Characterization of epithelial cells, connective tissue cells and immune cells in human upper airway mucosa by immunofluorescence multichannel image cytometry: a pilot study

Aris I Giotakis,Herbert Riechelmann,Ljilja Bektic-Tadic,Rudolf Glueckert,Daniel Dejaco,Leyla Pinggera,Jozsef Dudas,Julia Ingruber,Sarah A M Gabriel,Veronika Innerhofer,Felix Fleischer

doi:10.1007/s00418-020-01945-y

Aris I Giotakis, Herbert Riechelmann + Show 9 more

Open Access

https://doi.org/10.1007/s00418-020-01945-y

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Abstract

Epithelial, connective tissue and immune cells contribute in various ways to the pathophysiology of chronic rhinosinusitis (CRS). However, data of their distribution in upper airway mucosa are sparse. We aimed to provide quantitative, purely informative data on the distribution of these cell lineages and their coexpression patterns, which might help identifying, e.g., cells in the epithelium undergoing through epithelial–mesenchymal transition (EMT). For this purpose, we used immunofluorescence multichannel image cytometry (IMIC). We examined fixed paraffin-embedded tissue samples (FFPE) of six patients with chronic rhinosinusitis (CRS) and of three patients without CRS (controls). The direct-conjugated antibodies pancytokeratin, vimentin and CD45/CD18 were used for coexpression analysis in epithelial layer and lamina propria. Image acquisition and analysis were performed with TissueFAXS and StrataQuest, respectively. To distinguish positive from negative expression, a ratio between cell-specific immunostaining intensity and background was developed. Isotype controls were used as negative controls. Per patient, a 4.5-mm2 tissue area was scanned and a median of 14,875 cells was recognized. The most common cell types were cytokeratin-single-positive (26%), vimentin-single-positive (13%) and CD45/CD18-single-positive with CD45/CD18–vimentin-double-positive cells (29%). In the patients with CRS, CD45/CD18-single-positive cells were 3–6 times higher compared to the control patients. In the epithelial layer, cytokeratin–vimentin-double-positive EMT cells were observed 3–5 times higher in the patients with CRS than in the control patients. This study provided quantitative data for the distribution of crucial cell types in CRS. Future studies may focus on the distribution and coexpression patterns of different immune cells in CRS or even cancer tissue.

Highlights

Chronic rhinosinusitis (CRS) is a disabling disease affecting more than 10% of the general population (Hastan et al 2011)
A cell was considered positive if its individual cell/background ratio was higher than the upper bound of the 95% confidence interval (CI) of the median cell/background ratio
We provide the first relative distribution of epithelial, connective tissue and immune cells in fixed paraffin-embedded tissue samples (FFPE) human upper airway tissue samples

Summary

Introduction

Chronic rhinosinusitis (CRS) is a disabling disease affecting more than 10% of the general population (Hastan et al 2011). CRS is classified into CRS with nasal polyps. (CRSwNP) and CRS without nasal polyps (CRSsNP) (Fokkens et al 2020). Epithelial cells, connective tissue cells and immune cells contribute in various ways to the pathogenesis of CRS. Few data on the relative abundance of these cell lineages are available (Jiang et al 2013). Our aim was to provide quantitative, purely informative data of the distribution of these cell lineages and their coexpression patterns in healthy and diseased upper airway mucosa. Coexpression patterns might identify, e.g., cells in the epithelium undergoing through epithelial–mesenchymal transition (EMT) (Li et al 2019; Yan et al 2019). After nuclear and cytoplasm segmentation, an area free of nuclei was analyzed in each tissue sample after setting the nuclear segmentation parameters to 0. Cytoplasmic-immunostained tissue sample was examined for positive cells in the remaining two cytoplasmic immunostainings

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