Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4

Abstract

Although studies have been performed to characterize responses of macrophages from individual anatomical sites (e.g., alveolar macrophages) or of murine-derived macrophage cell lines to microbial ligands, few studies compare these cell types in terms of phenotype and function. We directly compared the expression of cell surface markers and functional responses of primary cultures of three commonly used cells of monocyte-macrophage lineage (splenic macrophages, bone marrow-derived macrophages, and bone marrow-derived dendritic cells) with those of the murine-leukemic monocyte-macrophage cell line, RAW 264.7. We hypothesized that RAW 264.7 cells and primary bone marrow-derived macrophages would be similar in phenotype and would respond similarly to microbial ligands that bind to either Toll-like receptors 2, 3, and 4. Results indicate that RAW 264.7 cells most closely mimic bone marrow-derived macrophages in terms of cell surface receptors and response to microbial ligands that initiate cellular activation via Toll-like receptors 3 and 4. However, caution must be applied when extrapolating findings obtained with RAW 264.7 cells to those of other primary macrophage-lineage cells, primarily because phenotype and function of the former cells may change with continuous culture.

Figure 1

Representative results of an experiment is which cells within each population were stained for CD11b. CD11b staining for each cell type is shown relative to its corresponding negative control.

Figure 2

TNFα concentrations (mean with range of values of three replicate experiments) in supernatants of RAW cells, Sp-Mφ, BM-Mφ, and BM-DC incubated in media alone (control) or media containing LPS, poly I:C (pIC), or Pam₃CSK₄ (PAM). All four of the cell populations produced significantly higher concentrations of TNFα after incubation with LPS, than when incubated with medium alone (indicated by “a”). TNFα production by RAW cells when stimulated with PAM was significantly above that produced by other cell types (indicated by #).

Figure 3

RANTES concentrations (mean with range of values of three replicate experiments) in supernatants of RAW cells, Sp-Mφ, BM-Mφ, and BM-DC incubated in media alone (control) or media containing LPS, poly I:C (pIC), or Pam₃CSK₄ (PAM). All four of the cell populations produced significantly higher concentrations of RANTES after incubation with LPS, than when incubated with medium alone (indicated by “a”). RANTES production by BM-Mφ when stimulated with PAM was significantly above that produced by other cell types (indicated by #).