High doses of LPS caused a distinct macrophage phenotype including protein expression of NKG2D ligands that trigger NK cell–mediated lysis. (A) Macrophages or macrophages incubated with 100 or 200 ng/mL LPS for 48 hours (denoted 0, 100, 200 respectively) were stained with anti-CD54 mAb or anti-CD44 mAb and analyzed by flow cytometry. (B) Cytokines secreted into the supernatant by macrophages stimulated with different doses of LPS for 48 hours. (C) Macrophages that were incubated with different amounts of LPS for 48 hours were probed for transcripts of different NKG2D ligand genes by RT-PCR. M indicates marker; 0, 50, 100, and 200, the amount of LPS in nanograms per milliliter; -, water; +, positive control. (D) Macrophages incubated with different amounts of LPS for 48 hours were tested for expression of ULBP3 and MICA by Western blotting. (E) Macrophages after incubation with 0 (green line), 100 (macrophage^{low LPS}; black), and 200 (macrophage^{high LPS}; blue) ng/mL LPS were stained for NKG2D ligands, class I MHC protein, or CD48 and analyzed by flow cytometry. Expression of each NKG2D ligand was confirmed by using alternative mAb as shown (clone names given in parentheses). Isotype-matched control mAbs were used for comparison (shown in each panel for macrophage^{high LPS}; gray filled histograms). (F) NK cells were preincubated with 20 μg/mL anti-NKG2D mAb or an IgG1 isotype-matched control for 45 minutes before testing their cytotoxicity against macrophages incubated with 200 ng/mL LPS for 48 hours. NK cells and target cells were incubated at the ratios indicated, and specific lysis was measured by standard ³⁵S release assays. In each experiment the spontaneous release of ³⁵S was less than 20% of the total. Proliferation (G) and IFN-γ secretion (H) of NK cells after 48 hours incubated alone (no APC) or coincubated with macrophages or macrophages activated with 100 ng/mL (macrophage^{low LPS}) or 200 ng/mL (macrophage^{high LPS}) LPS at a 10:1 ratio. NK cells were pulsed with anti-2B4 (4 μg/mL), anti-NKG2D (20 μg/mL), or isotype-matched control mAbs prior to coculture with APC for 48 hours. *P < .05, **P < .01 by Student t test for unpaired values. The results are expressed as the mean ± SE of at least 3 independent experiments using cells from different donors.