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Keywords:

  • allergic rhinitis;
  • asthma;
  • chronic sinusitis;
  • eosinophil;
  • natural killer T cells

Abstract

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

Background:  Chronic sinusitis (CS) with asthma generally exhibits a high degree of sinus tissue eosinophilia and recurrence often occurs even after surgical therapy. However, the cause has not yet been fully clarified.

Aims of the study:  To elucidate the pathogenesis of this refractory disease, we examined the infiltration of natural killer T (NKT) and type 1 helper T (Th1)/type 2 helper T (Th2) cells, and the cytokine expression in the sinus mucosa.

Methods:  Sinus mucosal specimens were obtained surgically from 16 CS patients with nasal polyps. The NKT cells, Th1/Th2 cells and the expression of IL-4, IL-5, IL-13 and IFN-γ were examined by a polymerase chain reaction or flow cytometry. Nasal mucosal specimens from six other patients with allergic rhinitis (AR) were examined in a similar manner.

Results:  The NKT cells were detected to varying degrees in the sinus mucosa from asthmatic CS patients, but neither in the nonasthmatics nor in the nasal mucosa from the patients with AR. The Th2 cells and Th2 cytokines were expressed at significantly higher levels in the sinus mucosa from the CS patients with asthma in comparison to those without asthma. However, the Th1 cell infiltration and IFN-γ expression were not different between these groups.

Conclusion:  Natural killer T cells may, therefore, play important roles in the enhanced Th2 cytokine expression and increased infiltration of Th2 cells and eosinophils observed in the sinus mucosa from asthmatic CS patients through MHC-independent mechanisms.

Abbreviations:
α-GalCer

α-galactosylceramide

AR

allergic rhinitis

CS

chronic sinusitis

CSE

chronic sinusitis with sinus tissue eosinophilia

CSN

chronic sinusitis with sinus tissue neutrophilia

NKT cell

natural killer T cell

Chronic sinusitis (CS) is a common disease that affects about 20% of the population, and is usually treated by conservative medical management (1). Many cases of CS with polyps in Japan predominantly exhibit an infiltration of neutrophils in the sinus and are generally responsive to macrolide therapy (2, 3). Although some cases of CS with sinus tissue neutrophilia (CSN) require surgical intervention, their prognosis is generally satisfactory (1, 4).

However, there is a severe subform of CS, which exhibits marked sinus infiltration with eosinophils and, to a lesser degree, neutrophils and other infiltrating cells. These cases of CS with sinus tissue eosinophilia (CSE) also exhibit the characteristics of multiple nasal polyps, relapse even after surgery, and a high degree of complication with asthma (5, 6). Although the involvement of fungal infection (7) and some bacterial toxins as allergens or superantigens has been reported (8), the exact mechanisms of development of CSE have not yet been ascertained. The involvement of a type 1 allergic reaction in CSE remains uncertain because it occurs infrequently with allergic rhinitis (AR) and CSE is more frequently linked to nonatopic rather than atopic asthma (9–11).

Human natural killer T (NKT) cells express the invariant Vα24JαQ paired with the Vβ11 antigen receptor and play important roles in innate immunity (12). Natural killer T cells are activated by a specific glycolipid antigen, α-galactosylceramide (α-GalCer), in a CD1d-dependent manner (13). CD1d is an HLA class I-like antigen priming molecule that does not exhibit any allelic polymorphism. Therefore, the NKT activity is not restricted to MHC antigens. Recently, the involvement of NKT cells in the development of airway hypersensitivity in mice (14) and the detection of NKT cells in bronchoalveolar-lavage fluid samples from patients with moderate to severe asthma (15) were reported.

In the present study, we examined NKT cells, as well as Th1/Th2 cells and the expression of various cytokines in the sinus mucosa from asthmatic and nonasthmatic CS patients.

Methods

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

Patients and samples

All patients included in this study complained of CS with nasal polyps, were operated at Chiba University Hospital from January to October 2006. Chronic sinusitis was clinically determined as the presence of a combination of major symptoms (such as nasal discharge, nasal obstruction, hyposimia, facial congestion, facial fullness) and computed tomography (CT) findings. The symptoms were scored in all patients. The symptom scoring range in this study had four options as reported previously (16, 17). The exclusion criteria included cystic fibrosis, immotile cilia syndrome, autoimmune diseases and being positive response to HIV, hepatitis C virus and for hepatitis B surface antigens. The diagnosis of asthma was based on the American Thoracic Society criteria (18) and patient with aspirin-intolerant asthma was proven to have histories of severe bronchoconstriction after the ingestion of at least two different nonsteroidal anti-inflammatory drugs.

All patients received various medications including anti-histamines, anti-leukotrienes, and local or systemic steroids but failed to show any significant improvements. All medications were withdrawn for 1 week prior to the operation, however, the one patient (#15 in Table 1) could not stop using oral steroids because of asthma. A preoperative examination included allergen specific IgE (mite, cedar, house dust, Candida, Alternaria by CAP radio-allergo sorbent test) and blood eosinophil counts by using a standard automated cell counter. Surgical biopsy samples from the ethmoidal sinus mucosa, including polyps, were obtained and examined for inflammatory cells by hematoxylin and eosin staining. The sinus mucosa specimens from each patient were immediately cut into small pieces and incubated with PBS containing 5% fetal calf serum (FCS), 1 mg/ml collagenase A (Roche, Mannheim, Germany) and 0.2 mg/ml DNase I (Sigma-Aldrich, St Louis, Mo, USA) at 37°C for 35 min. After incubation with 20 mM EDTA for 5 min at room temperature, the specimens were passed through a fine nylon mesh. Mononuclear cells were subsequently obtained by the Ficoll–Hypaque technique and were used for the polymerase chain reaction (PCR) assay. For the flow cytometric analysis, collagenase and DNase I were not used during the preparation of the mononuclear cells to avoid interference with the cell surface markers.

Table 1.   Profile of chronic sinusitis (CS) and allergic rhinitis (AR) patients
 PatientAgeSexBlood eosinophils (%)ARCS with polypsTissue eosinophils*Tissue neutrophils*E/N ratio†CS Score‡
  1. n.t., not tested.

  2. *Number of eosinophils and neutrophils/×400 magnification.

  3. †Number of tissue eosinophils/number of tissue neutrophils.

  4. ‡Severity scoring of CS major symptoms (from ref. 17).

CS without asthma154Mn.t.+5900.062.6
256M1.1+41200.032.4
355M2.6+61800.032.6
477M1.0+21100.022.6
538M3.7+124000.032.8
665M2.5+31400.022.6
771F1.8+2700.032.8
864Mn.t.+31200.032.4
932M3.4++61500.042.6
1057M6.6++135502.72.8
CS with asthma1143F1.8+421200.352.8
1261M11.2+>500110>4.53
1325M4.7+125502.52.6
1457F16.9+190553.52.8
1545M2.6+330804.12.6
1656F8.0+>500100>5.02.8
AR1753M4.9+    
1866M7.1+    
1925F14.7+    
2037M7.1+    
2154Fn.t.+    
2222M4.9+    

Another six patients with serious perennial AR due to Dermatophagoides pteronyssinus were also enrolled in this study. The diagnosis of AR was based on the criteria of Okuda et al. (19), namely, typical perennial allergic nasal symptoms and a positive CAP radio-allergo sorbent test result against D. pteronyssinus. The inferior nasal turbinate mucosa was obtained surgically and was examined in the same manner.

The study received prior approval from the Ethics Committee of the Chiba University. Informed consent was obtained from each patient.

PCR analysis

Total RNA was isolated from the mononuclear cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using oligo primers and Superscript II RT (Invitrogen). The specific primers for the detection of IL-4, IL-5, IL-13, IFN-γ CD3, GAPDH were purchased from Applied Biosystems (Foster City, CA, USA). For the detection of Vα24Jα18, the forward primer 5′-CCTCCCAGCTCAGCGATTC-3′, the reverse primer 5′-TATAGCCTCCCCAGGGTTGA-3′ and the probe FAM-5′-CCTCCTACATCTGTGTGGTGAGCGACA-3′-TAMtph were used (20). The samples were subjected to a real-time PCR analysis on an ABI PRISM7000 Sequence Detection system.

Flow cytometric analysis

The mononuclear cells were incubated on ice with the appropriate staining reagents: fluorescein isothiocyanate (FITC)-conjugated anti-CD4 monoclonal antibody (mAb), R-phycoerythrin (R-PE)-conjugated anti-CCR4 mAb, allophycocyanin (APC)-conjugated anti-CXCR3 mAb, or FITC-, RPE- or APC-conjugated isotype match control mAb (BD Bioscience, San Jose, CA, USA) (21).

The cells were subjected to a flow cytometric analysis using a flow cytometer (FACS Calibur: BD, Franklin Lakes, NJ, USA) and the results were analyzed using the FlowJo software program (Tree Star, Inc, Ashland, OR, USA).

Statistical analysis

A statistical analysis was performed using the Wilcoxon rank sum test or the Wilcoxon signed rank test for paired and unpaired data. P-values <0.05 were considered to be statistically significant.

Results

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

A total of 16 patients were analyzed. All patients had a severe loss of smell and nasal obstruction. Allergic fungal sinusitis with eosinophil granule-rich mucin and diagnostic clinical characteristics (17) was not detected in these patients. Six patients had asthma and two of them were aspirin-intolerant asthma (#12, 16 in Table 1). All these asthma patients were negative for specific IgE to inhalant allergens.

Based on the number of sinus mucosal eosinophils, the CS patients were divided into CSE [number of eosinophils/number of neutrophils (E/N ratio) >1.0] and CSN (E/N ratio <1.0). Of the 16 patients with CS, six were considered to be CSE and ten were CSN. Five of the six CSE patients had asthma and one had AR but not asthma. Of the 10 patients with CSN, one had asthma and one had AR.

IL-5 mRNA expression was observed in the mononuclear cells isolated from the sinus mucosa from all patients with asthma or with CSE and the expression was significantly higher than in patients without either asthma or with CSN. These findings were similar to the expression of IL-4 and IL-13 (Fig. 1). Although IFN-γ was detected in the sinus mucosa from each of the patients, there was no significant difference between patients with and without asthma (data not shown).

image

Figure 1.  IL-4, IL-5 and IL-13 mRNA expression in mononuclear cells isolated from sinus mucosa in chronic sinusitis patients. Significantly increased expression was observed in patients with asthma by real time PCR. This indicated that the T cells produced significant levels of IL-4, IL-5 and IL-13.

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In the flow cytometric analysis of the sinus mucosa, the number of CXCR3-positive T cells was not different between the patients with and without asthma, nor between CSE and CSN. However, a significantly greater number of CCR4-positive T cells detected in patients with asthma or with CSE in comparison to those without either asthma or with CSN (Fig. 2).

image

Figure 2.  The ratio of CXCR3-positive and CCR4-positive T cells to CD4-positive T cells in mucosal specimens from chronic sinusitis (CS) patients with or without asthma, or in nasal mucosa from patients with allergic rhinitis (AR). Although the ratio of CXCR3-positive T cells were not different among these groups of patients, CCR4-positive T cells were increased significantly in asthmatic CS and AR patients compared with the nonasthmatic CS patients.

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NKT cells were detected at various levels in the peripheral blood of all CS patients, however, in sinus mucosa NKT cells were only detected in the patients with asthma. Five out of six patients with CSE and only one patient with CSN exhibited NKT cells in the sinus mucosa (Fig. 3). The patient with CSE who did not exhibit NKT cells in the sinus mucosa had perennial AR due to mites, but not asthma. The patient with CSN exhibited NKT cells in the sinus mucosa, combined with asthma, and an expression of Th2 cytokines and infiltration of CCR4-positive T cells was observed in the sinus mucosa. The expression levels did not correlate with the severity of CS.

image

Figure 3.  The relative expression of natural killer T (NKT) cells to CD3 T cells. NKT cells were detected in the sinus mucosa from five of six chronic sinusitis with sinus tissue eosinophilia (CSE) patients and one of 10 chronic sinusitis with sinus tissue neutrophilia (CSN) patients. The one CSE patient (indicated by the arrow) who was negative for NKT cells did not have asthma, but exhibited allergic rhinitis. The single CSN patient (indicated by the arrow) who was positive for NKT cells had asthma.

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In the inferior nasal turbinate mucosa from the six patients with AR, the CCR4-positive T cells were as high as those in the CSE patients, and the CXCR3-positive T cells were similar to those of CSN and CSE. However, NKT cells were not detected in the nasal mucosa from any of the patients with AR (Fig. 3).

Discussion

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

In the present study, a significantly more number of Th2 cells, and consequently, expression of Th2 cytokines, was observed in the sinus mucosa from patients with asthma or with CSE. The low level of expression of Th2 cytokines and infiltration of Th2 cells in the sinus mucosa among the patients without asthma or with CSN, along with the fact that there was no difference in the Th1 cell infiltration among these sinus mucosa, altogether indicate that the Th2-predominant state in the sinus mucosa is characteristic of CS with asthma and of CSE.

A similar Th2-predominant state was observed in the nasal mucosa from patients with AR, which is a typical type I allergic disease mediated through allergen-specific IgE antibodies, although the pathogenesis of CSE is considerably different. The complication rate of AR in CSE is not high, and highly complicated asthma with CSE is nonatopic rather than atopic (9–11).

In this study, we detected NKT cells at various levels in the PBMCs from all patients, with no significant difference between the CSE and CSN patients. In the sinus mucosa, NKT cells were recognized in all the patients with asthma, in five out of the six patients with CSE and in one CSN patient. The patient with CSN who was positive for NKT cells in the sinus mucosa had asthma and exhibited some degree of infiltration of eosinophils and Th2 cells, despite the predominant infiltration of neutrophils. In contrast, the patient with CSE who was negative for NKT cells in the sinus mucosa had AR and a Th2-predominant state in the sinus mucosa, but this was not combined with asthma. Natural killer T cells were not detected in the sinus mucosa from the CS patients without asthma, or in the nasal mucosa from the AR patients. These results suggest that NKT cells are not directly related to the development of allergy, but they may play important roles in the development of sinus disease combined with asthma as ‘one airway, one disease (22)’, and substantially different mechanisms must exist between allergy and the accumulation of eosinophils in the sinus mucosa in CSE. In a recent study with mice, Meyer et al. (23) demonstrated eosinophil accumulation in the airway and the development of airway hyperreactivity following the respiratory administration of α-GalCer, a specific NKT cell-activating glycolipid antigen, and that eosinophil accumulation was observed in the respiratory tract even in MHC class II-deficient mice. In the present study, we clearly demonstrated the presence of NKT cells in the sinus mucosa from CS patients with asthma, but not from CSE patients without asthma, nor in the nasal mucosa from AR patients. Chronic sinusitis with sinus tissue eosinophilia may be composed of several diseases with different etiologies. Natural killer T cells were detected in the sinus mucosa from CS patients with asthma and are, therefore, suggested to participate in the local immune reactions of the sinus mucosa. The elucidation of the mechanisms of NKT cell activation in the sinus mucosa and its relationship to the process of tissue eosinophilia may, therefore, lead to the fundamental resolution of these refractory diseases, including asthma.

References

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