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Failure to induce nitric oxide production by human monocyte-derived macrophages. Manipulation of biochemical pathways
M Arias1, J Zabaleta, J I Rodríguez
1Laboratorio Central de Investigaciones, Facultad de Medicina, Universidad de Antioquía, Medellín, Colombia.
Abstract:
Production of nitrix oxide (NO-) by human macrophages is controversial. In the present study, the ability of human monocyte-derived macrophages (M phi) to produce NO- in response to M phi modulators was tested. M phi cultured for up to nine days and stimulated for 48 with different concentrations of LPS and/or IFN-gamma failed to produce significant amounts of NO2- compared to unstimulated cultures. Inhibition of the cyclo-oxygenase pathway with indomethacin did not increase NO2- production by LPS stimulated M phi. Since human M phi lack biopterin, needed for NO- synthesis by murine M phi, human M phi stimulated with LPS plus IFN-gamma were additionally cultured in the presence of neopterin or biopterin. These treatments did not induce NO2- production. Furthermore, simultaneous treatment with indomethacin and neopterin or biopterin also failed to induce NO2- production. However, human M phi, stimulated with IFN-gamma and LPs, produced TNF-alpha suggesting that the lack of increment in NO2- production was not due to an absence of response of M phi to the stimuli used. As an indirect approach to explore the NO- production, human M phi were infected with virulent Mycobacterium tuberculosis H37Rv and simultaneously treated with the competitive inhibitor NGmonomethyl-L-arginine (NGMMA). Mycobacterial intracellular replication was measured by 3H-uracil incorporation. NGMMA did not have any effect on mycobacterial replication. These results further suggest that human M phi do not produce NO- at least by the inducible pathway.
Insights
Human macrophages do not produce nitric oxide (NO) via the inducible pathway, even when stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Further tests confirmed this lack of NO production in response to Mycobacterium tuberculosis.
Area of Science:
- Immunology
- Cell Biology
Background:
- Nitric oxide (NO) production by human macrophages is a subject of ongoing research and debate.
- Understanding NO production is crucial for comprehending immune responses and host defense mechanisms.
Purpose of the Study:
- To investigate the capacity of human monocyte-derived macrophages (M phi) to produce nitric oxide (NO) in response to various stimuli.
- To explore potential factors influencing NO synthesis, including co-factors and inflammatory mediators.
Main Methods:
- Human M phi were cultured and stimulated with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-γ).
- Nitrite (NO2-) production was measured, and experiments included the use of indomethacin, neopterin, and biopterin.
- Macrophage response was assessed by measuring TNF-alpha production.
- Indirect NO production was evaluated by infecting M phi with Mycobacterium tuberculosis and treating with NG-monomethyl-L-arginine (NGMMA).
Main Results:
- Stimulated M phi failed to produce significant amounts of NO2- compared to unstimulated controls.
- Inhibition of the cyclo-oxygenase pathway or addition of biopterin/neopterin did not induce NO2- production.
- M phi produced TNF-alpha upon stimulation, indicating responsiveness to stimuli.
- NGMMA did not affect intracellular mycobacterial replication, suggesting no significant NO production.
Conclusions:
- Human monocyte-derived macrophages do not appear to produce nitric oxide (NO) through the inducible pathway.
- The lack of NO production was observed even when macrophages were stimulated with potent immune modulators and challenged with Mycobacterium tuberculosis.