Differential expression of interleukin-32 in chronic rhinosinusitis with and without nasal polyps

Abstract

Background: Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by local inflammation of the upper airways and sinuses and is frequently divided into polypoid CRS (CRSwNP) and nonpolypoid CRS (CRSsNP). However, the mechanism of inflammation in CRS has still not been fully elucidated. The aim of the study was to investigate the role of interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, in CRS.

Methods: We collected nasal epithelial cells and nasal tissue from patients with CRS and control subjects. We assayed mRNA for IL-32 by real-time PCR and measured IL-32 protein using ELISA, Western blot, and immunohistochemistry.

Results: The expression of mRNA for IL-32 was elevated in epithelial cells from uncinate tissue from patients with CRSsNP compared with patients with CRSwNP (P < 0.05), control subjects (P=0.06), and epithelial cells from nasal polyp (NP) tissue (P < 0.05). Production of IL-32 was induced by IFN-γ, TNF, and dsRNA in primary airway epithelial cells. In whole-tissue extracts, the expression of IL-32 protein was significantly elevated in patients with CRSwNP compared with patients with CRSsNP and control subjects. Immunohistochemistry data showed that IL-32 was detected in mucosal epithelial cells and inflammatory cells in the lamina propria. Levels of IL-32 were correlated with the levels of CD3 and macrophage mannose receptor in NP tissue. Immunofluorescence data showed IL-32 co-localization with CD3-positive T cells and CD68-positive macrophages in NPs.

Conclusion: Overproduction of IL-32 may be involved in the pathogenesis of CRS, although the role of IL-32 in the inflammation in CRSsNP and CRSwNP may be different.

Figure 1. Expression of IL-32 in epithelial scraping cells

Total RNA was extracted from epithelial scraping cells from uncinate tissue (UT) and nasal polyps (Polyp) and expressions of IL-32 were analyzed using real-time PCR. IL-32 mRNA levels were increased in epithelial scraping cells from UT in patients with CRSsNP compared to UT from controls (p=0.063), CRSwNP (p<0.05), and nasal polyps (p<0.05, n=25). *p<0.05

Figure 2. Induction of IL-32 in human primary epithelial cells

Primary nasal epithelial cells (PNEC, A) and primary normal human bronchial epithelial cells (NHBE, B and C) were incubated for 24 hours (A-C) with 100 ng/ml TNF, 10 ng/ml IFN-γ, 100 ng/ml IL-4, 100 ng/ml IL-17A, 5 μg/ml dsRNA. The expression of mRNAs for IL-32 was determined by real-time PCR (A-C). NHBE were stimulated with 50 ng/ml TNF, 10 ng/ml IFN-γ, 5 μg/ml dsRNA and their combinations for 48 hours and then protein expression in the cell lysates and supernatants was analyzed by western blot (D). Results shown are mean ± SEM of seven (A) and four (B and C) independent experiments. The results are representative of four separate experiments (D). *p<0.05.

Figure 3. Expression of IL-32 in sinus tissue

Representative immunostaining for IL-32 and control IgG1 in nasal polyp tissue from a patient with CRSwNP (A). IL-32 was detected in mucosal and glandular epithelium as well as in infiltrated inflammatory cells in nasal polyps. Total RNA was extracted from UT and nasal polyps (Polyp) and expression of IL-32nonA (IL-32) was analyzed by real-time PCR (B). IL-32 mRNA was significantly increased in nasal polyp tissue (p<0.001). The concentration of IL-32 in tissue homogenates of UT and Polyp was measured using ELISA (C). IL-32 protein was significantly increased in CRSwNP. IL-32 concentration was normalized to the concentration of total protein. *p<0.05.

Figure 4. Correlation of IL-32 with cell specific markers in nasal polyps

Total RNA was extracted from nasal polyp tissue and the expression of IL-32 and cell specific markers was analyzed by real-time PCR (n=26). The correlations were assessed by using the Spearman rank correlation.

Figure 5. Identification of IL-32 producing cells by immunofluorescence assay

The immunofluorescence assay was performed using anti-human IL-32 mAb (red fluorescence), anti-CD3 mAb (green fluorescence) for T cells (A), anti-CD68 mAb (green fluorescence) for macrophages (B), anti-tryptase mAb (green fluorescence) for mast cells (C) and control IgG (D). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue fluorescence). IL-32 was detected in CD3⁺ T cells and CD68⁺ macrophages in nasal polyps as demonstrated by co-localization (yellow fluorescence) of the antibody staining. The results are representative of three separate patients.