Differentiation and Functionality of Bone Marrow-Derived Mast Cells Depend on Varying Physiologic Oxygen Conditions

Abstract

Mast cells (MCs) are long-living multifunctional innate immune cells that originate from hematopoietic precursors and specifically differentiate in the destination tissue, e.g., skin, respiratory mucosa, intestine, where they mediate immune cell recruitment and antimicrobial defense. In vivo these tissues have characteristic physiological oxygen levels that are considerably lower than the atmospheric oxygen conditions (159 mmHg, 21% O2; 5% CO2) traditionally used to differentiate MCs and to study their functionality in vitro. Only little is known about the impact of physiological oxygen conditions on the differentiation process of MCs. This study aimed to characterize the differentiation of immature murine bone marrow-derived MCs under physioxia in vitro (7% O2; 53 mmHg; 5% CO2). Bone marrow-derived suspension cells were differentiated in the presence of interleukin-3 with continuous, non-invasive determination of the oxygen level using a Fibox4-PSt3 measurement system without technique-caused oxygen consumption. Trypan blue staining confirmed cellular viability during the specified period. Interestingly, MCs cultivated at 7% O2 showed a significantly delayed differentiation rate defined by CD117-positive cells, analyzed by flow cytometry, and reached >95% CD117 positive population at day 32 after isolation. Importantly, MCs differentiated under physioxia displayed a decreased transcript expression level of hif-1α and selected target genes vegf, il-6, and tnf-α, but an increase of foxo3 and vhl expression compared to MCs cultivated under normoxia. Moreover, the production of reactive oxygen species as well as the amount of intracellular stored histamine was significantly lower in MCs differentiated under low oxygen levels, which might have consequences for their function such as immunomodulation of other immune cells. These results show for the first time that physioxia substantially affect maturation and the properties of MCs and highlight the need to study their function under physiologically relevant oxygen conditions.

TAK1 signaling activity links the mast cell cytokine response and degranulation in allergic inflammation

“…Primary mast cell cultures were established by isolating bone marrow, rich in hematopoietic stem and progenitor cells, from the tibia and femur of wild‐type mice, with differentiation being induced by the addition of IL‐3 and PGE 2 29 . BMMC cultures were maintained at 37°C and 5% CO 2 as previously described 16 .…”

“…Primary mast cell cultures were established by isolating bone marrow, rich in hematopoietic stem and progenitor cells, from the tibia and femur of wild-type mice, with differentiation being induced by the addition of IL-3 and PGE 2 . 29 BMMC cultures were maintained at 37 • C and 5% CO 2 as previously described. 16 This concentration was selected based on successful TAK1 inhibition by OZ in various of cell types and models [30][31][32][33] and no impact on cell viability was detectable.…”

Mast cells—Good guys with a bad image?

“…The c-Kit receptor is not only expressed by MCs but also by mainly all hematopoietic progenitors in the bone marrow, by thymocyte progenitors as well as interstitial cells of Cajal in the digestive tract and skin melanocytes [50][51][52]. Whereas other cells downregulate the expression of CD117 during their development, the expression on MCs is constantly high during lifetime 53.…”

Detection of Mast Cells Expressing c-Kit Using Antibody Covalently Bound to Gelatin Elongated from Surface of Immunosensor Based on Surface Plasmon Resonance

“…3,7,8 Since the c-Kit is constantly and strongly expressed on the surface of mast cells, it is a good cell marker. [9][10][11] The mast cells play a key role in IgE-associated immune responses. 3,8 After the allergen is bound to the IgE, of which Fc domain had been bound to FcεRI receptor of the mast cell surface, chemical mediators such as histamine, leukotriene, and prostaglandin are released from the granules.…”

“…The mast cells are generally detected by using a microscope or a flow cytometer after reacting with fluorescence-labeled anti-c-Kit antibody. [9][10][11] On the other hand, an immunosensor based on surface plasmon resonance (SPR-immunosensor) was developed for real-time detection of the MEG01s cell line expressing c-Kit derived from human megakaryoblastic leukemia and of HEK293T cell line expressing a low amount of c-Kit derived from a human fetal kidney, without fluorescence label. 12 In this study, the mast cell however almost cannot be detected by their condition.…”