1,25-Dihydroxyvitamin D3 induces nitric oxide synthase and suppresses growth of Mycobacterium tuberculosis in a human macrophage-like cell line

Abstract

Inducible synthesis of nitric oxide (NO) by macrophages is an important mechanism of the host defense against intracellular infection in mice, but the evidence for significant levels of inducible NO production by human macrophages is controversial. Here we report that the human promyelocytic cell line HL-60, when differentiated to a macrophage-like phenotype, acquires the ability to produce substantial amounts of NO on stimulation with LPS or 1, 25-dihydroxyvitamin D3 (1,25-D3) in the absence of activating factors such as gamma interferon. Expression of the inducible nitric oxide synthase (NOS2) was confirmed by sequencing of the reverse transcription-PCR product from stimulated HL-60 cells. Kinetic studies after lipopolysaccharide stimulation show that NOS2 mRNA levels rise within 3 to 6 h, that conversion of [14C]arginine to [14C]citrulline is maximal at 5 to 6 days, and that levels of reactive nitrogen intermediates stabilize at around 20 microM at 7 to 8 days. We find that 1,25-D3 acts to suppress the growth of Mycobacterium tuberculosis in these cells and that this effect is inhibited by NG-monomethyl-L-arginine, suggesting that vitamin D-induced NO production may play a role in the host defense against human tuberculosis.

FIG. 1

RNI production by original HL-60 cells (a) and HL-60_hca cells (b). Cells (10⁵ in 100 μl of medium) were stimulated for 48 h with medium alone (), LPS (100 ng/ml) (□), PMA (10 ng/ml) (), or LPS (100 ng/ml) plus PMA (10 ng/ml) (). (c) Inhibitory effect of l-NMMA on RNI production by HL-60_hca cells after 6 days of stimulation with LPS (100 ng/ml) (•). ■, background level of RNI production by unstimulated cells. Data are means ± SEMs for three experiments in each case.

FIG. 2

(a) Accumulation of RNI with time in culture wells containing 3 × 10⁶ HL-60_hca cells in 2 ml of medium stimulated with LPS (100 ng/ml). Data represent means ± SEMs for nine experiments. (b) Daily rate of [¹⁴C]citrulline formation from [¹⁴C]arginine, using 1.5 × 10⁵ cells per well containing 100 μl of medium and stimulated with 100 ng of LPS per ml. Results are means ± SEMs for six experiments.

FIG. 3

(a) RT-PCR for NOS1 (lanes 1), NOS2 (lanes 2), NOS3 (lanes 3), or a GAPDH control (lanes G) with RNA extracted from HL-60_hca cells stimulated with LPS (100 ng/ml) for 0, 24, or 96 h and from unstimulated HUVEC controls. (b) Northern analysis of NOS2 gene expression in HL-60_hca cells after various durations of stimulation with LPS (100 ng/ml). Kinetics over the first 24 h are shown by the graph, representing the ratio of NOS2 mRNA to β-actin mRNA as quantitated on a digital image as described in text. Longer kinetics are depicted in the inset, which compares NOS2 mRNA (Northern blot) with total RNA stained with ethidium bromide (EtBr). The molecular size of the NOS2 band is approximately 4.5 kb.

FIG. 4

(a) Induction of RNI in HL-60_hca cells after 6 days of stimulation with 1,25-D₃ (□), ergocalciferol (○), or cholecalciferol (▵). Filled circles denote experimental controls with medium alone, LPS (100 ng/ml), or LPS (100 ng/ml) plus l-NMMA (1 mM). Data points are means ± SEMs from four experiments. (b) RT-PCR for NOS1 (lanes 1), NOS2 (lanes 2), NOS3 (lanes 3), or a GAPDH control (lanes G) with RNA extracted from HL-60_hca cells stimulated with 10⁻⁸ M 1,25-D₃ LPS for 0, 24, or 96 h and from unstimulated HUVEC controls.

FIG. 5

Electron transmission micrograph of HL-60_hca cells, demonstrating infection with M. tuberculosis after 3 days of culture. Magnifications, ×16,500 (left panel) and ×32,500 (right panel). N, HL-60_hca cell nucleus. Arrows point to mycobacteria.