The interaction of human natural killer cells with either unpolarized or polarized macrophages results in different functional outcomes

Abstract

The cross-talk among cells of the innate immunity can greatly affect both innate and adaptive responses. Here we analyzed the molecular interactions between human natural killer (NK) cells and autologous macrophages. Activated NK cells killed M0 and M2, whereas M1 macrophages were more resistant to lysis because of their higher expression of HLA class I molecules. Following exposure to LPS or bacillus Calmette-Guérin, M0 and M2, but not polarized (endotoxin tolerant) M1 macrophages, induced strong activation of resting NK cells. The expression of CD69 and CD25 activation markers and the acquisition of cytotoxicity against tumor cells and immature dendritic cells required soluble factors being mostly contact independent. On the contrary, IFN-γ production was contact dependent and required the interaction of DNAM-1 and 2B4 (on NK) with their ligands on macrophages as well as IL-18. IL-18 was involved also in the acquisition of CCR7 by NK cells. Interestingly, M0 and M2 cells expressed a membrane-bound form of IL-18, which was released in small amounts after LPS treatment. Our data indicate that, upon interaction with M0 macrophages exposed to microbial products, NK cells may amplify classical type 1 immune responses. In addition, M1-polarizing stimuli can rescue M2 macrophages from their immunomodulatory state and shape their functional behavior toward NK stimulatory capability.

Fig. 1.

Susceptibility of macrophages to lysis mediated by autologous NK cells. (A) Short-term-activated NK cells were analyzed at different E:T ratios for cytolytic activity against autologous M0, M1, M2, iDCs, and mDCs either in the absence (Left) or in the presence (Right) of mAb-mediated masking of HLA class I molecules. Average of 10 independent experiments. (B) Macrophages were analyzed by flow cytometry for the expression of HLA class I molecules. Average of 11 (HLA-I) and four (HLA-E) independent experiments. (C) Short-term-activated NK cells were analyzed for cytolytic activity against autologous M0, M1, and M2 in the presence of mAbs specific for different triggering NK receptors (E:T ratio 5:1). Average of seven independent experiments. SD is indicated. *P < 0.05.

Fig. 2.

Analysis of the cytolytic activity of NK cells cocultured with macrophages. After coculture with autologous M0, M1, and M2 (with or without LPS), NK cells were analyzed for cytolytic activity against K562 or autologous iDCs either in the presence of cell-to-cell contacts or in transwell (white bars). Average of six independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Analysis of CD69, CD25, and CCR7 expression in NK cells cocultured with macrophages. After coculture with autologous M0, M1, and M2 (with or without LPS), NK cells were analyzed by flow cytometry for the expression of CD69, CD25, and CCR7. The experiments were performed either in the presence of cell-to-cell contacts or in transwell (gray symbols). Average of 10 independent experiments. Squares and triangles indicate the MFI and the percentage of positive cells, respectively.

Fig. 4.

Analysis of mIL-18 expression and release by macrophages. (A) Macrophages either untreated or LPS treated were analyzed by flow cytometry for mIL-18 and CCR7 expression. (B) M0 were treated at the indicated time intervals with LPS in the absence (open symbols) or in the presence (black symbols) of monensin. Cell culture supernatants were analyzed by ELISA for the presence of IL-18 (triangle) and IL-12 (square). Average of three independent experiments (Left). The analysis was paralleled by the cytofluorimetric analysis of mIL-18 surface expression (Right). The mean MFI is indicated.

Fig. 5.

Molecular interactions involved in the macrophage-mediated induction of IFN-γ release by NK cells. (A) NK cells were cocultured with M0, M1, and M2 (with or without LPS) either in the presence of cell-to-cell contacts (Left) or in transwell (Right). Supernatants were analyzed by ELISA for the presence of IFN-γ. Average of 13 (contact) and four (transwell) independent experiments. (B) NK cells were cocultured with M0 and M2 in the presence of LPS and the indicated blocking/neutralizing antibodies. Supernatants were analyzed by ELISA for the presence of IFN-γ. Average of five independent experiments.