Original article
1-α,25-Dihydroxyvitamin D3 regulates inducible nitric oxide synthase messenger RNA expression and nitric oxide release in macrophage-like RAW 264.7 cells☆

https://doi.org/10.1016/j.lab.2003.08.002Get rights and content

Abstract

The expression of inducible nitric oxide synthase (iNOS) expression and release of nitric oxide (NO) from macrophages are markedly increased in granulomatous infections. Activation of macrophages 1α-hydroxylase results in an increase of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, the significance of this increased production is not completely understood. In this study, we analyzed 1,25(OH)2D3 and NO production in patients with tuberculosis infection and hypercalcemia and used lipopolysaccharide (LPS) to stimulate RAW 264.7 cells in an attempt to assess iNOS expression and gaseous NO production regulated by 1,25(OH)2D3. Peroxynitrite (OONO−) production and lactate dehydrogenase activity were also examined. Without additional stimulation, peripheral-blood mononuclear cells (PBMCs) from patients with tuberculosis converted more 25-hydroxyvitamin D3 to 1,25(OH)2D3 than did those from normal controls. These PBMCs released less NO than did those from control subjects, at baseline and in the stimulated state. We found that 1,25(OH)2D3 dose-dependently inhibited iNOS messenger RNA expression of the LPS-stimulated RAW 264.7 cells and also significantly reduced the gaseous NO release and OONO− production. Paralleling the 1,25(OH)2D3-induced inhibition of NO release were reductions in OONO− and LDH production. In conclusion, 1,25(OH)2D3 inhibited iNOS expression and reduced NO production by LPS-stimulated macrophages in the range of physiological doses. Inhibition of the NO surge was coupled with a reduction in OONO− and LDH production. Increased 1,25(OH)2D3 production and decreased release of NO from the PBMCs of patients with tuberculosis and hypercalcemia were also noted. We propose that 1,25(OH)2D3 production by macrophages may protect themselves against oxidative injuries caused by the NO burst. In the case of tuberculosis infection, increased 1,25(OH)2D3 synthesis may further contribute to the development of an unwanted phenomenon—hypercalcemia.

Section snippets

Ethical concerns

All experiments were designed and performed under the regulation of the Ethical Committee for Human and Animal Research of Kaohsiung Medical University.

Isolation of human PBMCs

Six healthy volunteers and 6 patients (all male) with newly reactivated pulmonary TB infections were included; all gave their informed consent to inclusion in the study. The mean plasma ionized calcium level was 4.72 ± 0.40 mg/dL in normal subjects and 5.84 ± 0.52 mg/dL in patients with TB. Daily urine calcium excretion was 65 ± 22 mg in normal

1,25(OH)2D3 levels from sera and PBMCs of normal subjects and and tuberculosis patients

Before preparing PBMCs, we subjected serum samples from controls and patients to measurement of 1,25(OH)2D3. Patients with tuberculosis had a significantly higher 1,25(OH)2D3 levels (57.1 ± 5.4 pg/mlL than did normal subjects (30.9 ± 1.9 pg/mL) (Fig 1, A). Fig 1, B, shows the 1,25(OH)2D3 synthetic capacity of the PBMCs after augmentation with 250 nmol/L 25-(OH)vitamin D3 as substrate and incubation for 6 hours. In PBMCs from normal subjects, 1.0 μg/mL LPS stimulation did not increase the

Discussion

The discovery of the universal presence of vitamin D receptors in more than 30 tissues initiated consideration of the possible functions of 1,25(OH)2D3 outside its classical role in calcium/bone homeostasis.22 To date, 1,25(OH)2D3 has been viewed as a pluripotent hormone that regulates cell proliferation/differentiation and expresses immunomodulatory activities in a paracrine fashion.9 Interaction between 1,25(OH)2D3 and immune system is important in Mycobacterium tuberculosis (TB) infection,

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    Supported by grant NSC 86-2314-B-037-040 from the National Science Council of Taiwan.

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