Allergy Asthma Immunol Res.  2016 Jan;8(1):69-78. 10.4168/aair.2016.8.1.69.

Alternative Method for Primary Nasal Epithelial Cell Culture Using Intranasal Brushing and Feasibility for the Study of Epithelial Functions in Allergic Rhinitis

• Park DY
• Kim S
• Kim CH
• Yoon JH
• Kim HJ

• KMID: 2166655
• DOI: http://doi.org/10.4168/aair.2016.8.1.69

Abstract

PURPOSE
Although differentiated normal human nasal epithelial (NHNE) cells can be used to study the role of human nasal epithelium, there is a need for effective culture models of nasal epithelium in sinonasal disease status, including allergic rhinitis (AR). We aimed to examine the feasibility of intranasal brushing for culture of nasal epithelial cells in AR patients and to verify the hypothesis that allergic nasal epithelial (ARNE) cells differ in histologic and physiologic characteristics.
METHODS
We established a system for isolating (via intranasal brushing) and culturing (with air-liquid interface, ALI) nasal epithelial cells from healthy volunteers (n=8) and AR patients (n=8). We used this system to compare the histologic findings and physiologic characteristics of NHNE and ARNE.
RESULTS
The histology results showed that fully differentiated ALI culture was obtained at least 14 days after confluence and that both ciliated and secretory cells were well differentiated in ALI culture using nasal brushing. The histology results of ARNE culture were significantly different from NHNE. The number of ciliated cells was lower, and secretory cells were more dominant in ARNE cell culture compared to NHNE cells. We also observed, by electron microscopy, loose tight junctions and short cilia in cultured ARNE cells. In addition, the mRNA level of TSLP which was one of the epithelial-derived allergic cytokines was significantly higher, and the expressions of genes involved in ciliogenesis were lower in cultured ARNE cells without allergen stimulation.
CONCLUSIONS
Our findings suggest that ALI culture of ARNE cells using intranasal brushing may be an alternative method for epithelial cell culture in AR patients and that cultured ARNE cells will be useful for in vitro studies of the mechanisms at play during AR because they maintain unique allergic characteristics.

Keyword

Intranasal brushing; human nasal epithelial cells; primary cell culture; allergic rhinitis

MeSH Terms

Cell Culture Techniques
Cilia
Cytokines
Epithelial Cells*
Healthy Volunteers
Humans
Microscopy, Electron
Nasal Mucosa
Primary Cell Culture
Rhinitis*
RNA, Messenger
Tight Junctions
Cytokines
RNA, Messenger

Figure

• 

  Fig. 1 Morphology of cultured nasal epithelial cells under light microscope, measurement of transepithelial electric resistance (TEER) and histologic appearance of ALI cultures of NHNE cells. (A) Primary nasal epithelial cells growing on subculture (left panel) and ALI culture periods (right panel) (original magnification ×20). (B) TEER of unstimulated NHNE cells cultured for 7, 14, 21, and 28 days. Values are expressed as mean±SD. (C) Cross section (5 µm) of NHNE cells, at 14 days after confluence, stained with H&E (upper panel), PAS (middle panel) and immunofluorescence (lower panel, red color: α-tubulin IV, blue color: DAPI) (original magnification ×200).

• 

  Fig. 2 Scanning electron microscopic (SEM) and Transmission electron microscopic (TEM) findings of cultured NHNE cells at 14 days after confluence. SEM findings of cultured NHNE cells showing well-differentiated ciliated and secretory cells (A, original magnification ×100) (B, original magnification ×200). TEM findings of a ciliated cell at the apical surface of the epithelium and a columnar secretory cell containing electron-lucent secretory granules (C, original magnification ×1,000) (D, original magnification ×2,000).

• 

  Fig. 3 Histologic appearance of human nasal mucosa from the inferior turbinate with H&E and PAS staining. H&E staining (A) showed the structure of human nasal epithelium, including ciliated and secretory cells and basement membrane. Well-stained secretory cells were observed in human nasal epithelium through PAS staining (B).

• 

  Fig. 4 Comparison of mean period for proliferation and differentiation in NHNE and ARNE cells. (A) Comparison of mean days taken for subculture in NHNE and ARNE cells. (B) Comparison of mean days taken for confluence in ALI culture of NHNE and ARNE cells. The results are presented here as the mean±SD (white bar: NHNE cells, black bar: ARNE cells). (C) Cross section (5 µm) of ARNE cells at 14 days after confluence stained with H&E (upper panel), PAS (middle panel) and immunofluorescence (lower panel, red color: α-tubulin IV, blue color: DAPI) (original magnification ×200). (D) Numbers of secretory cells expressed as a PAS-positive score 14 days after ALI confluence in NHNE and ARNE cells. (E) Numbers of ciliated cells per high power field 14 days after ALI confluence in NHNE and ARNE cells (white bar: NHNE cells, black bar: ARNE cells). The mRNA levels of Foxj1 and TEKTIN-1 are considerably lower in ALI culture of ARNE cells. Real-time PCR showed that Foxj1 (F) and TEKTIN-1 (G) mRNA levels are attenuated in ARNE cells 14 days after confluence. Results are presented here as the mean±SD from eight independent ALI cultures (* P<0.05 compared to mRNA levels in NHNE cells).

• 

  Fig. 5 Scanning electron microscopic (SEM) and transmission electron microscopic (TEM) findings of cultured ARNE cells at 14 days after confluence. SEM findings of cultured ARNE cells show smaller numbers of ciliated cells and higher numbers of secretory cells (A, original magnification ×100) (B, original magnification × 200). TEM findings of loose cell-to-cell tight junctions in nasal epithelium and increased number of columnar secretory cells containing electron-lucent secretory granules (C, original magnification ×1,000) (D, original magnification ×2,000). The mRNA levels of E-cadherin and ZO-1 were measured in ALI culture of NHNE and ARNE cells 14 days after confluence. Real-time PCR showed that both E-cadherin (E) and ZO-1 (F) mRNA level were significantly attenuated in ALI culture of ARNE cells (White dot: NHNE cells, Black dot: ARNE cells). Results are presented here as the mean±SD from eight independent ALI cultures (*P<0.05 compared to mRNA levels in NHNE cells).

• 

  Fig. 6 The gene expression of epithelium-derived cytokines in ALI culture of NHNE and ARNE cells. The mRNA levels of TSLP, IL-25, and IL-33 were measured in ALI culture of NHNE and ARNE cells 14 days after confluence. (A) Real-time PCR showed that the TSLP mRNA level was significantly higher in ALI culture of ARNE cells. However, IL-25 (B) and IL-33 (C) mRNA levels were not significantly different between NHNE and ARNE cells (White dot, NHNE cells; Black dot, ARNE cells). Results are presented here as the mean±SD from eight independent ALI cultures (*P<0.05 compared to mRNA levels in NHNE cells).

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