Allergic rhinitis and chronic rhinosinusitis (CRS) are two widely prevalent inflammatory diseases in upper airways. They severely impact the quality of life of affected individuals and are strongly associated with asthma . Allergic rhinitis is characterized by intense eosinophil infiltration and mucus hypersecretion that are orchestrated mainly by antigen-specific Th2 cells and their cytokines such as interleukin (IL)-4, IL-5, and IL-13. Primarily on the basis of the absence or presence of nasal polyps, CRS is currently divided into two types: CRS without nasal polyps (CRSsNP) and CRS with nasal polyps (CRSwNP) . In whites, CRSsNP is characterized by a Th1 milieu, whereas CRSwNP is featured by a Th2-biased eosinophilic inflammation . In contrast, in Asians, only about 50% of CRSwNP demonstrates Th2-skewed eosinophilic inflammation . Although the insights into the pathophysiology of allergic rhinitis and CRS have largely expanded over the last two decades, the pathogenic mechanisms of these two conditions remain to be elucidated. To date, one of the most important characteristics of inflammatory upper airway diseases is the exaggerated and/or prolonged inflammatory and immune reactions in local mucosa. Inflammation is essential for clearing out invading pathogens and allergens, but it can also be harmful to the host if appropriate regulation of the magnitude and duration of the response are disturbed. Therefore, inflammation is usually under tight control at multiple levels. Loss of this control due to the dysfunction of anti-inflammatory network can lead to hyperinflammatory states associated with inflammatory diseases.
Clara cell 10-kD protein (CC10), also known as Clara cell secretory protein, Clara cell protein 16, uteroglobin, progesterone-binding protein, human protein 1, urine protein 1, and polychlorinated biphenyl-binding protein, is a prototypical member of the newly recognized secretoglobin superfamily . It is a steroid-inducible and secretory protein with anti-inflammatory and immunomodulatory effects. CC10 has been shown to antagonize the activity of secretory phospholipase A2, diminish inflammatory cell chemotaxis, and suppress Th2 cell differentiation and Th2 cytokine production . CC10 is constitutively expressed by the mucosal epithelial cells lining all organs that encounter the outer environment. In airways, CC10 has traditionally been thought to be expressed by nonciliated Clara cells in rodents and humans, which locate at the distal airways . CC10 has been implicated in the regulation of pulmonary inflammation and immune responses. CC10 positive epithelial cells are decreased in small airways and CC10 levels are reduced in bronchoalveolar lavage fluid of patients with asthma . Compared with wild-type mice, CC10 knockout mice demonstrate exaggerated pulmonary inflammation provoked by allergic responses and bacterial and viral infections . Recently, studies from us and others indicated that CC10 can also be produced in upper airways and CC10 is involved in the development of allergic rhinitis and eosinophilic CRS, revealing a novel role of CC10 in inflammatory airway diseases.
CLARA CELL 10-KD PROTEIN PRODUCTION IN SINONASAL MUCOSA
In spite of the fact that Clara cells have been traditionally considered to be the site of CC10 production in airways, CC10 expression by goblet cells and other ill-defined cells in lower airways has been reported . In upper airways, although there are no Clara cells, CC10 has been found in human nasal lavage fluid by two-dimensional gel electrophoresis and peptide mass fingerprinting . CC10 mRNA expression in nasal mucosa has also been detected by DNA microarray and RT-PCR [10,11]. By means of immunohistochemistry and periodic acid-Schiff staining, we demonstrated that CC10 was mainly expressed by goblet cells and nonmucus and nonciliated cells in epithelium of human sinonasal mucosa . The identity of those nonmucus and nonciliated cells remains to be defined by more detailed studies in future, such as electronic microscopy. In mice, we demonstrated that CC10 was expressed by dome-shaped columnar epithelial cells, ciliated cells, and occasionally by goblet cells in epithelium in turbinate mucosa and by ciliated cells and occasionally by goblet cells in epithelium in septal mucosa, indicating a clear difference in the cell types secreting CC10 in upper airways between humans and rodents [12▪▪].
CLARA CELL 10-KD PROTEIN AND ALLERGIC RHINITIS
Using DNA microarray to profile the gene expression in nasal fluid cells from patient with symptomatic birch and/or grass pollen-induced intermittent allergic rhinitis, Benson et al. found that CC10 gene was the most downregulated anti-inflammatory gene in allergic rhinitis patients compared with controls. Additional studies revealed that CC10 levels in nasal fluid were lower in children with birch pollen-induced allergic rhinitis both before and during the pollen season than in those in controls . In intermittent allergic rhinitis patients challenged with allergen out of season, an inverse relation was found between nasal fluid CC10 levels and symptoms and signs of rhinitis . Most recently, an inverse relationship between CC10 levels and the occurrences of mast cells in nasal fluid was found in patients with persistent allergic rhinitis and intermittent allergic rhinitis in an allergen-free interval [16▪]. These studies suggest that CC10 may have a counter-regulatory role in relation to the inflammatory response in allergic rhinitis and the restoration of CC10 to normal levels contributes to normalization of signs and symptoms of allergic rhinitis. To truly prove the validity with respect to the role of CC10 in allergic rhinitis, we take advantage of CC10-deficiency mice. We found that compared with wild-type allergic rhinitis mice, Th2-dominated eosinophilic inflammation was markedly exaggerated in CC10-knockout allergic rhinitis mice that could be reversed by CC10 administration during both sensitization and challenge phases, confirming the inhibitory role of CC10 in the development of allergic rhinitis [17▪▪]. Moreover, we found that CC10 may exert its inhibitory effect through modulating osteopontin [17▪▪]. Osteopontin is a multifunctional extracellular matrix protein and immune modulator that has been implicated in the regulation of allergic airway inflammation recently . Osteopontin can inhibit the migration of plasmacytoid dendritic cells at sensitization phase and conventional dendritic cells at challenge phase, respectively, and promote eosinophil migration and airway remodeling . We found that osteopontin was upregulated locally in allergic rhinitis mice compared with control mice and CC10 downregulated osteopontin expression in nasal mucosa and splenocytes [17▪▪]. Moreover, we showed that CC10 could inhibit osteopontin-induced Th2 and proinflammatory cytokines production in human airway epithelial cell line, BEAS-2B cells [17▪▪]. Similarly to the findings in the murine model, we found that CC10 expression was significantly decreased, whereas osteopontin expression was significantly increased, in nasal epithelium in patients with dust mite-induced allergic rhinitis, and there was a negative correlation between CC10 and osteopontin expression [17▪▪], implicating a link between CC10 and osteopontin in human allergic rhinitis. Apart from Th2 responses, recent evidence suggests an effector role of IL-17A and Th17 cells in allergic airway diseases including allergic rhinitis . We found that CC10-knockout enhanced, whereas CC10 treatment decreased Th17 responses in allergic rhinitis mice (unpublished data). CC10 did not affect Th17 cell differentiation directly, but inhibited polarizing cytokine production by dendritic cells that resulted in the suppression of dendritic cell-induced Th17 cell differentiation (unpublished data).
CLARA CELL 10-KD PROTEIN AND CHRONIC RHINOSINUSITIS
Similarly to the discovery of the involvement of CC10 in allergic rhinitis, the potential role of CC10 in CRS was also first revealed by DNA microarray study [10,11]. CC10 gene was the most downregulated gene in nasal polyp tissues compared with normal sinus tissues and the most upregulated gene after nasal glucocorticoid treatment [10,11]. Although CC10 expression levels were also found to be lower in CRSsNP than in controls, this downregulation was significantly milder in comparison with CRSwNP [20▪]. In addition, we found that CC10 downregulation was more prominent in CRS patients with concomitant asthma and in CRS patients with eosinophilic inflammation [20▪,21▪▪], suggesting that CC10 may play a more important role in Th2-biased eosinophilic inflammation. Interestingly, we found that CC10 levels in sinonasal mucosa negatively correlated with local inflammatory cell infiltration, preoperative computed tomography scores, postoperative symptoms, and endoscopy scores, underscoring a regulatory function of CC10 in CRS and indicating that CC10 might be a potential biomarker to predict the patient's response to surgery [20▪]. To dissect the cause–effect relationship between CC10 and CRS, we established an eosinophilic CRS mouse model through prolonged ovalbumin challenge [21▪▪]. We found that compared with wild-type mice with eosinophilic CRS, a significantly greater extent of eosinophil infiltration and tissue remodeling including epithelium thickening and goblet cell hyperplasia was found in CC10-knockout mice with eosinophilic CRS, which was associated with significantly higher levels of proinflammatory cytokines, Th2 cytokines, and eotaxin-1 [21▪▪]. Using DNA microarrays, we compared the difference in gene expression profiles between control mice and eosinophilic CRS mice, and between wild-type eosinophilic CRS mice and CC10-knockout eosinophilic CRS mice. Genes identified in both the comparisons are believed to be important not only in the development of eosinophilic CRS but also in CC10-modulated response in eosinophilic CRS. By using this approach, chitinase 3-like 1 protein (CHI3L1) gene was found to be one of the top five upregulated genes both involved in the pathogenesis of eosinophilic CRS and associated with CC10's regulatory pathway [21▪▪]. Chitinase-like proteins do not have true enzyme activity like chitinases, but have strong binding affinity to chitin . By using CHI3L1 knockout and transgenic mice, Lee and Elias  found that CHI3L1 can promote Th2 responses possibly through inducing dendritic cell accumulation and alternative macrophage activation in allergic lower airway inflammation. We found that CHI3L1 may exaggerate chronic eosinophilic inflammation through inducing eotaxin-1 expression [21▪▪]. These findings indicate that CHI3L1 is a novel molecule involved in the development of eosinophilic airway inflammation. In human beings, we revealed that CHI3L1 protein expression was mainly located in epithelial cells in sinonasal mucosa and its expression increased significantly in eosinophilic CRSwNP compared with controls and noneosinophilic CRSwNP [21▪▪]. We further showed that CC10 could inhibit proinflammatory and Th2 cytokine-induced CHI3L1 expression in airway epithelial cell line, BEAS-2B cells, and reconstitution of CC10 expression in CC10-knockout mice was able to suppress the increase of CHI3L1 expression, and eosinophilic inflammation and tissue remodeling in nose [21▪▪], highlighting the involvement of CHI3L1 in CC10-modulated immune responses. Although CC10 expression is downregulated in both eosinophilic and noneosinophilic CRSwNP, only eosinophilic CRSwNP demonstrates increased CHI3L1 expression, suggesting that other molecules may be involved in CC10-mediated process in noneosinophilic CRS, which, however, remain to be defined.
THE EXPRESSION REGULATION OF CLARA CELL 10-KD PROTEIN IN NOSE
The CC10 gene is located on chromosome 11q12-13. This region is linked to atopy and contains candidate genes for allergy such as the high-affinity immunoglobulin E receptor. A single nucleotide polymorphism (SNP) at position 38 (A + 38G) downstream from the transcription initiation site within the noncoding region of exon 1 may alter transcription levels, thus impacting CC10 protein expression levels. This variant was reported to be associated with plasma CC10 levels, and asthma, wheezing, and bronchial hyperresponsiveness . However, no significant association of CC10 A + 38G polymorphism with allergic rhinitis or CRS was found by Benson et al. or us , respectively, indicating that this variant may not contribute to the decreased CC10 expression in human inflammatory upper airway diseases. However, the material investigated in our study and by Benson et al. was relatively small and it should be noted that disease-associated SNPs often show small differences between patients and controls. Thus, further studies of larger materials are needed to define the relevance of CC10 SNP and allergic rhinitis and CRS better. An alternative explanation for altered CC10 production could be the inflammatory cytokines. CC10 expression in lung tissue and pulmonary cell lines could be regulated by inflammatory cytokines, such as IL-4, interferon (INF)-γ, and tumor necrosis factor (TNF)-α. However, compared with lung tissue, CC10 is produced by different types of cells in the upper airways; therefore, it is possible that CC10 gene expression is regulated via different systems in sinonasal mucosa. We found that TNF-α, IL-1β, and IL-4 could inhibit CC10 production, whereas INF-γ and IL-10 could promote CC10 production in nasal mucosa after short-time stimulation [20▪]. The counter effect of Th1 and Th2 cytokines on CC10 production may explain the more prominent CC10 downregulation in eosinophilic CRSwNP compared with noneosinophilic CRSwNP. Moreover, the mutual regulatory axis between cytokines and CC10 may exaggerate and perpetuate the inflammatory reactions in upper airways (Fig. 1). Interestingly, the expression-inducing effect of IL-10 and glucocorticoids on CC10 gene may suggest that CC10 is potentially involved in the anti-inflammatory function of IL-10 and glucocorticoids. Analysis of the lung-specific expression of CC10 has resulted in the identification of several transacting factors, such as hepatocyte nuclear factor-3, thyroid transcript factor-1, CCAAT/enhancer-binding protein, and chicken ovalbumin upstream promoter transcription factor. In sinonasal mucosa, we could not detect the mRNA expression for CCAAT/enhancer-binding protein-α, chicken ovalbumin upstream promoter transcription factor I and II before or after cytokine stimulation [20▪], highlighting the possibility that different molecular systems may be involved in CC10 gene regulation in upper and lower airways. The development of chronic airway diseases is usually a long-term process. During this process, the phenotype of epithelial cells may also undergo significant change, apart from the alterations of CC10 gene transcriptional activity. Recently, we found that although goblet cells increased significantly in ovalbumin-induced allergic rhinitis mice, the total number of epithelial cells kept unchanged [12▪▪]. In turbinate mucosa, the number of CC10-positive cells (mainly dome-shaped cells) decreased, whereas the number of trefoil factor family 1 (TFF1)-positive cells (mainly ciliated cells) increased. In septal mucosa, the number of CC10-positive and TFF1-positive cells (mainly ciliated cells) decreased simultaneously, and the number of goblet cells increased [12▪▪]. Intermediate phenotypic goblet cells could express CC10 and TFF1 simultaneously [12▪▪]. These findings suggest that allergen exposure may lead to a transdifferentiation of CC10 secreting cells into TFF1 secreting cells and/or goblet cells in upper airways, which may contribute to the downregulation of CC10 expression and goblet cell metaplasia in allergic rhinitis. Our findings also suggest that similarly to Clara cells, the CC10-positive cells in the upper airways also possess significant plasticity and, likely, have a role as progenitors or reserve cells for other epithelial cells. Nevertheless, the significance of these findings needs to be further evaluated in human individuals.
Over recent years, novel insights have been gained into the role of CC10 in the inflammatory and allergic diseases of the upper airways. CC10 has a significant impact on Th2-biased eosinophilic inflammation in allergic rhinitis and CRS through modulating the newly recognized molecules in immune responses including osteopontin and CHI3L1. Moreover, we recently discovered that CC10 gene transfection can inhibit NF-κB activity in airway epithelial cells [26▪]. Because NF-κB pathway impacts a lot of biological processes and immune responses, our results indicate that CC10 may have a much broader role in airway diseases that goes beyond the regulation of eosinophilic inflammation. Given the lack of association between CC10 gene variant and CRS and allergic rhinitis, CC10 is more likely an endogenous modulator of disease process instead of an initiator of CRS and allergic rhinitis. Until now, despite the significant progress in characterizing the pathophysiological roles of CC10, the putative surface receptor of CC10 remains enigmatic, which hampers the study of the intracellular mechanisms of CC10's action. CC10 is relatively small, resistant to proteases, stable to extremes of heat and pH, and can be produced by recombinant methods. These characteristics make CC10 an excellent candidate for clinical development to treat inflammatory and allergic airway diseases. However, the only double-blinded and placebo-controlled trial with a small sample size failed to show that repeated nasal administration of recombinant human CC10 protein in a dose of 0.56 mg per nasal cavity per day, for 7 days, could improve allergen-induced morning, postchallenge, or evening symptoms compared with placebo . Therefore, additional randomized controlled trials with a larger sample size, various doses of CC10, and different treatment durations are urgently needed to evaluate the potential therapeutic effect of CC10 on allergic rhinitis and CRS.
This study was supported by National Natural Science Foundation of China (NSFC) grant 81020108018 to Z.L., NSFC grant 81200733 to H.W., and a grant from Ministry of Health of China (201202005).
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 120).
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