Abstract
Leukocytes are a part of the immune system that plays an important role in the host’s defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells’ types. To prove this hypothesis, UV−Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.
Highlights
Eosinophils (EOS), neutrophils (Ne) and basophils are granulocytes found in human peripheral blood and tissues
Even though the 785 nm excitation line was not close enough to electronic transitions of neutrophils and eosinophils cells for Raman spectroscopy (rR), we found low-intensity bands attributed to their peroxidases; i.e., at 756 (ν15 ), 1122 (ν22 ) and 1580 cm−1 (ν2 ) for eosinophil peroxidase (EPO) and at 756 (ν15 ), 933 (ν46 ), and 1588 cm−1 (ν2 ) for MPO
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]
Summary
Eosinophils (EOS), neutrophils (Ne) and basophils are granulocytes found in human peripheral blood and tissues They are small and round-shaped with specific granules in their cytoplasm, which contain characteristic proteins specific for each granulocyte type. Eosinophils are involved in the initiation and propagation of inflammatory responses [8], helminth infections and allergic diseases [7]. They are mainly known for their antiparasitic activity, but they constitute a prevalent cell population in the female reproductive tract [3]. Neutrophils are in turn azurophilic granulocytes with a diameter of 10–12 μm and multilobed nuclei [9] They are the most abundant leukocytes (54–62%)
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