Introduction
Macrophages are involved in inflammation from induction to resolution. Polarization
of macrophages along the M1 (classical) or M2 (alternative) axis occurs during inflammation
and can be at least partly categorized by the route of arginine metabolism within
the macrophage, balancing the activities of the arginase and nitric oxide synthase
(NOS) enzyme families (1, 2). Arginase activity is associated with tissue repair responses
(via ornithine production and pro-proliferative effects). In contrast, NOS2 generates
nitric oxide (NO) species with anti-proliferative effects that is necessary for protection
against pathogens and aberrant cells (2, 3). Other NOS enzymes produce NO that acts
in the regulation of smooth muscle tone and other cellular processes (4). Macrophages
preferentially expressing the arginase or NOS2 pathways enzymes also influence T-cell
activation, proliferation, signaling, and apoptosis in different ways (1).
While arginase and NOS enzymes can be used to ascertain the pathway of macrophage
activation in rodents, there has been debate as to whether they are present in macrophages
from humans and other mammals. The arginase and NOS enzymes are extensively conserved,
and the NOS forms found in mammals are similar to those in cnidarians, mollusks, and
other chordates (5, 6). These arginine-metabolizing enzymes are present in some human
leukocytes, and there is evidence that they are also present in macrophages from other
vertebrates, including chickens, rabbits, cows, and primates (7–12). However, comparisons
of tissue macrophages of different species are lacking, which limits our understanding
(13). Many studies in humans have principally focused on blood monocytes, leading
some researchers to question the suitability of rodents as model of macrophage activation,
as there is not always a direct correlation with human cells. Was Robert Koch correct
when he said “Gentlemen, never forget that mice are not humans,” or can the differing
results between species be explained, in part, by differences in the types of monocyte
or macrophage studied? Our purpose here is to examine this question.
Arginine Metabolism in Mammalian Cells
Many mammalian cells, including neutrophils, granulocytes, erythrocytes, hepatocytes,
cardiac myocytes, dendritic cells, myeloid-derived suppressor cells, foam cells, natural
killer cells, endothelial cells, and smooth muscle cells, have arginase (12, 14–16)
or NOS activity (8, 17–19), albeit to different degrees. Macrophages are the primary
circulating cells that can express either of these enzymes, depending on the inflammatory
circumstance. Experiments that detect NO, ornithine, or urea production (via NOS2
or arginase) have most often been performed on rodent macrophages. Macrophages from
some mouse strains (e.g., the M1-biased C57BL/6 strain) can be stimulated by lipopolysaccharide
(LPS) to produce considerable quantities of NO. Macrophages from others strains (e.g.,
M2-skewed BALB/c mice) produce much less NO (20) and produce more ornithine instead.
Some researchers did not detect any NO production in macrophages from humans, pigs,
and rabbits (8, 11, 14, 21–23), but others (including ourselves) have observed NOS
or arginase activity in macrophages from rabbits, humans, and other primates (4, 7,
10, 12, 17, 24–26).
Why is there Controversy?
One main difference between the studies from laboratories is that some use monocyte-derived
macrophages (MDM), while others study tissue macrophages directly. A number of groups
have detected NOS or arginase activity in human monocytes or macrophages (3, 27–29);
but others have not. Why is this so? Part of the explanation lies in the fact that
in vitro-derived macrophages can generate different responses from macrophages obtained
in vivo as discussed below (and shown in Table 1). Another explanation is that many
groups use the identification of enzyme protein rather than detection of enzyme activity
as evidence of enzyme expression. Failure to detect the presence of a protein is not
definitive evidence for absence of expression (especially when considering potentially
different detection thresholds of antibodies or the high V
max of arginase, i.e., very little enzyme is required for ornithine production).
Table 1
The presence of arginine-metabolizing enzymes in human monocytes and macrophages varies
with cell source, treatment and health status/stress level of the individual.
Cell origin
Cell
Treatment
NOS test
ARG test
Result
Reference
Blood monocytes
Monocyte, mono-mac
0, 2, 3, or 5d culture
RNA, citrulline, FC
RNA, urea
NOS, ARG1 and ARG2 levels vary between monocyte subpopulations
(27)
Blood monocytes
Monocyte, mono-mac
0, 3, or 7d culture or 7d M-CSF, 0.75d IFNγ/LPS, or IL4
Gene array
No difference (≤2-fold cut-off, therefore genes with smaller differences discounted)
(30)
Blood monocytes (filaria-infected)
Monocyte
1d culture
RNA
RNA
↑ARG1, ↓NOS2
(28)
Blood monocytes (burns victims)
Monocyte
2d culture
Urea
↑ARG1
(29)
Blood monocytes
Monocyte
2d microfilaria, M-CSF, IL4, or IFNγ/LPS
RNA
RNA
Most donors had low but detectable NOS2 and ARG1 RNA expression which did not change
with any treatment.
(31)
Blood monocytes
Mono-mac
3d IFNγ and/or IL4 (No M-CSF)
RNA
↓ARG1, but detectable in all conditions
(32)
Monocyte/macrophage cell line (U937)
Mono-mac
?d LPS and/or IFNγ
Transcription run-on assay
No induction of NOS2 gene transcription (for that particular region of the promoter
region)
(33)
Monocyte/macrophage cell line (U937)
Mono-mac
1d selenomethionine and 1d LPS and/or IFNγ
Griess, RNA Western
Selenomethionine ↓LPS-induced NOS2 expression (RNA and protein) and nitrite production
(34)
Blood monocytes, peritoneal macrophages
Mono-mac, macrophage
?d culture, 2d LPS, IFNγ, or TNFα/GM-CSF
Griess, amino acid HPLC
No nitrite, ornithine, citrulline production, no arginine consumption
(22)
Blood monocytes, peritoneal macrophages
Monocyte, mono-mac
0d or 3d LPS or cytokine
RNA, IB, ICC, biopterin, citrulline, Griess
NOS2 mRNA and protein present in monocytes, ↑peritoneal macrophages (↑with LPS). Both
cell types produce neopterin, nitrite/nitrate and citrulline (low levels)
(35)
Blood monocytes (MS sufferers)
Macrophage
6d GM-CSF 0.75d IL4, IFNγ, LPS, or TNFα
RNA, Griess
RNA, WB, urea
ARG1 and NOS2 mRNA and nitrite production in MS and controls, ↑with M1 or M2 cytokine
challenge. ARG1 protein and urea production present in controls, ↑in MS
(36)
Blood monocytes
Macrophage
8d M-CSF, 5d oxLDL
RNA
No change in ARG1 levels
(10)
Blood monocytes
Macrophage
10d M-CSF, 1d IL4, or IL10
Urea, WB arginine
No ARG1 after induction by IL4 or IL10
(14)
Blood monocytes
Macrophage
14d IFNγ/LPS
Griess
No nitrite production
(37)
Alveolar macrophages (volunteers)
Macrophage
IFNγ
Griess, citrulline
No NO production, no effect of NOS inhibitor
(21)
Alveolar macrophages
Macrophage
?d (short), 0.8d IL4, or forskolin (i.e., ↑cAMP)
Urea
Untreated macrophages have ARG activity similar to unstimulated RAW cells. ↑ARG with
IL4/forskolin but not IL4 alone
(38)
Alveolar macrophages (cancer suffers, volunteers)
Macrophage
0.75d IFNγ/LPS or IL-10
RNA, WB
RNA
↑ARG with IL10 stimulated cells, ↑NOS2 with IFNγ/LPS stimulated cells
(39)
Alveolar macrophages (TB patients, volunteers)
Macrophage
None
IHC, WB, RNA, diaphorase
45–49% of cells from TB patients have NOS2. Smoking controls had some NOS2-positive
macrophages, non-smoking controls have few NOS2-positive cells
(24)
Alveolar macrophages (TB patients)
Macrophage
None
IHC
Macrophages in TB granulomas stain for NOS1, NOS2 and nitrotyrosine (i.e., active)
(26)
Alveolar macrophages (TB patients)
Macrophage
None
IHC
IHC
ARG1 in macrophages in TB granulomas, few have Arg2. Some macrophages on outer margins
have both NOS2 and ARG1, some near center have NOS2, NOS3 and ARG1
(12)
Atherosclerotic plaque macrophages
Macrophage
None
ISH, IHC
NOS2 in macrophages and smooth muscle cells, co-localized with oxidized lipoproteins
and peroxynitrite (i.e., NOS is active)
(7)
Atherosclerotic plaque macrophages
Macrophage
None
IHC, WB
Fatty streaks: no NOS2. Advanced plaques: NOS2 present in macrophages near necrotic
core, associated with ceroid accumulation and nitrotyrosine (i.e., active)
(25)
Atherosclerotic plaque foamy macrophages
Macrophage
None
IHC
↑ARG1 in macrophages in superficial layers, ↓ARG1 in macrophages surrounding lipid
core
(10)
Atherosclerotic plaque
Macrophage
None
IHC, ISH
NOS2 and nitrotyrosine localized to smooth muscle cells, macrophages and foam cells
(i.e., active)
(17)
Oral macrophages
Macrophage
None
IHC, nitrate
NOS2 present in macrophages from gingivitis samples
(40)
Placental macrophages
Macrophage
None
FC
Some M2 macrophages have ARG1
(16)
Skin macrophages (wound)
Macrophage
None
IHC, HPLC
IHC, WB, ELISA, HPLC
NOS2 present in macrophages, some have ARG2, but none have ARG1. Controls: no ARG2
(41)
Tumor-associated macrophages
Macrophage
None
IHC
NOS2 present in some macrophages (bladder)
(42)
While changes in RNA expression of arginine-metabolizing enzymes have been used to
identify macrophage activation states, protein changes [such as western blotting (WB)
or immunohistochemistry (IHC)] are also useful. Nitric oxide synthase (NOS) activity
can be assessed directly [e.g., production of citrulline or NO (e.g., Griess assay)]
or by the presence of markers of NO production (such as peroxynitrite, nitrotyrosine
or ceroid, a complex of oxidized lipids and proteins). Arginase (ARG) activity can
be measured as urea or ornithine production (e.g., urea assays, amino acid HPLC).
d, number of days; ?d, unspecified number of days; FC, flow cytometry; M-CSF, macrophage
colony-stimulating factor; IFNγ, interferon-γ; LPS, lipopolysaccharide; IL, interleukin;
TNFα, tumor necrosis factor-α; GM-CSF, granulocyte-macrophage colony-stimulating factor;
IB, immunoblot; ICC, immunocytochemistry; MS, multiple sclerosis; oxLDL, oxidized
low density lipoprotein; TB, tuberculosis; ISH, in situ hybridization; Griess, Griess
assay for nitrite/nitrate production.
It should be noted that NO production below the detection levels of this relatively
insensitive assay may still have functional effects (43).
Macrophages produced in vitro
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 obtained in vivo
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.
Effect of disease and trauma on macrophage activation
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.
Conclusion
The modulation of macrophages to express NOS or arginase has clear benefits for treating
disease in humans (and other species). To do this, one needs to either determine suitable
signals to stimulate these pathways or obtain a sufficient number of human macrophages
(e.g., by tissue culture) that function like tissue macrophages.
Because macrophages from different inbred strains of mice vary greatly in their macrophage
NOS and arginase balance, one would predict similar variability to be found in humans
as well. In addition, the source of the macrophages being studied has been found to
be important. Several groups have reported that human monocytes from healthy volunteers
that have been differentiated or manipulated in vitro using current protocols tend
not to have detectable levels of arginase and NOS enzymes, whereas MDM from diseased
or stressed individuals or tissue macrophages obtained from normal, diseased, or stressed
individuals do express NOS and/or arginase. Together these observations suggest that
the current system of differentiating macrophages from human peripheral monocytes
in vitro needs further refinement before it can be considered to be an accurate model
of human macrophage behavior in vivo (63). In turn, we need to understand the differences
and similarities between the different species and the cells being studied to develop
experimental models that will answer some of the outstanding questions regarding macrophage
M1/M2 or other activation states: What regulates macrophage activation in tissues?
What mechanisms regulate macrophage plasticity and stability? How does plasticity
of phenotype affect tissue macrophages? What are the full in vivo ramifications of
the M1/M2 paradigm?
Further work is important to be sure that our observations of the human system in
vitro are real, and not due to our cell source, measurements, or manipulations. We
suggest that macrophages obtained from mice remain useful for investigating aspects
of these questions in humans/human macrophages. So, although mice are not men (as
Robert Koch observed), we agree with Rudolf Virchow that “Between animal and human
medicine there is no dividing line – nor should there be. The object is different
but the experience obtained constitutes the basis of all medicine” [Rudolph Virchow,
1821–1902].
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.