Copyright ©ERS Journals Ltd 2001 L-Arginine is not the limiting factor for nitric oxide synthesis by human alveolar macrophages in vitro1 Dept of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, 2 Dept of Pulmonology, University Hospital Groningen, Oostersingel 59, Groningen, and 3 Dept of Pharmacology and Pathophysiology, Utrecht University, Utrecht, the Netherlands CORRESPONDENCE: G. Folkerts, Dept of Pharmacology and Pathophysiology, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands. Fax: 31 302537420 Keywords: alveolar macrophage, human, monocyte, nitric oxide
Received: December 1, 2000
This study was funded by a research grant from the Netherlands Asthma Foundation no. 95.28.
Unlike murine mononuclear phagocytes, human macrophages do not release high amounts of nitric oxide (NO) in vitro despite the presence of nitric oxide synthase (NOS). To determine whether this limited NO synthesis in vitro is due to limited availability of the NOS substrate l-;arginine, and putative NOS inhibiting factors present in foetal serum preparations, both alveolar macrophages (AM) and monocyte derived macrophages (MDM) were incubated in various circumstances.
Nitrite production measured using stimulated AM was typically <5 pmol·min1·106 cells. A range of stimuli were tested, but without result. Furthermore, incubation of MDMs with normal human serum or purified bovine serum albumin instead of foetal calf serum failed to enhance NO production. Moreover, neither the use of arginase inhibitors nor the addition of surplus l-;arginine resulted in an increased NO synthesis. Interestingly, addition of the NOS intermediate N
It is concluded that the limited nitric oxide production of human macrophages in vitro can neither be explained by limited availability of l-;arginine, nor by nitric oxide synthase inhibiting substances in foetal serum. Furthermore, it is shown that nitrite release from N Nitric oxide (NO) is a well-documented effector molecule in rodents. Inflammatory cells, such as neutrophils and macrophages, release high amounts of NO upon immunostimulation 1, 2. Moreover, studies performed in inducible nitric oxide synthase (NOS2 or iNOS) knock-out mice reveal that NO plays a crucial role in the defence against the protozoan Leishmania major 3. Unlike rodent mononuclear phagocytes and granulocytes, both human macrophages and monocytes do not release significant amounts of NO in vitro when stimulated with the classical iNOS inducers interferon gamma (IFN-; ) and lipopolysaccharide (LPS). Despite this limited NO release, the induction of nitric oxide synthase (NOS) has been demonstrated by Weinberg et al. 4 using immunohistochemistry and Western blots. Furthermore, the formation of 3-;nitrotyrosine, a footprint of NO derived oxidants, has been demonstrated in human inflammatory disorders, e.g. asthma 5 and inflammatory bowel disease 6. For an extensive review about iNOS induction and NO production by human mononuclear phagocytes, the reader is referred to Weinberg et al. 7. At present, it is unclear why human mononuclear phagocytes do not release high amounts of NO in vitro despite the fact that iNOS is inducible and low amounts of NO formation are detectable 4. Theoretically, this lack of high output NO synthesis could be due to an in vitro artefact, since in vivo NO production during inflammatory processes seems evident 8.
There are several possible explanations for the limited NO output by human inflammatory cells in vitro. Theoretically, essential immunomodulating factors could be missing in the incubation medium. In contrast, the medium could very well contain factors that prevent NO synthesis. An example is docosahexaenoic acid, which has been demonstrated to inhibit the induction of iNOS by murine macrophages 9. Concentrations
According to Murphy and Newsholme 11, the lack of NO release by human mononuclear phagocytes is due to an increased breakdown of the NOS substrate l-;arginine by the enzyme arginase. Murphy and Newsholme 11 claim that pre-incubating monocyte derived macrophages (MDM) with low levels of l-;arginine prevents the induction of arginase. Resupplying the cells with l-;arginine at the time of immunostimulation would thus result in enhanced NO synthesis, since breakdown of l-;arginine by arginase is prevented (fig. 1
In this study, an attempt was made to induce NO output by human mononuclear phagocytes. For this purpose, both freshly isolated alveolar macrophages (AM) and MDM were used. Firstly, a variety of stimuli were screened on their ability to induce NO release. Secondly, the incubation circumstances (i.e. serum source and concentration, coating of the incubation plates) were varied. Moreover, whether the lack of high output NO synthesis in vitro can be explained by a limited availability of the NOS substrate l-;arginine was tested.
Alveolar macrophages Nonsmoking, healthy volunteers underwent bronchoalveolar lavage (BAL), as described previously 12. In short, four aliquots of 50 mL sterile phosphate-buffered saline (PBS) of 37°C were instilled into the lateral segment of the right middle lobe and recovered by gentle suction (40 cmH2O) after each aliquot. The BAL fluid was collected and immediately placed in ice. After collection, the BAL fluid was centrifuged for 5 min at 400xg (4°C). After washing (three times with PBS) the cells were resuspended in K-;medium (which comprised of Roswell Park Memorial Institute 1640 medium supplemented with 10% foetal bovine serum, 10 mM Hepes, 4 mM glutamate, 2 mM pyruvate, 50 µg·mL1 gentamicin, penicillin/streptomycin and 100 µM ß-;mercapto-ethanol (all from Gibco-;BRL, Merelbeke, Belgium) and incubated at 37°C with 5% carbon dioxide (CO2) for 2 h in plastic petri dishes. Thereafter, nonadherent cells were washed away (three times with PBS); the adherent cells were scraped off and incubated in sterile 96-;well plates (costar) at a concentration of 1x106 cells·mL1. The cells were incubated overnight with and without stimuli, as described in the results section, at 37°C at 5% CO2. Finally, the supernatant was collected and kept at 20°C until nitrite measurement.
Monocyte derived macrophages
Murine macrophages
l-;Arginine depletion
Denitrification of N
Nitrite measurements
Effect of various stimuli on nitrite accumulation by alveolar macrophages and monocyte derived macrophages Various immune and nonimmune stimuli were tested for their ability to induce NO synthesis in both human AM and MDMs. None of the tested stimuli were able to increase nitrite production in human cells (table 1 /LPS resulted in high concentrations of nitrite in the culture medium.
Effect of different serum concentrations and source on nitrite production by monocyte derived macrophages Addition of different concentrations of either normal HS or FCS to cells stimulated with a combination of IFN-; (200 U·mL1), tumour necrosis factor (TNF)-; (200 U·mL1) and interleukin (IL)-;1ß (50 U·mL1) with and without LPS did not induce nitrite accumulation (table 2
Effect of arginase inhibition on nitrite accumulation by alveolar macrophages and monocyte derived macrophages Figure 2 -;hydroxy-;l-;arginine (100 µM) to stimulated AMs resulted in a significant (p<0.05, n=5) production of nitrite. This release, however, could not be inhibited by the NOS inhibitor aminoguanidine (1 mM). Similar results were obtained using MDMs (n=2). Furthermore, the arginase inhibiting amino acid l-;valine (10 mM) failed to enhance nitrite accumulation (data not shown).
Hydrogen peroxide releases nitrite from N -;hydroxy-;l-;arginineHydrogen peroxide dose-dependently released nitrite from 100 µM of N -;hydroxy-;l-;arginine after 2 h of incubation in a cell-free system (fig. 3
Effect of l-;arginine depletion/repletion on nitrite synthesis Pre-incubation of monocytes in l-;arginine free medium for 48 h did not result in an enhanced NO synthesis upon stimulation. Furthermore, replenishment of l-;arginine after this period had no effect (table 3
At present, it is still unclear why human macrophages and monocytes fail to produce NO in levels comparable to murine cells, especially since NO production during human inflammatory processes in vivo seems evident 5, 8. Despite the lack of NO production in vitro, previous work by Weinberg et al. 4 clearly demonstrated that the NO producing enzyme iNOS can be brought to expression in both human macrophages and monocytes, as shown by Western blots and immunohistochemistry. Interestingly, the same group showed that the essential iNOS cofactor tetrahydrobiopterin (BH4) is absent in human mononuclear phagocytes. Replenishment of intracellular BH4 levels, however, also failed to activate the enzyme. However, it cannot be excluded that, as such, NO is a less important defence mechanism in humans than in rodents. Thus, NO release by human inflammatory cells could also be limited in vivo, despite the presence of iNOS.
According to Murphy and Newsholme 11, the limited NO production by human mononuclear phagocytes is due to an increased breakdown of the NOS substrate l-;arginine by arginase. They suggest that incubation of human mononuclear phagocytes with l-;arginine concentrations as high as 1 mM (which is normal in culture media) induces the enzyme arginase. Arginase in turn breaks down l-;arginine, putatively depleting the substrate for NOS synthesis. In the present study, however, culturing the cells without l-;arginine, using the same procedure as described by Murphy and Newsholme 11, did not result in enhanced NO synthesis. Moreover, addition of the arginase inhibitors l-;valine or N Foetal or neonatal calf serum is a common constituent of cell culture media. Foetal serum contains docosahexaenoic acid, which has been demonstrated to inhibit the induction of NOS by murine macrophages 9. In theory, compounds like these could account for the limited NO production by human mononuclear phagocytes. The present data show that neither this compound nor other constituents of FCS account for the lack of high output NO release, since incubation of MDMs without any serum, with fatty acid free bovine serum albumin or with adult human serum did not boost NO synthesis. Despite the fact that neither human mononuclear phagocytes nor granulocytes release high amounts of NO, there is a report stating that these cells release peroxynitrite, the coupling product of NO and superoxide. The evidence in this paper, however, is based solely on oxidation of the aspecific peroxynitrite probe 123-;dihydrorhodamine without evidence for NO production 14. Considering the fact that 123-;dihydrorhodamine oxidation by human neutrophils is completely dependent on myeloperoxidase activity 15, it is doubtful that human neutrophils release peroxynitrite.
Interestingly, incubating both AM and MDMs with the NOS intermediate N In summary, the present data show that the limited nitric oxide release by human mononuclear phagocytes can neither be explained by a limited availability of the nitric oxide synthase substrate l-;arginine, nor by putative inhibiting factors present in foetal calf serum. It is concluded that great care should be taken whilst interpreting results concerning nitric oxide and its metabolites (i.e. peroxynitrite) obtained by the use of human granulocytes and mononuclear phagocytes in vitro.
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||