Phenotypic non-equivalence of murine (monocyte-) macrophage cells in biomaterial and inflammatory models

Abstract

Cells of the mononuclear phagocytic system including monocytes and macrophages (e.g., pooled human monocytes, bone marrow-derived macrophages, etc.) are often employed for in vitro assessment of novel biomaterials and to assay anti-inflammatory drug activity. In this context, numerous macrophage cells are treated interchangeably in the literature despite a lack of demonstrated equivalence among immortalized cell lines and further, between cell lines and primary-derived macrophages of different species. Three murine (monocyte-) macrophage cell lines (IC-21, J774A.1, and RAW 264.7), commonly utilizedin biomaterial and pharmaceutical screening research, have been compared with primary-derived murine bone marrow macrophages. Significant differences were discovered in the expression of cell surface proteins requisite for cell adhesion and activation among cell lines and primary-derived cells as well as between the different cell lines. Results demonstrate activation but with reduced cytokine expression to chemical stimulus (lipopolysaccharide) by cell lines compared with that of primary-derived macrophages. Limited correlation between cultured primary and immortalized cells in cytokine production, phenotype and intrinsic activation states has relevance to fidelity for in vitro testing. These differences warrant justification for selection of various cell lines for specific assay purposes, and merit caution if comparisons to primary cell types (i.e., for biocompatibility) are required.

Figure 1.

Morphology of macrophage-lineage cell types. Phase contrast microscopy images of RAW 264.7, J774A.1, IC-21, and BMMΦ cells on tissue culture-treated polystyrene (TCPS).

Figure 2.

Phenotypic characteristics of primary-derived macrophages and macrophage cell lines. Flow cytometric mean percent positive and mean fluorescent channel (MFC) data for macrophage cell-surface markers F4/80, CD14, FcR, CD11b, CD18, CD11c, CD54, CD40, TLR-4, and MMR, ± the standard error (data are representative of at least three experiments, statistical significance of p ≤ 0.05 is indicated by an asterisk). Cells were cultured on TCPS for 24 h in serum containing media as described in Materials and methods section.

Figure 3.

Flow cytometric analysis of inflammatory cytokines. Cells were incubated with LPS and/or a complex to stop Golgi complex export of cytokines for 8 h prior to intracellular immunostaining and flow cytometry analysis. White bars indicate basal cytokine expression on TCPS and black bars indicate cytokine expression after treatment with LPS. Error bars represent standard error. The induction of cytokine expression varies greatly from cell type to cell type. (Data are representative of at least three experiments, statistical significance of p ≤ 0.05 is indicated by an asterisk).

Scheme 1.

Flowchart of experimental procedures. Methods for comparison of cell types included culture with various stimuli (model surfaces, lipopolysaccharide), followed by data collection at both the mRNA transcript (PCR, MPCR) and the protein (flow cytometry) level.