A good correlation is also seen between the TGF-β and IL-10 mRNA expression after 1 year of therapy (r = 0.61, P = 0.0002). No correlations were observed between IL-5 and IL-10. No differences between time points or treatment groups were seen in PPD-induced cytokine mRNA expressions (results not shown). PPD stimulation resulted in slightly decreased IL-10 mRNA expression when compared with unstimulated in all groups at all time points.
Figure 2. In vitro allergen-induced interleukin (IL)-10 and transforming growth factor (TGF)-β mRNA expression in peripheral blood mononuclear cell (PBMC) cultures of the study patients assessed with real time kinetic reverse transcription polymerase chain reaction (RT-PCR; TaqMan®). The expression is shown as TaqMan index, indicating fold increase in comparison with no stimulus. The mean and SEM are shown separately for patients treated in dose group 2 (black square), dose group 1 (grey triangle) and placebo (white circle) before the treatment and after 1 and 2 years of therapy. The P-values refer to non-parametric unpaired comparison with placebo group at each time point (Mann–Whitney U-test).
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Figure 3. The correlations of the observed change in cytokine expression from baseline to 1 year. Change of transforming growth factor (TGF)-β mRNA expression is compared with that of interleukin (IL)-5 and IL-10 mRNA expression. The expression is shown as TaqMan index, indicating fold increase in comparison with no stimulus. The plot symbols are shown separately for patients treated in dose group 2 (black square), dose group 1 (grey triangle) and placebo (white circle).
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The comparison of the IL-5 mRNA expression and the symptom score at 2 years is illustrated in Fig. 4. A good correlation is seen only in the dose group 2 (r = 0.69, P = 0.037). Least symptoms and lowest IL-5 levels are seen in the actively treated group. The correlations between IL-5 mRNA expression and LPSR at different time points are illustrated in Fig. 5. A good correlation is established at 2 years (r = 0.40, P = 0.045) mainly because both LPRS and IL-5 are low in the actively treated groups while they are high in the placebo group.
Figure 4. The correlations of the symptom score index and interleukin (IL)-5 after second study year. IL-5 mRNA expression is expressed as TaqMan index, indicating fold increase in comparison with no stimulus. The correlations are shown separately for patients treated in dose group 2 (black square), dose group 1 (grey triangle) and placebo (white circle).
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Figure 5. The correlations of the late phase skin response and interleukin (IL)-5 at commencement, after the first study year and after the second study year. IL-5 mRNA expression is expressed as TaqMan index, indicating fold increase in comparison with no stimulus. The correlations are shown separately for patients treated in dose group 2 (black square), dose group 1 (grey triangle) and placebo (white circle).
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Based on the telephonic survey 5 years after the 30 study subjects could be divided into two groups, one group with no signs of bronchial asthma (n = 16) and another group being on inhaled steroids and/or having had asthmatic wheezing during the past 12 months. Some patients on inhaled steroids were asymptomatic. The IL-4/IFN-γ mRNA expression ratio, IL-5 mRNA expression and the LPSR at the three time points, the symptoms and medication at 2 years and the number of patients in each treatment group are shown in Table 2. It appears that a significant decrease of IL-4/IFN-γ ratio (P = 0.03) is seen in the non-asthma group at 2 years and an increase in IL-5 mRNA expression is seen in the asthma group at 1 (P = 0.048) and 2 (P = 0.036) years. No differences were seen in TGF-β and IL-10 mRNA expression. LPSR decreased in both groups but less in the asthma group so that a significant difference between the groups is seen at 1 (P = 0.02) and 2 (P = 0.003) years. Both symptom (P = 0.02) and medication (P = 0.02) scores at 2 years are higher in the asthma group than in the non-asthma group. The majority of the subjects in the dose group 2 (the highest dose group, eight of 10) are in the non-asthma group while the majority of the subjects in the low dose and placebo groups (12 of 20) are in the asthma group (P = 0.038, chi-square test).
Table 2. Comparison of study parameters according to later (2003) reported bronchial asthma
| ||Year||No asthma||WSR||Asthma||WSR||MWU|
|IL-4/IFN-γ*||1998||2.56 (0.60)|| ||1.31 (0.25)|| ||0.20|
| ||1999||1.51 (0.32)||0.23||1.20 (0.22)||0.98||0.59|
| ||2000||1.26 (0.29)||0.03||1.52 (0.44)||0.78||0.84|
|IL-5†||1998||546 (186)|| ||528 (234)|| ||0.71|
| ||1999||1201 (489)||0.33||1613 (570)||0.05||0.18|
| ||2000||934 (230)||0.12||1828 (564)||0.04||0.23|
|LPSR||1998||3120 (551)|| ||3220 (425)|| ||0.63|
| ||1999||1671 (384)||0.01||3246 (407)||0.94||0.02|
| ||2000||785 (192)||0.002||2151 (381)||0.02||0.003|
|Symptoms‡||2000||1.66 (0.33)|| ||4.29 (0.84)|| ||0.02|
|Medication§||2000||2.17 (0.96)|| ||5.69 (1.49)|| ||0.02|
|Group|| || || || || || |
| Dose group 2|| || || || || || |
| 200 000¶ (n = 10)|| ||8|| ||2|| || |
| Dose group 1|| || || || || || |
| 24 000¶ (n = 10)|| ||4|| ||6|| || |
| Placebo (n = 10)|| ||4|| ||6|| || |
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- Material and methods
Our study demonstrated that SLIT had systemic effects demonstrated in PBMC. In addition, a clear dose–response effect was demonstrated. The important role of regulatory cytokine IL-10 in the highest dose SLIT and in addition the inhibition of upregulation of IL-5 was demonstrated, but no changes were seen in the actual IL-4/IFN-γ ratio.
The importance of the regulatory cytokine IL-10 in VIT and SCIT for pollen allergy has been demonstrated in several studies (18–22). For the first time, we have demonstrated that IL-10 has a role in SLIT of pollen allergic children. Recently an increase in PHA-induced IL-10 production was demonstrated in house dust mite SLIT in young adults (25). Data taken together with earlier studies suggest that in SLIT for respiratory allergy, as in VIT and subcutaneous immmunotherapy, upregulation of regulatory cytokines, especially IL-10 is essential, and the regulatory elements in peripheral blood may be similar in different form for immunotherapy (15, 18–29). Interleukin-10 is a T cell-derived cytokine that downregulates both Th1 and Th2 type responses and appears to suppress both IgE-mediated inflammation and delayed type hypersensitivity (DTH)-type inflammation (3–5). It can also, together with IFN-γ, decrease the release of histamine and other mediators from mast cells and basophils (7, 8). Interleukin-10 has been regarded as the main factor in the peripheral tolerance observed in VIT (18, 19). Also this peripheral tolerance is an important phenomenon in subcutaneous injections allowing the repeated injections of high allergen doses (20, 21). During SCIT, an early IL-10 response has been associated with good therapeutic outcome, whereas in SLIT no systemic changes could be seen in cytokine responses during dose build-up phase or early maintenance period (30). The timing of the regulatory response might thus differ between the two routes of immunotherapy.
From several studies in animal models, it is well known that the oral route is ‘tolerogenic’ and can redirect the Th1/Th2 differentiation (10, 31, 32). By using radiolabelled purified allergens, it has been shown that the contact of the allergen with the oral mucosa is critical and that the allergen is not directly absorbed in the mouth but is retained for up to 40 h in the mouth. On the contrary, the chemically modified allergen (monomeric allergoid) was detected unmodified in traces in plasma, whereas in the gut the allergen seems to be absorbed as peptides, not as native protein (33, 34).
Transforming growth factor-β is an essential cytokine in the immunosuppression induced by oral tolerance and it antagonizes both Th1 and Th2 type inflammatory responses (9–11). There were no consistent significant changes in the TGF-β mRNA expression during the study. Yet, it has been recently suggested that, in addition to IL-10, increased TGF-β is associated with the tolerance regulation in SLIT and the cytokines co-operate in the regulation (20). It has been shown that IL-10 enhances TGF-β receptor II expression and consequently T-cell responsiveness to TGF-β (35). The regulation of these two cytokines are thus connected together, with IL-10 having a regulatory role over TGF-β responses. We demonstrated a significant correlation between the changes in IL-10 and TGF-β mRNA expression, mainly because in the lower dose and placebo groups, the IL-10 and TGF-β mRNA levels decreased or showed little increase. In the highest dose group there were clear increases of IL-10 and TGF-β mRNA expressions that furthermore showed good correlation with each other. TGF-β is a cytokine that is secreted by monocytes, macrophages and lymphocytes as a latent isoform that forms a complex with latent TGF-β-binding protein. Bioactive TGF-β is generated by release of TGF-β from the complex. Macrophages and plasmin and urokinase are essential in this activation process (36). The secretion and extracellular activation of the latent isoform of TGF-β is essential for its function. Therefore, the slight changes observed in the expression of TGF-β mRNA may be significant.
There is no prospective data available on allergen-induced IL-5 or TGF-β mRNA expression as a function of age in children with AR. Our data suggest that AR could imply gradually increasing levels of allergen-induced IL-5 mRNA expression. The increased IL-5 expression was clearly inhibited by high-dose SLIT while only a trend but no statistically significant decrease was seen in IL-4/IFN-γ ratio. A relatively small sample size and lower levels of allergen-induced IL-4 mRNA expression when compared with IL-5 may explain the results partly. Decreased serum levels of IL-13, another Th2 type cytokine, have been observed during SLIT (37). Interleukin-5 is a key cytokine in eosinophil differentiation and activation and subsequent development of mucosal inflammation in AR and asthma (14, 16, 38). It is known that SCIT performed at early age prevents later development of bronchial asthma (39). It is possible that the mechanism behind this preventive effect is the inhibition of a ‘natural’ increase of IL-5 levels and consequent eosinophil maturation and accumulation on bronchial mucosa. Previously the anti-inflammatory effects of SLIT have been shown by observation that SLIT decreases the mucosal expression of the intracellular adhesion molecule (ICAM-1) and, as a consequence, the inflammatory infiltration (40). There was a negative correlation between the observed change of IL-5 and TGF-β mRNA expression mainly because of increased IL-5 and decreased TGF-β mRNA observed in the dose group 2 and decrease or no change in TGF-β and increase or no change in IL-5 in the dose group 1 and placebo group. Transforming growth factor-β mRNA appeared to decrease by age and the highest dose SLIT inhibited this decrease. Even though the function of bioactive TGF-β can only be distantly associated with mRNA expression, the observed minimal decrease of TGF-β mRNA expression and associated increase of IL-5 at a certain age may be phenomena in the development of bronchial asthma in children with AR. It seems that inhibition of IL-5 mRNA expression and thereby inhibition of eosinophil maturation and activation in the bronchial mucosa may be behind the prevention of bronchial asthma observed in our patients. The results from our non-validated telephonic survey of the asthma symptoms and medication use 5 years after showed that signs of asthma were seen in patients who, at the time of the study, had most symptoms and medication and who remained with elevated levels of LPSR, IL-5 mRNA expression and IL-4/IFN-γ ratio.
During SLIT using a high concentration of SQ-standardized allergen there is increased expression of regulatory cytokine IL-10 mRNA and in addition an inhibitory effect on IL-5 mRNA expression increase that is associated with TGF-β mRNA expression. These cytokine responses may contribute to downregulation of the allergic inflammation decreasing symptoms and LPSR by treatment. It remains to be investigated whether these phenomena eventually decrease the risk for later outcome of bronchial asthma.