Distinct immunopathologic characteristics of various types of chronic rhinosinusitis in adult Chinese

Article Outline

• Abstract
• Methods
  • Subjects
  • Histologic analysis
  • Immunohistochemistry
  • Quantitative PCR
  • Statistical analysis
• Results
  • Histopathologic examination
  • Immunohistochemical examination
  • Quantitative PCR
• Discussion
• Table E1. 
• Table E2. 
• References
• Copyright

Background

Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP) is reported to be different in inflammatory patterns of the sinonasal mucosa in white patients. Studies in nonwhite populations may further be helpful to understand the pathogenic mechanisms of CRS.

Objective

To investigate the immunopathologic profiles of CRSwNP and CRSsNP in adult Chinese.

Methods

Histologic characteristics of surgical samples were analyzed in 50 controls, 94 CRSsNP patients, and 151 CRSwNP patients. Tissue samples from 17 controls, 36 CRSsNP patients, and 45 CRSwNP patients were stained for CD3, CD4, CD8, CD20, CD68, myeloperoxidase, and dendritic cell lysosome-associated membrane protein. Expression profiles of transcription factors of T-cell subsets in relation to cytokines and a marker of natural killer T cell (Vα24) were examined by means of quantitative RT-PCR.

Results

Over half of CRSwNP patients presented noneosinophilic inflammation. CRSwNP had a higher number of eosinophils, plasma cells, and CD3⁺, CD8⁺, CD20⁺, and CD68⁺ cells and a lower myeloperoxidase expression rate than CRSsNP. Expression levels of transcription factors and cytokines of T_H1/T_H2/T_H17 were increased, whereas the expression rate of Forkhead box p3 and TGF-β1 was decreased in both CRSsNP and CRSwNP compared with controls. Comparing CRSsNP and CRSwNP, CRSsNP had higher levels of IFN-γ expression, whereas only eosinophilic CRSwNP demonstrated an enhanced expression of GATA-3 and IL-5. Compared with noneosinophilic CRSwNP, an exaggerated T_H2/T_H17 reaction and Vα24 expression were found in eosinophilic CRSwNP.

Conclusion

Both Chinese CRSsNP and CRSwNP patients demonstrate impaired regulatory T cell function and enhanced T_H1/T_H2/T_H17 responses. CRSsNP is confirmed to be a predominant T_H1 milieu, whereas T_H2 skewed inflammation with predominant T_H17 reactions, and infiltration of natural killer T cells can be demonstrated only in eosinophilic CRSwNP, but not in noneosinophilic CRSwNP.

Key words: Chronic rhinosinusitis, nasal polyps, eosinophils, T_H1, T_H2, T_H17, regulatory T cells, natural killer T cells

Abbreviations used: CRS, Chronic rhinosinusitis, CRSsNP, Chronic rhinosinusitis without nasal polyps, CRSwNP, Chronic rhinosinusitis with nasal polyps, DC-LAMP, Dendritic cell lysosome-associated membrane protein, FOXP3, Forkhead box P3, HE, Hematoxylin-eosin, NKT, Natural killer T, HP, High power, LP, Lamina propria, NP, Nasal polyp, PAS, Periodic acid-Schiff, RORC, Retinoic acid–related orphan receptor C, T-bet, T-box transcription factor, Treg, Regulatory T

Chronic rhinosinusitis (CRS) is characterized by inflammation of the mucosa of the nose and paranasal sinuses. It remains a significant health problem with a considerable socioeconomic burden and is still increasing in prevalence and incidence.¹, ² Over the last 2 decades, increasing evidence suggests that CRS is a heterogeneous group of sinus disorders that may represent an umbrella covering different disease entities.¹, ², ³ Studies from white patients showed that CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP) display distinct features on the basis of histomorphology and the expression of inflammatory and remodeling mediators.¹, ⁴, ⁵, ⁶, ⁷ CRSwNP was characterized by a T_H2-skewed eosinophilic inflammation, whereas CRSsNP represented a predominant T_H1 milieu.³, ⁷ However, until now, only few studies have been conducted in Chinese patients with CRS, and histologic and immunologic features of CRS in Chinese remain poorly defined.

Chronic rhinosinusitis with nasal polyps is the most commonly studied type of CRS. Eosinophilic inflammation is found to be a distinctive characteristic in 65% to 90% of total nasal polyp (NP) cases in white subjects. ¹, ², ⁸ Recently, a limited number of studies have suggested that NPs in Asians present different immunopathologic features compared with those in white patients,⁹, ¹⁰, ¹¹ implying that distinct mechanisms may underlie the pathogenesis of NPs between these 2 ethnic groups. Kim et al⁹ found that eosinophilic NP accounted for only 33.3% of the 30 NP samples obtained from Koreans. Zhang et al¹⁰, ¹¹ discovered that NP samples from southern Chinese demonstrated a T_H1/T_H17 cell pattern with a minor eosinophilic inflammation. Although these studies indicated the differences in immunopathologic characteristics of NPs between Asian and white patients, the characteristics of CRSsNP in Asians and whether there exists a difference between CRSwNP with different pathologic features (for example, eosinophilic versus noneosinophilic) have not yet been investigated. Further understanding of immunopathologic features of CRS in nonwhite populations will be of great value to elucidate the pathogenesis of CRS. The purposes of the current study were (1) to characterize the histologic features of CRSsNP and CRSwNP in a large sample of adult Chinese patients, and (2) to investigate further the pattern of inflammatory cell infiltration, the expression profiles of T_H1/T_H2/T_H17/regulatory T (Treg)–specific transcription factors in relation to cytokines, and a role of natural killer T (NKT) cells in different subgroups of patients with CRS comprehensively.

Back to Article Outline

Methods 

Subjects 

This study was approved by the Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology and was conducted with written informed consent from patients.

Ninety-four patients with CRSsNP, 151 patients with CRSwNP, and 50 controls were enrolled for histologic study, and an additional 36 CRSsNP, 45 CRSwNP, and 17 control subjects were recruited for immunologic study. Clinical data of patients are summarized in this article's Table E1 in the Online Repository at www.jacionline.org. The diagnosis of CRS including CRSsNP and CRSwNP was made according to the current European EAACI Position Paper on Rhinosinusitis and Nasal Polyps² and American guidelines.¹ All patients with CRS came from central China and had bilateral CRS. They had failed to respond to medical therapy and subsequently underwent endoscopic sinus surgery. Diseased ethmoid sinus mucosa tissues from the most hypertrophied and hyperemic regions and NP tissues from the apex region of polyps were collected during surgery. Control subjects were patients undergoing septoplasty because of anatomic variations and did not have sinus disease. One biopsy was taken from the inferior turbinate mucosa of control patients during septal plastic surgery. The atopic status was evaluated by skin prick test to a standard panel of aeroallergens. The diagnosis of asthma and aspirin sensitivity was based on history and physician diagnosis. Oral glucocorticoid and intranasal steroid sprays were discontinued at least 3 months and 1 month before surgery, respectively. Before surgery, all patients with CRS received 3 to 5 days of antibiotics. Subjects who had an antrochoanal polyps, cystic fibrosis, fungal sinusitis, primary ciliary dyskinesia, or gastroesophageal reflux disease were excluded from the study.

In histologic study, the samples were fixed in formaldehyde solution and later embedded in paraffin for hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and Masson staining. For immunologic study, freshly obtained tissue was divided into 2 parts. One part was fixed in formaldehyde solution for HE and immunohistochemical staining, and the other part was immediately snap-frozen in liquid nitrogen and stored at –80°C for later quantitative RT-PCR experiments.

Histologic analysis 

Paraffin sections (5 μm) were stained with HE, PAS, and Masson dye. Afterward, the stained sections were observed by 2 independent physicians who were blind to the clinical data. In case of disagreement (the 2 counts differed by >10%), a consensus was reached by reviewing the specimen at a multihead microscope by our research team. Sections stained with HE were used to determine the general pathologic features of tissues at ×40 and ×100 magnification. The number of eosinophils, mononuclear cells, plasma cells, total inflammatory cells, and glands in the lamina propria (LP) was counted at high power (HP) magnification (×400), and 10 HP fields were randomly selected and analyzed. Results were expressed as cells or glands per HP field of LP. Sections stained with PAS were used to evaluate the number of goblet cells. Three sites containing intact epithelium were randomly selected and the results were expressed as cells per millimeter of epithelium. Sections stained with Masson were used to determine the degree of fibrous hyperplasia. Referring to the method of Wenzel et al,¹² both CRSsNP and CRSwNP were classified as eosinophilic when percent eosinophils exceeded twice the SD of the mean of controls (4.77% + 2 × 2.47% = 9.71%; therefore, 10% was chosen as the cutoff).

Immunohistochemistry 

After deparaffinization and rehydration, sections were subjected to heat-induced antigen retrieval using Target Retrieval Solution (Dako, Carpinteria, Calif). For endogenous peroxidase inhibition, 3% hydrogen peroxidase was used. The normal serum of secondary antibody was used to block nonspecific binding. Sections were stained with mAb of CD3, CD4, CD8, CD20, and CD68 (Zhongshan Golden Bridge Biotechnology, Beijing, China) and dendritic cell lysosome-associated membrane protein (DC-LAMP; R&D; Minneapolis, Minn), and polyclonal antibody of myeloperoxidase (Zhongshan Golden Bridge Biotechnology), respectively. CD3, CD4, CD8, CD20, CD68, and myeloperoxidase were detected by using the polyglucosan-horseradish peroxidase complex method (Changdao Biotechnology, Shanghai, China) according to the manufacturer's instructions. DC-LAMP was detected by using the streptavidin-peroxidase complex method with a histostain-plus kit (Boster Biotechnology, Wuhan, China) as described previously.¹³ Color development was achieved with 3′,3′-diaminobenzidine, which rendered positive cells brown. Finally, sections were counterstained with hematoxylin and mounted. Species-matched and isotype-matched antibodies were used as negative controls. The number of positive cells per HP field was analyzed. Five fields were randomly selected and scored by 2 independent observers as in the histologic study.

Quantitative PCR 

Total RNA was extracted from tissue samples by using TRI reagent (Invitrogen, Carlsbad, Calif) and treated by using a DNA-free kit (Fermentas, Hanover, Md) to remove contaminating DNA. One microgram total RNA was reverse-transcribed to cDNA with random hexamer primer as previously described.¹⁴ The PCR of transcription factors (T-box transcription factor [T-bet], GATA-3, forkhead box P3 [FOXP3], and retinoic acid–related orphan receptor C [RORC]), cytokines (IFN-γ, IL-4, IL-5, IL-17A, IL-22, IL-23, IL-10, and TGF-β1), and Vα24 (a marker of NKT cell) was performed by using the SYBR Premix Ex Taq kit (TaKaRa Biotechnology, Dalian, China) with appropriate primers constructed from published sequences (see this article's Table E2 in the Online Repository at www.jacionline.org). cDNA equivalent to 40 ng total RNA was used to perform quantitative PCR, as mentioned elsewhere.¹³ Relative gene expression was calculated by using the comparative CT method. A inferior turbinate sample was used as a calibrator.¹³, ¹⁴ Glyceraldehydes-3-phosphate dehydrogenase was used as a housekeeping gene for normalization, and a no template sample was used as a negative control.

Statistical analysis 

For continuous variables, results are expressed as medians and interquartile ranges, or in bar charts the represent medians and interquartile ranges. In continuous variables, when comparisons were made between groups, the Kruskal-Wallis H test was used to assess significant intergroup variability. The Mann-Whitney U 2-tailed test was used for between-group comparison. Differences in proportions between groups were tested by the χ² test. The Spearman test was used to determine correlations. Significance was accepted at P < .05.

Back to Article Outline

Results 

Histopathologic examination 

On the basis of the features of tissue remodeling of LP,¹⁵, ¹⁶ it was found that edematous type was the commonest histopathologic type in CRSwNP (66%), whereas glandular hyperplastic type was the commonest histopathologic type in CRSsNP (48%). As a parallel, more glands were found in CRSsNP than in CRSwNP. Moreover, a higher number of glands was found in noneosinophilic CRSwNP compared with eosinophilic CRSwNP (Table I, Table II). In comparison with controls, all types of CRS demonstrated a similar extent of goblet cell hyperplasia with no difference between CRSsNP and CRSwNP, or between eosinophilic and noneosinophilic CRSwNP (Table I, Table II). As to inflammatory cell infiltration, compared with controls, a significantly increased number of total inflammatory cells, mononuclear cells, and plasma cells was found in both CRSsNP and CRSwNP, whereas the number of eosinophils was increased only in CRSwNP. Comparing CRSsNP and CRSwNP, a greater extent of inflammatory cells infiltration was found in CRSwNP (Table I). Eosinophilic inflammation was found in 46.4% of CRSwNP versus 12% of CRSsNP (P < .001). Eosinophilic CRSwNP had a greater number of total inflammatory cells besides eosinophils compared with noneosinophilic CRSwNP (Table II). Because CRSsNP and noneosinophilic CRSwNP presented a similar extent of eosinophil infiltration, we made further comparisons between them. We found that they also had a similar number of total inflammatory cells and mononuclear cells, but CRSsNP had fewer plasma cells and more glands (Table II). Almost all the patients with concomitant asthma showed evident eosinophilic inflammation in sinonasal mucosa, but further statistical analysis was not performed because of a limited number of subjects with asthma in our current study. Comparing patients with and without allergy, no significant difference in eosinophil infiltration was revealed in both CRSsNP and CRSwNP groups (data not shown). Analyzing the relationship between the number of eosinophils and other parameters, we found that the number of eosinophils correlated positively with the number of total inflammatory cells (r = 0.48; P < .001) and mononuclear cells (r = 0.26; P = .001), but negatively with the number of mucosal glands (r = –0.33; P < 0.001).

Table I. General histologic analysis in control, CRSsNP, and CRSwNP

Values of goblet cells are expressed as number per millimeter of intact epithelium, whereas values of other parameters are expressed as number per HP field of LP. Mann-Whitney U test was used for unpaired comparisons. P <.05 was considered statistically significant. NS, Not significant.

Table II. General histologic analysis in eosinophilic and noneosinophilic CRSwNP

Values of goblet cells are expressed as number per millimeter of intact epithelium, whereas values of other parameters are expressed as number per HP field of LP. Mann-Whitney U test was used for unpaired comparisons. P < .05 was considered statistically significant. NS, Not significant.

Immunohistochemical examination 

We found that CD3⁺ T cells, especially CD8⁺ T cells, were the predominant inflammatory cells in both CRSsNP and CRSwNP. CD3⁺ T cells accounted for 44.9% and 45.2%, and CD8⁺ T cells accounted for 37.5% and 41.7%, of total inflammatory cells in CRSsNP and CRSwNP, respectively. Statistical analysis demonstrated that both CRSsNP and CRSwNP had a significantly greater number of CD3⁺, CD4⁺, and CD8⁺ T cells, naive B cells (CD20⁺), macrophages (CD68⁺), and dendritic cells (DC-LAMP⁺) than controls (Table III). CRSwNP showed more severe CD3⁺ and CD8⁺ T-cell, naive B-cell, and macrophage infiltration than CRSsNP (Table III). Neutrophils (myeloperoxidase⁺) were found in only some subjects. The frequency of neutrophil infiltration was higher in patients with CRSsNP than in patients with CRSwNP and controls (CRSsNP vs CRSwNP vs controls: 72.2% vs 42.2% vs 29.4%; P = .007 for CRSsNP vs CRSwNP and P = .003 for CRSsNP vs controls), and no difference was found between CRSwNP and controls. When comparing eosinophilic and noneosinophilic CRSwNP, noneosinophilic CRSwNP showed a higher frequency of neutrophil infiltration (eosinophilic vs noneosinophilic: 21% vs 57.7%; P = .014); however, no difference in other immune cells was found between them (Table IV). Interestingly, no difference in immune cells infiltration was found between noneosinophilic CRSwNP and CRSsNP except for naive B cells, which showed more severe infiltration in noneosinophilic CRSwNP (Table IV). The number of eosinophils was significantly correlated only with the number of CD3⁺ T cells (r = 0.40; P = .006).

Table III. Immunohistochemistry analysis in control, CRSsNP, and CRSwNP

Values are expressed as number per HP field of LP. Mann-Whitney U test was used for unpaired comparisons. P < .05 was considered statistically significant. NS, Not significant.

Table IV. Immunohistochemistry analysis in eosinophilic and noneosinophilic CRSwNP

Values are expressed as number per high power field of lamina propria. Mann-Whitney U test was used for unpaired comparisons. P < .05 was considered statistically significant. NS, Not significant.

Quantitative PCR 

Significantly higher levels of T_H1 (T-bet and IFN-γ), T_H2 (GATA-3 and IL-5), and T_H17 (RORC, IL-17A, IL-22, and IL-23) transcription factors and cytokines were found in both CRSsNP and CRSwNP patients than controls (Fig 1). In most study samples, IL-4 mRNA expression was undetectable. When comparing patients with CRSsNP and CRSwNP, no significant difference in levels of transcription factors and cytokines was found except for IFN-γ, which was markedly increased in patients with CRSsNP (Fig 1). In comparison with noneosinophilic CRSwNP, eosinophilic CRSwNP was found to have increased levels of T_H2 and T_H17 transcription factors and cytokines (Fig 1). Compared with CRSsNP, eosinophilic CRSwNP showed markedly elevated levels of T_H2 transcription factors and cytokines, although CRSwNP as a whole group did not. The only difference found between CRSsNP and noneosinophilic CRSwNP was that CRSsNP had higher levels of INF-γ expression (Fig 1). Intriguingly, except for T-bet, no difference in T_H1, T_H2, and T_H17 transcription factors and cytokines was found between noneosinophilic CRSwNP and controls (Fig 1). The proportions of CRSwNP and CRSsNP patients, including patients with eosinophilic and noneosinophilic CRSwNP, having positive mRNA expression of Treg cell transcription factor FOXP3 and Treg cell cytokine TGF-β1 were lower than those of controls. Nevertheless, no significant difference was detected between CRSsNP and CRSwNP, or between eosinophilic and noneosinophilic CRSwNP (Fig 1). On the contrary, compared with controls, IL-10 levels were markedly enhanced in all types of CRS except for noneosinophilic CRSwNP. No difference in IL-10 levels was found between CRSsNP and CRSwNP, and CRSsNP and noneosinophilic NP; however, a significant difference was found between eosinophilic and noneosinophilic CRSwNP (Fig 1). Compared with controls, Vα24 expression levels were significantly enhanced only in eosinophilic CRSwNP, and there was a significant difference in Vα24 expression between eosinophilic and noneosinophilic CRSwNP (Fig 1).

• 
  • View Large Image
  • Download to PowerPoint

• Fig 1. 

  mRNA expression levels or expression rate of transcription factors and cytokines of T cell subsets, and a marker of NKT cell (Valpha24) in various types of CRS. TH1 cells transcription factor T-bet (A) and cytokine IFN-gamma (B); TH2 cells transcription factor GATA-3 (C) and cytokine IL-5 (D); TH17 cells transcription factor RORC (E) and cytokines IL-17A (F), IL-22 (G) and IL-23 ( H); Treg cells transcription factor FOXP3 (I) and cytokines TGF-beta1 (J) and IL-10 ( K) and NKT cell marker Valpha24 (L) were measured by quantitative RT-PCR. EOS CRSwNP, Eosinophilic CRSwNP; Non-EOS CRSwNP, noneosinophilic CRSwNP. ^∗P < .05; ^∗∗P < .01.

Back to Article Outline

Discussion 

In this study, we investigated the patterns of sinus mucosal inflammation in adult Chinese patients with CRS. A large sample size histologic study showed that Chinese CRSwNP had a higher degree of inflammatory cells infiltration than CRSsNP, which is further supported by a immunohistochemical study that demonstrated higher levels of cell infiltration with CD8⁺ T cells, B cells, and macrophages in CRSwNP. Nevertheless, as to neutrophils, a higher frequency of infiltration was found in CRSsNP than in CRSwNP. Most importantly, in marked distinction from white CRSwNP,¹, ², ⁸ more than a half (53.6%) of Chinese CRSwNP presented noneosinophilic inflammation in the current histologic study. Further comparisons between eosinophilic and noneosinophilic CRSwNP revealed that noneosinophilic CRSwNP had a lower degree of total inflammatory cells infiltration and a higher frequency of neutrophil infiltration than eosinophilic CRSwNP. These differences reflect a distinguished eosinophil and neutrophil granulocyte activation bias in eosinophilic CRSwNP, and noneosinophilic CRSwNP and CRSsNP, respectively, suggesting different underlying pathogenic processes between CRSsNP and CRSwNP, and between eosinophilic and noneosinophilic CRSwNP. However, it should be noted that all patients with CRS in our study received antibiotics preoperatively, which might have biased the results of neutrophil measurement.

In our study, it is obvious that T cells, especially CD8⁺ T cells, were the predominant inflammatory cells in both CRSsNP and CRSwNP, which is consistent with reports from white patients.¹⁷, ¹⁸, ¹⁹, ²⁰ Moreover, increased mature dendritic cells were discovered in both CRSsNP and CRSwNP, suggesting that a T-cell–mediated immune response may play an important role in Chinese CRS. Recently, 2 studies from Van Bruaenue et al⁷ and Zhang et al¹¹ explored the T-cell regulation in white CRS and Chinese NPs, respectively. They found that in white subjects, CRSsNP was characterized by a predominant T_H1 milieu and adequate Treg cell functions, whereas CRSwNP showed a T_H2 skewed eosinophilic inflammation with defective functions of Treg cell.⁷ As to Chinese, NP samples from southern Chinese showed a T_H1/T_H17 cell pattern with impaired Treg cell functions.¹¹ Markedly different from these reports, in this study, we found mixed T_H1/T_H2/T_H17 reactions with impaired Treg cell function in both CRSsNP and CRSwNP. Our data confirms T_H1 predominant responses in Chinese CRSsNP because there existed a difference only with higher levels of INF-γ in CRSsNP than CRSwNP. On the other hand, comparing with CRSsNP, T_H2-dominated reactions can only be found in eosinophilic CRSwNP instead of all CRSwNP. Interestingly, when dividing CRSwNP into eosinophilic and noneosinophilic CRSwNP, we found that an increased T_H2 and T_H17 cell function is predominant in eosinophilic CRSwNP but not in noneosinophilic CRSwNP compared with controls, suggesting that T_H responses may exert different impacts on the pathogenesis of eosinophilic and noneosinophilic CRSwNP.

T_H17, a newly defined T-cell lineage, has been shown to be deeply involved in the development of autoimmunity, cancer, and allergy. Although a role of T_H17 in CRSsNP and CRSwNP has not been found in white patients,⁷, ¹¹ we found that an enhanced T_H17 response existed not only in CRSsNP with neutrophil-biased inflammation but also in eosinophilic CRSwNP with a T_H2 skewed inflammation. Previous studies have shown that IL-17 can induce the recruitment of neutrophils through induction of Cysteine-X-Cysteine chemokines.²¹ Recently, a number of studies have shown that T_H17 cells can also upregulate T_H2 cell–mediated eosinophilic airway inflammation.²² Therefore, the exact role of T_H17 responses in different subgroups of Chinese CRS needs further investigation.

Regulatory T cells, indispensible in maintaining self-tolerance, regulate immune responses in both physiologic and disease statuses. Contrast to the report from white subjects,⁷ compared with controls, we found a defective function of Treg cells with a lower mRNA expression rate of FOXP3 and TGF-β1 not only in Chinese CRSwNP but also in Chinese CRSsNP, which may account for the increased T_H1/T_H2/T_H17 mixed reactions in Chinese patients with CRS. Paradoxically, although exaggerated T_H2/T_H17 reactions was found in eosinophilic CRSwNP compared with noneosinophilic CRSwNP, no difference in FOXP3 and TGF-β1 expression was found between them. Obviously, more studies are needed to elucidate this discrepancy. Besides TGF-β1, IL-10 is another important cytokine secreted by inducible Treg cells. However, contrary to TGF-β1, IL-10 levels were significantly increased in both CRSsNP and CRSwNP in our study. The elevated IL-10 levels in CRS may result from the production by other cell types, such as CD8⁺ T cells, macrophages, B cells, and epithelial cells, and IL-10 may play a negative feedback role in the inflammatory processes in CRS.

Natural killer T cells play an important role in innate immunity.²³ One recent publication reported that NKT cells were detected only in the sinus mucosa from patients with CRS with concomitant asthma,²⁴ which was commonly considered with abundant eosinophils. Sen et al²⁵ demonstrated that NKT cells from patients with allergic asthma can induce a T_H2 response. In this study, compared with controls, the maker of NKT cells, Vα24, was significantly increased only in eosinophilic CRSwNP with a T_H2-skewed inflammation, suggesting an essential role for NKT cells in sinonasal eosinophilic inflammation.

In contrast to previous results reported for white subjects,¹, ², ⁷ Chinese CRSsNP and CRSwNP display different patterns of sinus mucosal inflammation, suggesting different underlying pathogenic mechanisms for Chinese patients in which Treg and T_H17 cells may play a critical role. Currently, as to the pathogenesis of CRS, no matter the fungal or superantigen hypothesis, they all aimed at eosinophil activation.²⁶, ²⁷ The distinct eosinophilic and noneosinophilic subtypes discovered by our current study indicate that other novel factors that remain to be defined may trigger the development of noneosinophilic inflammation. On the other hand, interestingly, CRSsNP and noneosinophilic CRSwNP share a number of similarities in granulocyte activation and T_H responses, which prompts us to wonder whether they have some common pathogenic pathways and what factors eventually lead to the different clinical phenotypes. Addressing these questions fully in the future will ultimately provide new insight into the pathogenesis and treatment of CRS.

One limitation of our study is that protein levels of cytokines were not detected. However, protein levels of some of these cytokines were analyzed in the tissue homogenates in our previous study and the results are coincident with our current study of mRNA expression.²⁸

In conclusion, this study demonstrates impaired Treg cell function and enhanced T_H1/T_H2/T_H17 responses both in Chinese patients with CRSsNP and CRSwNP. CRSsNP is confirmed to be a predominant T_H1 milieu, whereas a distinct pattern of T_H2 skewed characteristics with predominant T_H17 reactions and infiltration of NKT cells can be demonstrated in eosinophilic CRSwNP, but not in noneosinophilic CRSwNP, which consists of at least half of the total Chinese CRSwNP. Further studies are needed to investigate in-depth immunopathologic mechanisms involved especially in noneosinophilic CRSwNP, because it could be regarded as a paradigm of chronic upper airway inflammation.

Back to Article Outline

Table E1. 

Patients' clinical data

Back to Article Outline

Table E2. 

Primers used for quantitative PCR analysis of transcriptional factors and cytokines gene expression

Back to Article Outline

References 

1. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas RA, et al. Rhinosinusitis: establishing definitions for clinical research and patient care. J Allergy Clin Immunol. 2004;114:155–212
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (1221 KB)
   • CrossRef
2. Fokkens W, Lund V, Mullol J European Position Paper on Rhinosinusitis and Nasal Polyps Group. European position paper on rhinosinusitis and nasal polyps 2007. Rhinol Suppl. 2007;20:1–136
   • View In Article
3. Van Zele T, Claeys S, Gevaert P, Van Maele G, Holtappels G, Van Cauwenberge P, et al. Differentiation of chronic sinus diseases by measurement of inflammatory mediators. Allergy. 2006;61:1280–1289
   • View In Article
   • MEDLINE
   • CrossRef
4. Malekzadeh S, Hamburger MD, Whelan PJ, Biedlingmaier JF, Baraniuk JN. Density of middle turbinate subepithelial mucous glands in patients with chronic rhinosinusitis. Otolaryngol Head Neck Surg. 2002;127:190–195
   • View In Article
   • MEDLINE
   • CrossRef
5. Berger G, Kattan A, Bernheim J, Ophir D. Polypoid mucosa with eosinophilia and glandular hyperplasia in chronic sinusitis: a histopaththological and immunohistochemical study. Laryngoscope. 2002;112:733–745
   • View In Article
6. Pozlehl D, Moeller P, Riechelmann H, Perner S. Distinct features of chronic rhinosinusitis with and without nasal polyps. Allergy. 2006;61:1275–1279
   • View In Article
   • MEDLINE
   • CrossRef
7. Van Bruaene N, Pe'rez-Novo CA, Basinski TM, Van Zele T, Holtappels G, DeRuyck N, et al. T-cell regulation in chronic paranasal sinus disease. J Allergy Clin Immunol. 2008;121:1435–1441
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (760 KB)
   • CrossRef
8. Tos M, Mogensen C. Mucous glands in nasal polyps. Arch Otolaryngol. 1977;103:407–413
   • View In Article
   • MEDLINE
9. Kim JW, Hong SL, Kim YK, Lee CH, Min YG, Rhee CS. Histological and immunological features of non- eosinophilic nasal polyps. Otolaryngol Head Neck Surg. 2007;137:925–930
   • View In Article
   • CrossRef
10. Zhang N, Holtappels G, Claeys C, Huang G, van Cauwenberge P, Bachert C. Pattern of inflammation and impact of Staphylococcus aureus enterotoxins in nasal polyps from southern China. Am J Rhinol. 2006;20:445–450
    • View In Article
    • MEDLINE
    • CrossRef
11. Zhang N, Van Zele T, Perez-Novo C, Van Bruaene N, Holtappels G, DeRuyck N, et al. Different types of T-effector cells orchestrate mucosal inflammation in chronic sinus disease. J Allergy Clin Immunol. 2008;122:961–968
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (343 KB)
    • CrossRef
12. Wenzel SE, Schwartz LB, Langmack EL, Halliday JL, Trudeau JB, Gibbs RL, et al. Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics. Am J Respir Crit Care Med. 1999;160:1001–1008
    • View In Article
    • MEDLINE
13. Liu Z, Lu X, Wang H, You XJ, Gao QX, Cui YH. Group II subfamily secretory phospholipase A2 enzymes: expression in chronic rhinosinusitis with and without nasal polyps. Allergy. 2007;62:999–1006
    • View In Article
    • CrossRef
14. Liu Z, Kim J, Sypek JP, Wang IM, Horton H, Oppenheim FG, et al. Gene expression profiles in human nasal polyp tissues studied by means of DNA microarray. J Allergy Clin Immunol. 2004;114:783–790
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (338 KB)
    • CrossRef
15. Hellquist HB. Nasal polyps update: histopathology. Allergy Asthma Proc. 1996;17:237–242
    • View In Article
    • MEDLINE
    • CrossRef
16. Kakoi H, Hiraide F. A histological study of formation and growth of nasal polyps. Acta Otolaryngol. 1987;103:137–144
    • View In Article
    • MEDLINE
    • CrossRef
17. Sánchez-Segura A, Brieva JA, Rodríguez C. T lymphocytes that infiltrate nasal polyps have a specialized phenotype and produce a mixed TH1/TH2 pattern of cytokines. J Allergy Clin Immunol. 1998;102:953–960
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (142 KB)
    • CrossRef
18. Hao J, Pang YT, Wang DY. Diffuse mucosal inflammation in nasal polyps and adjacent middle turbinate. Otolaryngol Head Neck Surg. 2006;134:267–275
    • View In Article
    • MEDLINE
    • CrossRef
19. Liu CM, Shun CT, Hsu MM. Lymphocyte subsets and antigen-specific IgE antibody in nasal polyps. Ann Allergy. 1994;72:19–24
    • View In Article
    • MEDLINE
20. Stoop AE, van der Heijden HA, Biewenga J, van der Baan S. Clinical aspects and distribution of immunologically active cells in the nasal mucosa of patients with nasal polyps after endoscopic sinus surgery and treatment with topical corticosteroids. Eur Arch Otorhinolaryngol. 1992;249:313–317
    • View In Article
    • MEDLINE
21. Laan M, Cui ZH, Hoshino H, Lötvall J, Sjöstrand M, Gruenert DC, et al. Neutrophil recruitment by human IL-17 via C-X-C chemokine release in the airways. J Immunol. 1999;162:2347–2352
    • View In Article
    • MEDLINE
22. Wakashin H, Hirose K, Maezawa Y, Kagami SI, 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
    • View In Article
    • CrossRef
23. Taniguchi M, Harada M, Kojo S, Nakayama T, Wakao H. The regulatory role of Valpha14 NKT cells in innate and acquired immune response. Annu Rev Immunol. 2003;21:483–513
    • View In Article
    • MEDLINE
    • CrossRef
24. Yamamoto H, Okamoto Y, Horiguchi S, Kunii N, Yonekura S, Nakayama T. Detection of natural killer T cells in the sinus mucosa from asthmatics with chronic sinusitis. Allergy. 2007;62:1451–1455
    • View In Article
    • CrossRef
25. Sen Y, Yongyi B, Yuling H, Luokun X, Li H, Jie X, et al. Vα24-invariant NKT cells from patients with allergic asthma express CCR9 at high frequency and induce Th2 bias of CD3+ T cells upon CD226 engagement. J Immunol. 2005;175:4914–4926
    • View In Article
    • MEDLINE
26. Inoue Y, Matsuwaki Y, Shin SH, Ponikau JU, Kita H. Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils. J Immunol. 2005;175:5439–5447
    • View In Article
    • MEDLINE
27. Patou J, Gevaert P, Van Zele T, Holtappels G, van Cauwenberge P, Bachert C. Staphylococcus aureus enterotoxin B, protein A, and lipoteichoic acid stimulations in nasal polyps. J Allergy Clin Immunol. 2008;121:110–115
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (493 KB)
    • CrossRef
28. Liu Z, Lu X, Zhang XH, Bochner BS, Long XB, Zhang F, et al. Clara cell 10-kd protein expression in chronic rhinosinusitis and its cytokine-driven regulation in sinonasal mucosa. Allergy. 2009;64:149–157
    • View In Article
    • CrossRef