Research Article: The Identification of Markers of Macrophage Differentiation in PMA-Stimulated THP-1 Cells and Monocyte-Derived Macrophages

Date Published: January 13, 2010

Publisher: Public Library of Science

Author(s): Marc Daigneault, Julie A. Preston, Helen M. Marriott, Moira K. B. Whyte, David H. Dockrell, T. Mark Doherty.

Abstract: Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells.

Partial Text: Differentiated tissue macrophages arise from monocytes recruited from the blood [1]. Once differentiated, macrophages become long-lived cells and develop specialised functions. Cell numbers are maintained by resistance to constitutive apoptosis [2], by recruitment of further monocytes from the blood and/or replication of local intermediates depending on the prevailing stimulus and anatomical location [3], [4].

Macrophage heterogeneity is influenced by differentiation state, with marked differences between monocytes and macrophages [1], [40]. Commonly used protocols induce macrophage differentiation in monocytic cell lines but derivation towards a highly differentiated macrophage phenotype has not been a major focus of prior studies. In this study we have identified a number of macrophage characteristics associated with differentiation which vary in THP-1 cells treated with different protocols. These characteristics include the expansion of the cytoplasm and of cytoplasmic organelles (such as mitochondria and lysosomes), the capacity to upregulate the anti-apoptotic protein Mcl-1 and resist apoptotic stimuli and the responsiveness to TLR2 or 4 ligands. We demonstrate that a protocol of treating THP-1 cells with PMA followed by a period of further culture without PMA drives cells towards a differentiated macrophage phenotype that more closely resembles MDM than does differentiation with PMA or VD3 at doses commonly used in the literature [8]. Since MDM represent a good surrogate for tissue macrophages, such as alveolar macrophages, this suggests the PMAr cells may also represent a useful model with which to study tissue macrophages [16].



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