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Keywords:

  • allergic rhinitis;
  • interleukin-10;
  • nasal epithelium;
  • provocation

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References

Background:  Despite constant exposure to micro-organisms and other immunogenic environmental factors, relatively very few immunological responses are initiated in the nasal mucosa. Although several mechanisms could play a role in maintaining this immune suppressive milieu, none of them have been validated. Previous data from our group suggested that locally produced interleukin (IL)-10 could be involved in maintaining local homeostasis.

Methods:  To investigate the role of epithelial IL-10 expression in the manifestation of allergic symptoms, we used immunohistochemistry to study the expression of IL-10 in the nasal epithelium of healthy individuals and house dust mite allergic patients. In the allergic patients, we determined potential correlations of epithelial expression with allergic symptoms, both at baseline and after allergen provocation.

Results:  IL-10 is expressed in the basal and differentiated epithelial cells of both healthy individuals and allergic rhinitis patients. In the allergic individuals, there is a strong negative correlation at baseline between the epithelial expression level of IL-10 and rhinorrhoea and sneezing, but not between that expression level and nasal blockage or peak nasal inspiratory flow (PNIF). This correlation disappears with steroid treatment or after allergen provocation, although the expression at baseline seems to predict PNIF scores after provocation.

Conclusions:  Our data not only reveals IL-10 expression by human nasal epithelial cells, but also suggests that nasal epithelial IL-10 regulates allergic symptoms. Targeting the regulation mechanisms affecting IL-10 or targeting the regulation mechanism affected by IL-10 could constitute new options for the treatment of allergic disease.

The incidence of allergic diseases in the western world is continuously increasing (1–5). Although this has led to extensive research, many aspects of the pathogenesis of allergic diseases are still unclear. In particular, there is no clear understanding of the factors that predict or are linked to the severity of symptoms in patients.

The local mucosal immune system, as present in the airways or the gastro-intestinal tract, guards major entry sites of the body against intrusion by foreign antigens. It, therefore, must be well-equipped to regulate the outcome of the immune response according to the type of antigen encountered. The concept of the inappropriate control of a local response to a harmless antigen as an important contributor to the pathogenesis of allergy is intriguing. As the nasal epithelium is an integral structural part of the local mucosal immune system it should also be seen as a functional part of a local regulation mechanism. The major function of the respiratory epithelium was thought to be primarily that of a physical barrier. However, the importance of the airway epithelial cells in regulating many of the inflammatory responses seen in respiratory diseases is increasingly being recognized (6–8).

Previous investigations by our group suggested that nasal epithelial cells expressed the immunomodulatory cytokine interleukin-10 (IL-10) (9). Given the immune regulatory roles described for IL-10 it is tempting to hypotheses that locally produced IL-10 may contribute to the regulation of the immune response and affect the manifestation of allergic symptoms. IL-10 production by various cells of the immune system has been found earlier to mediate immune suppression in dendritic cells (10, 11) and T lymphocytes (12–15) and to control IgE-isotype switching in B-cells (16, 17). Furthermore, IL-10 promotes IgA production (18, 19), cytolysis, neutrophil activation, and inhibits other aspects of effector responses, like chemokines, cytokines, and PGE2 production (20). The evidence that IL-10 contributes to the tolerogenic phenotype generally observed in the gut is undisputed (21, 22).

This study used the immunohistochemical staining of nasal biopsies from healthy and allergic individuals. The location of epithelial IL-10 expression at the border of the external and internal milieus suggests a role in the maintenance of mucosal homeostasis. To investigate this possibility, we studied the expression of IL-10 in the nasal epithelium of allergic patients at baseline and after allergen provocation, and correlated IL-10 expression levels to the severity of allergic symptoms in the allergic individuals. We also investigated the effect of local steroid treatment on these potential correlations.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References

Healthy individuals

After obtaining informed consent and Medical Ethical Commission approval, five healthy nonallergic subjects for whom rhinoplasty or septum correction had been planned were recruited for the study. These subjects had no history of allergic disease or prior use of nasal medication, did not have a concurrent respiratory tract infections, and had their nonallergic status confirmed with skin prick test for common aeroallergens (ALKabello, Nieuwegein, the Netherlands). The screening panel included tree mixture, grass, weed mixture, dust mite (Dermatophagoides pteronyssinus and Dermatophagoides farinae), cat, and dog. Before surgery, two biopsies were taken from the inferior turbinate with a Fokkens/Gerritsma forceps (Explorent, Tubingen, Germany), embedded in Tissue-Tek (Sakura, Zoeterwoude, the Netherlands) snap frozen in liquid N2, and stored at −80°C until further use.

Allergic patients

The study included 21 persistent rhinitis patients (median age 42, range 17–63) with a positive house dust mite (HDM) skin-prick test or radioallergosorbent test (RAST), and symptoms for over one year were included (Table 1). The medical ethics committee of the Erasmus MD approved the study and all patients gave their written informed consent.

Table 1.   Overview of the inclusion and the exclusion criteria
InclusionExclusion
House dust mite allergyNasal surgery 6 months prior to the study
Age between 16 and 65 yearsNasal polyps
NonsmokingSignificant septum deviation
HealthyImmunotherapy for house dust mite
Informed consentAsthma requiring inhaled corticosteroids
Pregnancy/lactation
Systemic corticosteroids 2 months prior to the study
Intranasal corticosteroids, antihistamines or other anti-allergic medication 1 month prior to the study

Study Design

At visit one, a baseline nasal mucosa biopsy was obtained and baseline symptom scores were recorded. After randomization, ten patients received fluticasone propionate aqueous nasal spray (FPANS) (200 μg in two actuations per nostril daily for seven days) and ten placebo. Patients reported to the clinic for visit two on day six of treatment, at which time and symptoms scores and peak nasal inspiratory flow (PNIF) were measured.

After bilateral nasal provocation, with a nasal spray delivering a fixed volume of 0.089 ml of 1000 biological units (BU)/ml HDM (total amount 2 × 89 BU) (ALK-Abellò, Nieuwegein, the Netherlands) symptom scores and PNIF were recorded every 15 min. After 90 min, patients left the clinic and recorded the symptoms and PNIF on an hourly basis for up to 8 h at home. On day seven, after administration of the study drug at home, patients re-entered the clinic for visit three. After the assessment of symptom scores and PNIF and the application of local anesthesia to the inferior turbinate, a nasal biopsy was obtained (24 hours after provocation). All the visits took place between February and May, outside the Dutch grass pollen season and neither vehicle provocation nor local anesthesia induced clinical symptoms. All biopsy specimens of nasal mucosa were taken from the inferior turbinate by the same investigator according to the previously described method (7, 23).

Assessment of symptoms

Visual analog scores (VAS): Rhinorrhoea, sneezing, and nasal blockage were recorded by placing a vertical mark on horizontal 100-mm line. Total VAS was calculated as the sum of the three VAS scores by symptom (total range 0–300) (24).

Peak nasal inspiratory flow

Peak nasal inspiratory flow (in-check inspiratory meter with facemask; Clement Clarke, Harlow, UK) was used to measure nasal airflow as described before (25). After initial instruction and training, for each assessment, the highest value of three repeat measurements was taken.

Immunohistochemistry

Specimens were snap-frozen and stored for immunohistology. In brief, each tissue specimen was cut into serial, 5-μm-thick sections on a Micron HM 560 Frigocut and transformed onto APES (amino-phosphate-ethylsilane) coated microscope slides (Sigma Chemical Co., St Louis, MO, USA), dried and stored at −80°C. The slides were stained within 3 months of sectioning. For staining, slides were brought up to room temperature and subsequently dried and fixed in acetone for 10 min at room temperature. The slides were then rinsed in phosphate buffer saline (PBS; pH 7.8) and placed in a semiautomatic strainer (Sequenza; Shandon, Sewickley, PA, USA) and incubated with 10% (v/v) normal goat serum (CLB, Amsterdam, the Netherlands) for 10 min. For block endogenous avidine and biotin all antibodies were diluted in 1% (w/v) blocking reagent (Roche, Basel, Switzerland). The sections were then incubated for 60 min with a mouse anti-human antibody against cytokeratin-14 (1 : 400) or appropriate isotype control at room temperature. Subsequently, the sections were rinsed with PBS for 5 min and incubated for 30 min with a biotinylated goat anti-mouse (1 : 50) immunoglobuline antiserum (Biogenics, Klinipathe, Duiven, the Netherlands), rinsed in PBS and incubated with streptavidin ss-AP (1 : 50) (Biogenics, Klinipathe, Duiven, the Netherlands) for 30 min at room temperature. Slides were then rinsed with PBS and Tris buffer (0.2 mol/l, pH 8.5) and incubated for 30 min with New Fuchsine (Chroma, Kongen, Germany) substrate (containing levamisole to block endogenous alkaline phosphatase [AP] enzyme activity). Finally, the sections were rinsed in distilled water, counterstained with Gill’s hematoxylin, and mounted in glycerine gelatine (Vectamount, Vector Laboratories, Peterborough, UK).

Staining with anti IL-10 took place using tyramide signal amplification. After incubation with biotinylated goat anti-mouse Ig serum, endogenous peroxidase was blocked using 0.2% (w/v) azide, 0.02% (v/v) hydrogen peroxide and 50% (v/v) methanol in PBS. Slides were then incubated with streptavidin conjugated peroxidase (NEN, Boston, MA, USA] for 30 min, biotinyl tyramide in Tris–HCl buffer for 10 min for amplification of the signal, alkaline-phosphatase conjugated goat-anti-biotin, and New Fuchsin substrate for 20 min.

Microscopically assessment of histochemical staining

The biopsies were coded and scored blind by three independent observers. Analysis included both baseline biopsies and biopsies after provocation. Basal epithelial cells were scored separately from differentiated epithelial cells. The intensity of staining epithelial cells was scored separately from quantity of positive epithelial cells. The following scoring system was used for intensity: high intensity staining = 3, intermediate intensity staining = 2, low intensity staining = 1, no staining = 0. For quantity: all epithelial cells in the relevant layer stain positive = 3, many cells stain positive = 2, few cells stain positive = 1, no cells stain = 0. Two biopsies were scored in every sample. This lead to a maximum score for each sample of 12 per observer and a maximum score of 36 in total per cell layer.

Statistical analysis

For changes in IL-10 expression over time, we used the paired Wilcoxon test. Spearman’s correlation test was performed to test correlations between the ranks of IL-10 expression and the ranks of symptom scores. The areas under the curve (AUCs) were calculated for the first hour after provocation (early phase) and for the remaining 23 h after provocation (late phase). P-values < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References

IL-10 is expressed in healthy and allergic nasal epithelium

We witnessed a very distinct pattern of IL-10 expression, with the cells lining the basal membrane staining very brightly for IL-10 along the entire length of the epithelium (Fig. 1A). The location of these cells – directly on top of the basal membrane – strongly suggests that these cells are the basal epithelial cells. To confirm the identity of these cells, we stained sequential sections of nasal biopsies for cytokeratin-14, which is exclusively expressed by basal epithelial cells (26). IL-10 expressing cells (Fig. 1A) and cytokeratin-14 expressing cells (Fig. 1B) do indeed co-localize. In addition to basal epithelial cells the differentiated epithelial cells also expressed. Upon comparisson IL-10 (not shown) with the healthy subjects, a similar pattern of IL-10 expression emerged in nasal biopsies of allergic patients, with staining of the basal epithelial cells (Fig. 1C) and the differentiated epithelial cells (Fig. 1D).

image

Figure 1.  (A) IL-10 expression in basal epithelial cells of a healthy individual. (B) Cytokeratine-14 expression in basal epithelial cells. (C) IL-10 expression in basal epithelial cells of allergic individual. (D) IL-10 expression in differentiated epithelial cells of allergic individual.

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At baseline, high nasal epithelial IL-10 expression in allergic patients correlates with low clinical symptoms

Allergic patients reported widely differing symptoms at the start of the study (Table 2) and the severity of these symptoms was significantly related to the level of epithelial IL-10 expression at baseline (Fig. 2). High levels of IL-10 correspond to low level of symptom severity and, conversely, low levels of IL-10 correspond to high symptom severity. This correlation could be observed between epithelial IL-10 scores for cells at baseline with the VAS score for rhinorrhoea at baseline (r = −0.704, P = 0.001 for the basal cells and r = −0.602, P = 0.005 for the differentiated cells; Fig. 2A). Epithelial IL-10 scores for cells at baseline also correlated, albeit less strongly, with VAS scores for sneezing at baseline (r = −0.490, P = 0.028 for the basal cells and r = −0.523, P = 0.018 for the differentiated cells; Fig. 2B). At baseline, nasal blockage and PNIF scores were not significantly correlated with epithelial IL-10 expression.

Table 2.   Characteristics and baseline values of the patients, median, minimum and maximum values
 PlaceboFPANS
  1. HDM, house dust mite; VAS, Visual analog scores;PNIF, peak nasal inspiratory flow.

Characteristics
 Number of patients 1010
 Age30.5 (17–45)44.0 (19–62)
 Sex (male/female)5/56/4
 Wheal size HDM (mm)5 (4–20)5 (4–9)
Baseline values
 Total VAS (mm)55 (6–184)52.5 (13–155)
 VAS rhinorrhoea17 (0–60)21.5 (0–54)
 VAS sneezing13,5 (0–50)0 (0–51)
 VAS nasal blockage19 (6–74)24 (0–86)
 PNIF (l/min)137.5 (70–260)125 (60–120)
 IL-10 expression basal epithelial cells32 (21–36)30 (19–35)
 IL-10 expression differentiated  epithelial cells11 (0–20)18 (4–29)
image

Figure 2.  IL-10 scores for basal and differentiated epithelial cells at baseline correlate with symptom scores for (A) Visual analog scores (VAS) rhinorroea at baseline with basal epithelial cells (r = −0.692, P = 0.001), (B) VAS rhinorroea at baseline with differentiated epithelial cells (r = −0.520, P = 0.032), (C) VAS sneezing at baseline with basal epithelial cells (r = −0.490, P = −0.028), and (D) VAS sneezing at baseline with differentiated epithelial cells (r = −0.523, P = 0.018).

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In allergic patients, nasal provocation leads to up-regulation of epithelial IL-10

Concomitant with the increase of signs and symptoms, there was a significant rise in IL-10 expression in the differentiated epithelial cell after allergen provocation (P = 0.032) compared with baseline. After provocation, the median of IL-10 scores was doubled (median before 12, after provocation 24) (Fig 3B). Whereas IL-10 expression in differentiated cells increased after provocation, no such increase was seen in IL-10 expression by the basal epithelial cells (Fig. 3A).

image

Figure 3.  In the placebo group, nasal provocation did not lead to significant change in IL-10 expression by the basal epithelial cells (3A, Wilcoxon P = 0.396), but was accompanied by a rise in IL-10 scores for the differentiated epithelial cells (3B, Wilcoxon P = 0.043). In the FPANS group, we observed no significant difference between pre- and postprovocation epithelial IL-10 levels for basal cells (3C, Wilcoxon P = 0.512) or differentiated cells (3D, Wilcoxcon P = 0.779).

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In allergic patients, the level of epithelial IL-10 does not only reflect rhinorrhoea and sneezing symptoms at baseline, but also predicts PNIF scores directly after provocation

Earlier, we showed that nasal HDM provocation led to significant up-regulation of allergic signs and symptoms in this patient group (7). In the placebo group, high IL-10 scores at baseline correlated with high AUC for the PNIF during the early phase response after allergen provocation, both for basal cells (r = 0.833 and P = 0.003) and for the differentiated epithelial cells (r = 0.699 and P = 0.034) (Fig. 4). This correlation was only seen in the placebo group for the early phase PNIF response and not for the late response, nor for any of the other early or late VAS AUC scores (rhinorrhoea, sneezing, and nasal blockage).

image

Figure 4.  In the placebo group, epithelial IL-10 scores correlate with the PNIF area under the curve of the early response after provocation for (A) basal cells; r = 0.833 and P = 0.003 and (B) differentiated cells; r = 0.699 and P = 0.034.

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Steroid treatment prevent up-regulation of IL-10 expression in differentiated cells

There were no differences in allergic patients allocated to the two treatment groups (placebo or FPANS) in terms of characteristics or symptoms at the start of the study (Table 2). In the group receiving active treatment, FPANS prevented the up-regulation of epithelial IL-10 after nasal provocation in the differentiated epithelial cells (Fig. 3D), but had no effect on the basal epithelial cells (Fig. 3C). Moreover, the link between PNIF and the expression of IL-10 in epithelial cells was no longer present in the FPANS-treated group (not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References

The study shows for the first time that the potent immunosuppressive cytokine IL-10 is expressed by human nasal epithelium and that its expression level is negatively correlated with allergic symptoms.

The link between the signs and symptoms of disease and a cellular or inflammatory marker is a research topic of great interest. The identification of such a marker is highly relevant as such a marker would be an important target for treatment. Although allergen-specific IgE is required for symptoms to develop, the level of IgE has no predictive value for the level of symptoms (27). Indeed, a substantial part of the population has IgE for a given allergen, but shows no symptoms at all (28–30). In these individuals (and also in allergic individuals) there is no clear relationship between the amount of mediators released from mast cells and the level of symptoms (27, 31). Our data show that in a group of patients with persistent rhinitis and proven HDM allergy, individuals with relatively high levels of IL-10 expression in basal epithelium suffer less from allergy, conversely, those with relatively low IL-10 expression levels suffer more from their allergy. Given the statistical correlation at baseline between low symptom scores for rhinorrhoea and sneezing with high IL-10 levels it is tempting to speculate that there is a causal relationship between the two.

Because of the continuous all-year-round exposure to HDM it is impossible to establish a clear, symptom-free baseline in perennial rhinitis patients, as is evidenced by the varying level of symptoms in our patient group. Consequently, the observed pattern of variable IL-10 expression and disease symptoms might be the result of low-grade and variable exposure to allergen. Allergen provocation in our model (and also in real life) leads to an increase in signs and symptoms. In our model, this is accompanied by an increase in the expression of IL-10 in the epithelium. Furthermore, we found nasal epithelial IL-10 expression and symptom VAS scores at baseline to be inversely related. Combining these two observations, we can begin to conclude that allergic symptoms and IL-10 levels are not regulated independently of each other. Moreover, since high allergen exposure evidently does not result in a fall in IL-10 levels, high symptom scores can not be held responsible for low IL-10 levels at baseline. Conversely, low symptom scores cannot be responsible for high IL-10 expression. At baseline, then, high IL-10 expression must be responsible for low allergic symptoms. Our data, therefore, provides a strong argument for the idea that epithelial IL-10 expression protects people from allergic symptoms.

This hypothesis raises questions about a possible mechanism. A hint comes from the observation that not all symptom scores were correlated with epithelial IL-10 expression. Whereas rhinorrhoea and sneezing do correlate with epithelial IL-10 expression, nasal blockage does not. Interestingly, as medication for allergic rhinitis patients, antihistamines can suppress symptoms of rhinorrhoea and sneezing, but are usually less efficient in treating nasal blockage (32, 33). This indicates that there is a mechanism in which IL-10 influences allergic symptoms via histamine. In vitro data supports the concept that IL-10 might reduce symptoms by inhibiting histamine. Royer et al. studied the effects of IL-10 on the release of pro-inflammatory mediators by activated mast cells and found that histamine release was inhibited in the presence of recombinant IL-10 (32, 34, 35).

The relationship between IL-10 and histamine is complex, as histamine has been shown to increase the synthesis and the release of IL-10 from inflammatory cells (36). Not only in macrophages, but also in T cells, IL-10 production was up-regulated after histamine stimulation (37, 38). As nasal epithelial cells have histamine receptors (39–41), it is conceivable that expression of epithelial IL-10 is also up-regulated in response to histamine. In this way, histamine, induced by repeated allergen challenge, appears to induce a complex negative feedback loop by up-regulating IL-10. However, the expression of IL-10 could also be a direct effect of epithelial cells being exposed to the HDM antigen. Epithelial cells have been shown to recognize allergenic mixtures through protease activated receptors (PARs) (42). This could be particularly relevant to HDM where a number of the dominant allergens (DerP1 and DerP2) exhibit protease activity (43, 44). To distinguish between a direct and an indirect effect of allergen exposure upon IL-10 expression it would be relevant to study the IL-10 response to HDM in nonallergic individuals.

After allergen provocation, the inverse linear relationship between symptoms and IL-10 levels is completely lost in the placebo group. Currently we can only speculate that allergen loads in our provocation model are sufficiently high to induce maximum symptom levels and, directly or indirectly, induce similarly maximum IL-10 levels. Furthermore, in the FPANS group, there is no correlation between IL-10 levels and symptoms. In this group, the levels of symptoms are lower than in the placebo group, which renders an argument inplausible for the placebo group after provocation. Our current model is not suitable for addressing the issue of corticosteroid influence on IL-10 expression, histamine release, and allergic symptoms. Steroids may directly affect IL-10 or histamine levels or the levels of their respective receptors. In vitro experiments using primary epithelial cells exposed to allergen, histamine, or steroids are suggested to resolve this rather complex interplay.

Our data not only reveal IL-10 expression by human nasal epithelial cells, but also show that nasal epithelial IL-10 both regulates and responds to allergic symptoms. This effect may well be mediated through histamine. These observations suggest that IL-10 may not only regulate the onset of the immune response, but also influence the effector phase of the allergic response. In this concept, targeting the regulation mechanisms affecting IL-10 or targeting the regulation mechanism affected by IL-10 might constitute new options for the treatment of allergic disease.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
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