Raman imaging highlights biochemical heterogeneity of human eosinophils versus human eosinophilic leukaemia cell line

Abstract

Summary Eosinophils are acidophilic granulocytes that develop in the bone marrow. Although their population contributes only to approximately 1–6% of all leucocytes present in the human blood, they possess a wide range of specific functions. They play a key role in inflammation‐regulating processes, when their numbers can increased to above 5 × 109/l of peripheral blood. Their characteristic feature is the presence of granules containing eosinophil peroxidase (EPO), the release of which can trigger a cascade of events promoting oxidative stress, apoptosis or necrosis, leading finally to cell death. Raman spectroscopy is a powerful technique to detect EPO, which comprises a chromophore protoporphyrin IX. Another cell structure associated with inflammation processes are lipid bodies (lipid‐rich organelles), also well recognized and imaged using high resolution confocal Raman spectroscopy. In this work, eosinophils isolated from the blood of a human donor were analysed versus their model, EoL‐1 human eosinophilic leukaemia cell line, by Raman spectroscopic imaging. We showed that EPO was present only in primary cells and not found in the cell line. Eosinophils were activated using phorbol 12‐myristate 13‐acetate, which resulted in lipid bodies formation. An effect of cells stimulation was studied and compared for eosinophils and EoL‐1.

Eosinophils and Neutrophils—Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy

“…As discussed in the introduction, eosinophils and neutrophils are mostly differentiated based on the nucleus shape, granule content as well as the presence of lipid bodies (LBs) observed in optical and fluorescence microscopy as we found in Raman images depicted in Figure 3 [6,13,52]. In turn the fluorescence images of EOS and Ne stained with the Hoechst dye and combined with the Columbus analysis showed that these organelles mainly differed in their symmetry whereas the radius and compactness were almost identical.…”

“…We have previously reported the sensing capability of normal Raman scattering imaging for the detection of LBs in primary eosinophils and eosinophilic cell line EoL-1 [6]. Nevertheless, LBs are not formed in each cell under normal conditions and without additional stimulation, and thus the collection of Raman images of single cells to detect LBs is time consuming.…”

“…The prosthetic group of EPO is a high spin, hexa-coordinated, ferric protoporphyrin IX. Major functions of EPO include the catalysis of oxidation of halides, pseudohalidase and nitric oxide to highly reactive oxygen species with cytotoxic properties [6]. MPO is also built of two monomers, which are linked through cysteine bridges [14].…”

“…Raman spectroscopic imaging is a powerful tool in the analysis of LB composition but a high through-put requires scanning of a large number of cells since LBs occur in a few percent of granulocytes only [6]. Therefore, the use of other methods, like fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopies, is desired to visualize the distribution of LBs in granulocytes.…”

“…The abundance of EOS in the peripheral blood is about 1–6% of all white blood cells and their lifespan in blood is about 18 h [ 4 ]. Approximately 200 granules with a diameter of 0.9–1.3 μm present in the EOS cytoplasm contain a pull of proteins including major basic protein (MBP), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP), and—eosinophil peroxidase (EPO) [ 6 , 7 ]. Eosinophils are involved in the initiation and propagation of inflammatory responses [ 8 ], helminth infections and allergic diseases [ 7 ].…”