Multinucleated giant cell phenotype in response to stimulation

Abstract

Macrophages fuse into multinucleated giant cells (MGC) in many pathological conditions. Despite MGC correlations with granulomas, their functional contribution to inflammation is relatively unknown. An in vitro mouse model of IL-4-induced bone marrow-derived macrophage fusion and microfiltration were used to generate enriched MGC and macrophage populations. Phenotypes were compared in response to well-known inflammatory stimuli, including lipopolysaccharide and crocidolite asbestos. Surface markers were assessed by flow cytometry: CD11b, CD11c, F4/80, and MHC II. Secreted cytokines were assessed by multiplex immunoassay: IFN-γ, IL-1β, IL-6, TNF-α, IL-10, IL-13, and IL-33. Results show that MGC maintained macrophage surface protein expression but lost the ability to produce a cytokine response. This suggests a potentially beneficial role of MGC in isolating the host from a foreign body without contributing to excessive inflammation. This study and future research using other stimulants and environments are important to gaining a fundamental MGC cell biology understanding. This will inform approaches to controlling the foreign body response to particle exposure, medical implants, and many diseases associated with granulomas.

Keywords: Cytokine; In vitro; Macrophage; Mouse; Multinucleated giant cell; Phenotype; Surface marker.

Fig. 1.. Enrichment by microfiltration.

Representative images of purified cell populations resulting from microfiltration separation. (A) Cells that passed through the filter were macrophage-enriched. (B) Cells blocked by the filter were MGC-enriched. After filtration, cell concentrations were adjusted to seed 96-well plates with equal numbers of nuclei/well for subsequent experiments. Scale is same for both images, bar 100 μm.

Fig. 2.. Surface markers before normalization.

Cells were gated according to nuclear fluorescence into three groups: one nucleus (1N; black bars), two nuclei (2N; gray), and three or more nuclei (≥3N; white). Median fluorescence intensity (MFI) of all surface markers increases in binucleated macrophages (2N) and MGC (≥3N), which may lead to the conclusion that marker expression is increased in these cells. However, the increase in fluorescence may simply be a result of larger cell size, with actual membrane density distribution of markers remaining unchanged. This demonstrates the importance of data normalization.

Fig. 3.. Surface markers after normalization.

Normalized protein expression by macrophages (gray bars) and MGC (white bars) that were unstimulated (control) or stimulated with LPS or LPS plus asbestos for 24 h. No significant differences in surface markers were observed between macrophages and MGC. Asterisks(*) indicate significant treatment effects versus corresponding cell-type control.

Fig. 4.. Cytokine secretion.

Supernatant protein concentrations from enriched populations of macrophages (gray bars) and MGC (white bars) that were unstimulated (control) or stimulated with LPS or LPS plus asbestos for 24 h. The immunoassay included cytokines generally associated with classical (M1; left column) and alternative (M2; right column) activation. Asterisks(*) indicate significant treatment effects versus corresponding cell-type control. Daggers(†) indicate other significant contrasts as shown by bars.