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Differentiation of THP-1 and U937 Monocytes into Macrophages
As already mentioned before, nearly all THP-1 cells start to adhere to culture plates and differentiate into macrophages after exposure to PMA. Also U937 pro-monocytes differentiate into mature monocytes or into macrophages upon PMA-treatment. Typical exposure to PMA is for 48 h (Zhang et al. 2010; Gillies et al. 2012; Moreno-Navarrete et al. 2009; Cam and de Mejia 2012). The temperature and atmosphere, if described, was the same for each study, namely 37 °C and 5 % CO2 respectively. Different stimuli were used, depending on the aim of the study.
Subsequently, macrophages can be further differentiated into subsets. Typical markers for M1-type macrophages are transcription or production of TNF-α, IL-1β, IL12-p40, IL-6, IL-8 and LOX-1, and for M2-type macrophages MRC-1, dectin-1, and DC-SIGN (Chanput et al. 2010).
Based on literature and on our experience, 0.5 × 106 THP-1 monocytes fully differentiate into macrophages after 48 h incubation at a minimal concentration of 100 ng/ml PMA (162 nM), followed by washing twice with culture media without PMA and a resting period of 24 h (Chanput et al. 2014), resulting in macrophages with a high phagocytic capacity for latex beads and expressing cytokine profiles that resembled PBMC monocyte-derived macrophages after exposure to TLR ligands (Chanput et al. 2014).
Human promonocytic leukaemia U937 cells differentiate into monocytes and macrophages by use of various agents such as retinoic acids, 1,25-dihydroxyvitamin D3 (VD3; at 100 nM, i.e. 42 ng/ml, Rots et al. 1999), and 12-O-tetradecanoylphorbol13-acetate (TPA; at 20 ng/ml, ca. 32 nM) (Chun et al. 2001).
Differentiation of THP-1 and U937 Monocytes into Dendritic Cells
THP-1 monocytes are described to differentiate into mature dendritic cells by transferring them to serum-free medium, and subsequently treating them with a mixture of IL-4, GM-CSF, TNF-α and ionomycin. These hematopoietic cell line-derived DCs are highly pure and monotypic, and display the morphologic, phenotypic, molecular, and functional properties of DCs generated from human donor-derived monocytes or CD34+ hematopoietic progenitor cells (see also Chap. 17). During differentiation into mature DCs, the cells exhibit de novo cell-surface expression of CD83, CD80, CD86, CD40, CD206, CD209, CD120a, CD120b, and intracellular synthesis of IL-10, increase their endocytotic capacity, and acquire the characteristic stellate morphology (Berges et al. 2005). In the THP-1 monocytes, mRNAs of tight junction molecules, occludin, tricellulin, JAM-A, ZO-1, ZO-2 and claudin-4, -7, -8, and -9 were detected by RT-PCR. In mature DCs that had elongated dendrites, mRNA and protein of JAM-A were significantly increased compared to the monocytes (Ogasawara et al. 2009).
Exposure of U937 to a self-peptide from apolipoprotein E, Ep1.B, induces DC-like morphology and surface marker expression in U937 (Stephens et al. 2008).
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