Effects of Mycobacterium bovis BCG infection on regulation of L-arginine uptake and synthesis of reactive nitrogen intermediates in J774.1 murine macrophages

Abstract

The generation of nitric oxide (NO) by activated macrophages is believed to control mycobacterial infection in the murine system. In this study we examined the effect of Mycobacterium bovis BCG infection on the L-arginine-dependent NO pathway in J774.1 murine macrophages. We have confirmed previous results by demonstrating that stimulation of J774.1 with lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) results in an increase in the uptake of 3H-labeled L-arginine and a concomitant increase in the production of NO. We have also shown that BCG can mimic LPS treatment, leading to enhanced L-[3H]arginine uptake by IFN-gamma-stimulated macrophages. Lipoarabinomannan, a component of the BCG cell wall that is structurally similar to LPS, is not responsible for the uptake stimulation in IFN-gamma stimulated macrophages. Although we demonstrated that there was a 2.5-fold increase in NO production by macrophages 4 h after LPS-IFN-gamma stimulation, BCG infection (with or without IFN-gamma stimulation) did not lead to the production of NO by the macrophages by 4 h postinfection. At 24 h postinfection, the infected macrophages that were stimulated with IFN-gamma produced amounts of NO similar to those of macrophages stimulated with LPS-IFN-gamma. This suggests that there are multiple regulatory pathways involved in the production of NO. Finally, our data suggest that increased expression of the arginine permease, MCAT2B, after 4 h of LPS-IFN-gamma treatment or BCG infection-IFN-gamma treatment is not sufficient to account for the increases in L-[3H]arginine uptake detected. This suggests that the activity of the L-arginine transporter(s) is also altered in response to macrophage activation.

FIG. 1

Uptake of l-[³H]arginine (A, B, and C) and l-[³H]proline (D) by resting (■), LPS–IFN-γ-stimulated (⧫), LPS-treated (○), IFN-γ-treated (▵), BCG-infected (▴), BCG-infected–IFN-γ-treated (●), heat-killed BCG-infected–IFN-γ-treated (◊), and heat-killed M. tuberculosis–IFN-γ-treated (□) J774.1 macrophages. Uptake is expressed as nanomoles of substrate per milligram of total protein (see Materials and Methods). Two independent experiments with each amino acid were performed in duplicate for each strain.

FIG. 2

Uptake of l-[³H]arginine by IFN-γ-primed J774.1 murine macrophages treated with either BCG LAM (■) or M. tuberculosis Erdman LAM (⧫) for 4 h.

FIG. 3

RT-PCR of MCAT1 and MCAT2B from resting (lane 1), LPS–IFN-γ-stimulated (lane 2), BCG-infected (lane 3), or IFN-γ-stimulated–BCG-infected (lane 4) 1774.1 macrophages. β-Actin was used as a quantitative control (see Materials and Methods). Fold differences in expression of MCAT1, MCAT2B, and β-actin are listed below each sample and are normalized to resting macrophage expression levels.

FIG. 4

Fold increase in the production of NO as measured by the DAN assay (see Materials and Methods) by resting (white bars), LPS–IFN-γ-stimulated (black bars), BCG-infected (dotted bars), and BCG-infected–IFN-γ-treated (vertically lined bars) J774.1 macrophages at 4 (A) or 24 (B) h poststimulation.

FIG. 5

Representative immunocytochemical staining of LPS–IFN-γ-stimulated J774.1 macrophages with α-MCAT1, α-MCAT2B, and α-iNOS antibodies. Concurrent phase-contrast images (Phase) are also shown.

FIG. 6

Fold increase in expression of MCAT1, MCAT2B, and iNOS in resting (white bars), LPS–IFN-γ-stimulated (black bars), BCG-infected (dotted bars), and BCG-infected–IFN-γ-treated (vertically lined bars) J774.1 macrophages as determined by FACS analysis.

FIG. 7

Uptake of l-[³H]arginine by resting (■), LPS–IFN-γ-stimulated (⧫), resting–cycloheximide-treated (▴), LPS–IFN-γ-stimulated and cycloheximide-treated (●) J774.1 macrophages. Uptake is expressed as nanomoles of substrate per milligram of total protein (see Materials and Methods). Two independent experiments with each amino acid were performed in duplicate for each strain.