Macrophages have been produced in vitro in a number of ways. Cells from bone marrow have been isolated and “differentiated” in culture medium containing high levels of cytokines (such as colony stimulating factors, CSFs) to produce bone marrow-derived macrophages (BMDM) (13, 23, 44–46). Macrophages have also been produced by isolating and culturing monocytes from blood, to produce MDM (10, 13, 22, 30, 37, 47, 48). Production of these in vitro-derived macrophages is cheap, simple, and reproducible, but they may not be a full representative of tissue macrophages, as the preparation and culture procedures may not be sufficient to induce cell activation (4). The differences between tissue macrophages and in vitro-derived macrophages are at least partly dependent on cell source, time in culture, and the degree of manipulation in culture. Each research group will use different types and sources of culture media and sera, which vary greatly in the concentrations of factors that influence NOS2 or arginase expression, such as transforming growth factor β (TGFβ) (4, 20, 49). Another confounding issue is that circulating monocytes and tissue macrophages arise from different stem cell populations (50), although some macrophages found at sites of infection or inflammation may derive from infiltrating monocytes (51). Together, these factors may account for many of the differences observed in NO and urea production in these macrophages (8, 20).

Monocyte-derived macrophages or BMDM from different strains of mice can differ in their response to interferon-γ (IFNγ), LPS, and tumor necrosis factor-α (TNFα) (4, 8), and differences in the rodent background can result in differences in macrophage gene expression (13, 20, 49). Human in vitro-derived macrophages also show variability in their responses to LPS (4, 22, 46). It may be that the same stimulus is able to generate quite different responses in genetically diverse individuals, as it does between mouse strains (38, 49, 52). In general, human macrophages are not as responsive to LPS as mouse macrophages, possibly because of the lower environmental exposure of humans to LPS. It is also possible that human monocytes may be more effectively stimulated to become M1-activated macrophages by cytokines other than IFNγ and LPS/TNFα (e.g., IFNα) (4, 18, 43). Human macrophages take longer time to respond to the stimulatory factors in vitro than mouse macrophages, and some experiments using human MDM may have ended before a response was detected (48). There are other indications that the timing and length of the exposure of the cells to varying cytokines in vitro are important. For example, when M1-polarizing cytokines were removed from the culture medium, NOS2 levels in mouse BMDM were reduced and NO production (measured as nitrite) ceased (45). In addition, whichever arginase or NOS enzyme was induced earliest, the alternative enzyme decreased in expression and activity, unless arginine was present in excess (15, 45, 53). Macrophages require the local environment to continuously give appropriate activation cues. Changes in environmental cues can stimulate macrophage populations in vitro to express varying percentages of M1 or M2 dominant activity (54). When activation cues are reduced or removed, macrophages may become deactivated (e.g., M2c) or indeterminate (e.g., have features of M1 and M2).

Macrophages can be identified in whole tissues and organs or isolated in large numbers from in vivo sources such as the peritoneum or granulomas, and either examined immediately or used ex vivo. Macrophages obtained in vivo or made from monocytes can respond differently to the same stimulus (35, 47). In one study, monocytes and tissue macrophages were obtained from patients with an inflammatory disease (either rheumatoid or psoriatic arthritis). Compared with tissue macrophages, the MDM had a blunted response to the M2 cytokines interleukin-4 (IL-4) and IL-13, at least partly due to a reduction in some of the receptor elements for these cytokines (47). These results suggest that the response of the macrophages to M2 cytokines may be source specific, but it is possible that these cytokines alone were not sufficient to fully stimulate the MDM (38). Several lines of evidence suggest that macrophages in vivo express functional NOS2. Blood monocytes and peritoneal macrophages obtained from women during laparoscopic procedures contained NOS2 mRNA and protein. The macrophages had higher NOS levels than the monocytes, and this could be increased by treatment with LPS. The monocytes and macrophages also produced neopterin, nitrite/nitrate, and citrulline (suggesting that the enzyme was active). Although the production of NO from these macrophages was low, it would probably have been sufficient to cause functional changes (35).

Macrophages can also be obtained from alveolar aspirates, skin, and the placenta (10, 16, 21, 38, 39, 55, 56). For example, sponges placed subcutaneously into mice, rats, or rabbits attract large numbers of macrophages. The sponges can be removed from the animal and the macrophages were isolated and purified (10, 55, 56). It is a little more difficult to obtain and purify macrophages from other tissues, such as atherosclerotic vessels (44), but intact biopsy, surgical, or cadaveric specimens can also be investigated. It should be noted that resident macrophages from different tissues observed at different times (and different health states) may not necessarily have identical properties (51, 57).

In order to perform their full range of functions, macrophage populations exhibit “plasticity” of phenotype (52, 58), regardless of whether they are found in vivo or derived in vitro. As macrophages adapt or change their functions, they can simultaneously express markers of M1 and M2 activation, including NOS2 and arginase-1 (12, 59, 60). For example, tissue macrophages (and MDM) from Mycobacterium tuberculosis-infected cynomolgus macaques have been observed to co-express functional NOS and arginase enzymes (12). We suggest that macrophages display a spectrum of activation phenotypes, and it is the relative (and not absolute) proportion of M1 or M2 markers that we can use as a ‘handle’ to determine the type of activation state.

Blood monocytes from healthy volunteers do not usually need to produce NOS or arginase, so it is not surprising that many studies have not detected NOS or arginase in these cells (10, 14, 21, 22, 29, 30, 37). However, studies performed on tissue or cells from people undergoing stress, trauma [e.g., burns (29)], pregnancy (16), or disease {such as infection [e.g., tuberculosis (12, 24, 26) or filarial infection (28)], atherosclerosis (7, 10, 17, 25), autoimmune diseases (27, 36) and cancer (42, 61)} demonstrate that human macrophages (and sometimes monocytes) can produce active forms of the arginine-metabolizing enzymes (Table 1).

Trauma results in a pattern of gene expression in macrophages that is consistent with a wound-healing response, with an initial increase in NOS followed by decreased NOS production and activity, elevated IL-4, IL-10, and TGFβ levels, and increased arginase expression and activity, resulting in decreased plasma arginine levels (28, 29, 62).

Disease, however, causes different patterns of gene expression. For example, monocytes from multiple sclerosis sufferers not only have higher levels of arginase-1 and increased urea production, but also have increased NOS2 mRNA and nitrite production (particularly when stimulated by M1 cytokines or LPS) (36). Macrophages from patients with inflammatory diseases, such as tuberculosis, malaria, or rheumatoid arthritis, have increased levels of NOS2 mRNA and active protein (4, 8, 24, 26, 63), which may contribute to elevated plasma NO levels (64). Atherosclerosis is another inflammatory disease with a considerable macrophage contribution, with oxidized low-density lipoproteins taken up by macrophages during their transformation into foam cells. Plaque macrophages express NOS2 RNA and protein, as well as markers of NOS activity (including the presence of nitrotyrosine or ceroid) (4, 7, 17, 25). Plaque macrophages and foam cells express arginase-1 (10), and macrophages laser-dissected from plaque have upregulated levels of arginase-2 and NOS2 (65). Macrophages present in some neoplastic diseases also produce active NOS2 (4, 42, 66). Reducing the local levels of arginine has been proposed as a treatment for these diseases, by reducing inflammation-triggered immune dysfunction, tumor escape, fibrosis, and immunosuppression (61). Possible pharmacological interventions include treatment with arginine degrading enzymes, NOS competitors and inhibitors, asymmetric dimethylarginine, NO-releasing aspirins, cyclooxygenase, and phosphodiesterase or arginase inhibitors (8, 61). These studies suggest that an inflammatory environment is necessary in order to observe NOS or arginase in human monocytes and macrophages. The in vitro experiments that do not demonstrate arginase or NOS expression may simply be lacking the additional cues needed for expression rather than demonstrating an inability to actually express these factors.