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Rapid Magnetic-microbead Method for Efficient Purification of Low-density Neutrophils

Neutrophils, important leukocytes of innate immunity, have traditionally been considered a homogeneous cell population. Nevertheless, recent evidence has ...

Universidad Nacional Autónoma de México

Neutrophils are the most abundant leukocytes in human peripheral blood, comprising about 70% of all leukocytes. They are regarded as the first line of defense of the innate immune system, but neutrophils have also the ability of regulating the adaptive immune response. Recently, However, multiple phenotypes and functional states of neutrophils have been reported, particularly in inflammation, autoimmunity, and cancer. One possible subtype of neutrophils, the so-called low-density neutrophils (LDN) is found among mononuclear cells (MNC), monocytes and lymphocytes, after separating the leukocytes from blood by density gradient centrifugation. LDN increase in numbers during several pathological conditions. However, LDN present in healthy conditions have not been investigated further. Therefore, in order to confirm the presence of LDN in blood of healthy individuals and to explore some of their cellular functions, neutrophils and MNC were isolated by density gradient centrifugation. Purified neutrophils were further characterized by multicolor flow cytometry (FACS) and then, using the same FACS parameters cells in the MNC fraction were analyzed. Within the MNC, LDN were consistently found. These LDN had a normal mature neutrophil morphology and displayed a CD10, CD11b, CD14, CD15, CD16b, CD62L, CD66b, and CXCR4 phenotype. These LDN had an enhanced reactive oxygen species (ROS) production and increased phagocytic capacity and were able to produce neutrophil extracellular traps (NET) similarly to neutrophils. These data confirm the presence of a small number of LDN is blood of healthy individuals and suggest that these LDN represent mature cells with a primed phenotype.

Low-Density Neutrophils in Healthy Individuals Display a Mature Primed Phenotype.

[This corrects the article DOI: 10.3389/fimmu.2021.672520.].

Corrigendum: Low-density neutrophils in healthy individuals display a mature primed phenotype.

Receptors for FcγR on human neutrophils constitute an important mechanism for the recognition of opsonized microorganisms and for cell activation. Human neutrophils express 2 FcγR: FcγRIIa and FcγRIIIb. Previously, it has been reported that activation of each FcγR induces different neutrophil responses by triggering distinct signal transduction pathways, although what particular signal transduction pathway is triggered by each FcγR has not been completely elucidated. It has also been reported that PKC is important for FcγR signaling and that each FcγR may activate different PKC isoforms. Therefore, we explored whether FcγRIIa or FcγRIIIb activates different PKC isoforms in human neutrophils and whether activation of these PKC isoforms results in different neutrophil responses. Hence, either FcγRIIa or FcγRIIIb was selectively cross-linked by monoclonal antibodies in the presence or absence of pharmacological inhibitors for various PKC isoforms. Inhibition of PKCα or PKCδ blocked FcγRIIa-induced reactive oxygen species productions. In contrast, inhibition of PKCα and/or PKCβ blocked FcγRIIIb-induced reactive oxygen species production. Also, inhibition of all PKC isoforms did not affect the FcγRIIa-induced increase in intracellular calcium concentration ([Ca2+]i), while inhibition of PKCα blocked FcγRIIIb-induced increase in [Ca2+]i. Additionally, inhibition of PKCδ blocked FcγRIIa-induced ERK phosphorylation, while inhibition of PKCα prevented FcγRIIIb-induced ERK phosphorylation. These results suggest that both FcγRIIa and FcγRIIIb activate unique PKC isoforms and that activation of these PKC isoforms can selectively regulate different neutrophil functions. These findings also reinforce the idea that each FcγR in human neutrophils triggers distinct signal transduction pathways.

Article	Year
Low-Density Neutrophils in Healthy Individuals Display a Mature Primed Phenotype. Frontiers in immunology\| PubMed ID: 34276661	2021
Corrigendum: Low-density neutrophils in healthy individuals display a mature primed phenotype. Frontiers in immunology\| PubMed ID: 39286251	2024
Fc gamma receptors activate different protein kinase C isoforms in human neutrophils. Journal of leukocyte biology\| PubMed ID: 39946245	2025

Carlos Blanco-Camarillo

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