Clara cell 10-kD protein suppresses chitinase 3-like 1 expression associated with eosinophilic chronic rhinosinusitis

Abstract

Rationale: Clara cell 10-kD (CC10) protein, an antiinflammatory molecule, is involved in inflammatory upper airway diseases, but its regulatory role is unclear, particularly in the process of chronic rhinosinusitis (CRS).

Objectives: To investigate the regulatory mechanisms of CC10 in eosinophilic CRS (ECRS) using an allergic mouse model.

Methods: Homozygous CC10-knockout mice were used to establish an allergic ECRS model. Phenotypic changes were examined by histology, cytokine ELISA, and gene microarray analysis. Differential expression of chitinase 3-like 1 (CHI3L1) was verified by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. The functional role of CHI3L1 in vivo was assessed by the use of anti-CHI3L1 antibody in ECRS mice. CHI3L1 gene expression regulated by inflammatory cytokines and CC10 protein was performed using BEAS-2B cell line.

Measurements and main results: Compared with wild-type mice, a significantly greater extent of inflammatory cell infiltration and tissue remodeling was found in CC10-knockout ECRS mice, which was associated with significantly higher levels of various cytokines and eotaxin-1. CHI3L1 was up-regulated in ECRS mice with a significant further increase in CC10-knockout mice. Anti-CHI3L1 treatment markedly ameliorated eosinophilic inflammation. Furthermore, nasal mucosal CC10 gene transfer in CC10-knockout mice attenuated eosinophilic inflammation and suppressed the levels of CHI3L1. Moreover, significantly up-regulated expression of CHI3L1 was noted in human ECRS. IL-1beta, tumor necrosis factor-alpha, and IL-13 were found to up-regulate CHI3L1 expression in BEAS-2B cells, whereas CC10 inhibited such up-regulation.

Conclusions: These results suggest that CHI3L1 is a novel molecule involved in ECRS and that CC10 plays a regulatory role in ECRS, presumably by attenuating CHI3L1 expression.

Figure 1.

The expression of Clara cell 10-kD protein (CC10) in eosinophilic chronic rhinosinusitis (ECRS) mice. (A) Representative photomicrographs of CC10 immunohistochemical staining of normal murine nose and lung tissue sections. Original magnification ×400. (B) CC10 mRNA expression in the normal murine nose and lung tissues by semiquantitative reverse transcriptase-polymerase chain reaction. (C) Representative photomicrographs of CC10 immunohistochemical staining of sinonasal tissue sections from ECRS mice at various time points. Original magnification ×400. (D) The number of CC10-positive cells decreased gradually during the development of ECRS. *P < 0.05 and **P < 0.01 for control mice (solid dots) challenged with saline compared with corresponding ECRS mice (open dots); n = 3 to 5 mice per group. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

An exaggerated inflammation in Clara cell 10-kD protein (CC10)-knockout mice compared with wild-type mice after establishment of eosinophilic chronic rhinosinusitis (ECRS). (A) The analysis of the number of eosinophils, mononuclear cells, total inflammatory cells, and goblet cells, and the extent of fibrosis and epithelial thickening in sinonasal mucosa. (B) Cytokine and chemokine levels in nasal lavage fluid assessed by ELISA. #P < 0.05 and ##P < 0.01 for ECRS mice compared with corresponding control mice challenged with saline. $P < 0.05 and $$P < 0.01 for CC10-knockout ECRS mice compared with wild-type ECRS mice; n = 3 to 5 mice per group. Open columns = control mice; solid columns = ECRS mice. INF = interferon; TNF = tumor necrosis factor.

Figure 3.

Increased chitinase 3-like 1 protein (CHI3L1) expression in eosinophilic chronic rhinosinusitis (ECRS) mice. (A) CHI3L1 mRNA expression levels in mice. (B) The number of CHI3L1-positive cells in mice. Open columns = control mice; solid columns = ECRS mice. (C) Representative photomicrographs of CHI3L1 immunohistochemical staining of sinonasal tissue sections from (a) wild-type mice challenged with saline, (b) wild-type ECRS mice, (c) Clara cell 10-kD protein (CC10)-knockout mice challenged with saline, and (d) CC10-knockout ECRS mice. Original magnification ×400. Arrows, CHI3L1-positive infiltrating cells. #P < 0.05 and ##P < 0.01 for ECRS mice compared with corresponding control mice challenged with saline. $P < 0.05 and $$P < 0.01 for CC10-knockout ECRS mice compared with wild-type ECRS mice. n = 3 to 5 mice per group.

Figure 4.

Anti–chitinase 3-like 1 protein (CHI3L1) antibody treatment ameliorates the inflammation in sinonasal mucosa. (A) The analysis of the number of eosinophils, mononuclear cells, total inflammatory cells, and goblet cells, and the extent of fibrosis and epithelial thickening in sinonasal mucosa. (B) Cytokine and chemokine levels in nasal lavage fluid assessed by ELISA. #P < 0.05 and ##P < 0.01 for wild-type eosinophilic chronic rhinosinusitis (ECRS) mice compared with corresponding control mice challenged with saline. $P < 0.05 and $$P < 0.01 for wild-type ECRS mice treated with anti-CHI3L1 compared with wild-type ECRS mice treated with preimmune rabbit IgG. n = 3 to 5 mice per group. Open columns = control mice; solid columns = ECRS mice. INF = interferon; TNF = tumor necrosis factor.

Figure 5.

Clara cell 10-kD protein (CC10) gene reconstitution inhibits the inflammation in CC10-knockout eosinophilic chronic rhinosinusitis (ECRS) mice. (A) The analysis of the number of eosinophils, mononuclear cells, total inflammatory cells, and goblet cells, and the extent of fibrosis and epithelial thickening in sinonasal mucosa. (B) Cytokine and chemokine levels in nasal lavage fluid assessed by ELISA. #P < 0.05 and ##P < 0.01 for ECRS mice compared with corresponding control mice challenged with saline. $P < 0.05 and $$P < 0.01 for CC10-knockout ECRS mice with mock plasmid transfer compared with CC10-knockout ECRS mice with pCC10 transfer. n = 3 to 5 mice per group. Open columns = control mice; solid columns = ECRS mice.

Figure 6.

Clara cell 10-kD protein (CC10) protein inhibits cytokine-induced chitinase 3-like 1 protein (CHI3L1) mRNA expression in BEAS-2B cells. (A) CC10 inhibits CHI3L1 production induced by individual cytokine. *P < 0.05 compared with medium controls, #P < 0.05 compared with the corresponding condition with CC10 treatment. Gray columns = with CC10; solid columns = without CC10. (B) CC10 inhibits CHI3L1 production induced by TNF-α alone or TNF-α combined with Th1 or Th2 cytokines. *P < 0.05 compared with the condition with TNF-α alone, # P < 0.05 compared with the corresponding condition with CC10 treatment. n = 7 to 13. Solid columns = medium; light gray columns = TNF-α; dark gray columns = TNFα + CC10.

Figure 7.

Increased chitinase 3-like 1 protein (CHI3L1) expression in human eosinophilic chronic rhinosinusitis (ECRS). (A) CHI3L1 mRNA expression rate in control subjects and patients with CRS. (B) The number of CHI3L1-positive cells in control subjects and patients with CRS. (C) Representative photomicrographs of CHI3L1 immunohistochemical staining of sinonasal tissue sections from (a) control, (b) CRS without NPs, (c) noneosinophilic CRS with NPs, and (d) ECRS with NPs. No obvious positive staining in a and b. Original magnification ×400. Arrows, CHI3L1-positive infiltrating cells. (D) Clara cell 10-kD protein (CC10) expression levels in control subjects and patients with CRS. *P < 0.05 and **P < 0.01.