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Critical Reviews™ in Immunology, 32(06):463-488 (2012)

M1 and M2 Macrophages: Oracles of


Health and Disease
Charles D. Mills
BioMedical Consultants, 16930 197th St. N, Marine, MN, 55047, Phone: 651 600 6519, Fax: 651 600 6519, Mills002@umn.edu

ABSTRACT: The purpose of immunology is simple. Cure or prevent disease. M1/M2 is useful because it
is simple. M1/M2 describes the two major and opposing activities of macrophages. M1 activity inhibits cell
proliferation and causes tissue damage while M2 activity promotes cell proliferation and tissue repair. Remark-
ably, the molecules primarily responsible for these “Fight” (NO) or “Fix” (Ornithine) activities both arise from
arginine, and via enzymatic pathways (iNOS and arginase) that down regulate each other. The names M1 and
M2 were chosen because M1 and M2 macrophages promote Th1 and Th2 responses, respectively. Products of
Th1 and Th2 responses (e.g., IFN-γ, IL-4) also down regulate M2 and M1activity, respectively. Thus, M1/M2
demonstrated the importance of Innate Immunity and how it is linked to Adaptive Immunity in a beautifully
counterbalanced system. “Civilization” and increased longevity present new disease challenges such as cancer
and atherosclerosis that do not display classical “foreign” antigens. And, these diseases are often associated
with (or caused by) M1- or M2- type responses that were formerly useful for fighting infections, but now are
inappropriate in our increasingly “germ-free” societies. In turn, there is considerable potential for modulating
M1 or M2 Innate responses in modern diseases to achieve better health. Finally, since M1 and Th1 (or M2
and Th2) often work in concert to produce characteristic immune responses and disease pathologies, it is rec-
ommended that Immune Type 1 or 2 (IT1, IT2) would be a simpler and unifying terminology going forward.

KEY WORDS: Cancer, M1, M2, Macrophages, Atherosclerosis, Innate Immunity, Autoimmunity, Stress, Obesity

ABBREVIATIONS: ACTH: Adrenocorticotropic Hormone; DAMP: Damage Associated Molecular Patterns; EGF:
Epidermal Growth Factor; Human Immunodeficiency Virus; IFN-g: Interferon Gamma; IL: Interleukin; NO: Nitric
Oxide; PAMP: Pathogen Associated Molecular Patterns; SCID: Severe Combined Immunodeficient; SLE: Systemic Lu-
pus Erythematosus; TGF-b: Transforming Growth Factor Beta; TNF-a: Tumor Necrosis Factor Alpha; VEGF: Vascular
Endothelial Growth Factor

I. SCOPE AND GOAL of clinical applications. Others have expertly


reviewed the molecular biology, surface markers
The greatest successes of immunology (i.e., and other aspects of macrophages.3-6 Therefore,
smallpox and polio) occurred in a simpler time, my goal is somewhat different. It is to explain
when little was known of how the immune Innate Immunity mainly from an M1/M2 per-
system operated. On the other hand, now the spective, and to identify diseases where changing
immune system (and macrophages in particular) the M1/M2 balance has therapeutic potential.
often seems vexingly complex. M1/M2 changed M1/M2 is, of course, an oversimplification of
that by describing Innate Immunity in simpler the many other cells and processes involved in
terms, and helped show that Innate Immunity Innate Immunity. However, I felt the usefulness
controls Adaptive Immunity, not vice versa.1,2 of describing Innate Immunity in terms of its
These were fundamental changes in under- core “Fight” or “Fix” functions would outweigh
standing the immune system and have a myriad what is lost by omission.

11040-8401/12/$35.00  © 2012 by Begell House, Inc. 463


464 Mills

II. HISTORY: VACCINES AND THE FALL attention was paid to the “simpler” cells (macro-
AND RISE OF MACROPHAGES phages) that were mainly viewed as “Servants”,
performing tasks (such as killing pathogens and
A. B.C. (Before Cells): Simple - Curative cleaning up debris) under the direction of the
“Masters” (T cells). It was a time of an “Adaptive
“Those who don’t know history are destined to repeat it.” Dictatorship.”
Edmund Burke (1769)
My own belief in the magic of Adaptive
Immunology began with a simple observation. Immunity was typical and instructive. I (like many
Edward Jenner noticed during outbreaks of others in the ’70s and ’80s), thought the next great
smallpox that milkmaids often survived.7 The cows immunologic triumph would be a vaccine against
had skin lesions that resembled those dying of our biggest health problem—cancer. In my case,
smallpox (later found to be a similar virus – cow- I studied mouse tumors that were immunogenic
pox). So, he took some material from these skin (elicit a tumor-specific response in the host), and
lesions and put it on the skin of a boy without found that elevating tumor-specific cytolytic T
smallpox. Later, the boy was immune to smallpox. cell responses could cause tumor regression.8,9
Skin is an important word here because if Jenner Others tried to identify antigens that are specific
had administered Compox orally, or injected it to human tumors that could be recognized by T
intravenously, immunity would not have resulted cells.10 Much effort has also been expended trying
or death would have occurred. Smallpox eradica- to identify monoclonal antibodies that specifically
tion became a triumph of immunology. Unknow- recognize cancer cells.11 Whereas potential exists
ingly, Jenner had discovered that humans have an in these areas, the problem in cancer and many
effective system for recognizing pathogens on the other diseases goes beyond classical T or B cell
skin (Innate Immunity) that can result in specific specificity.
protection (Adaptive Immunity). As will become
apparent later, the ability of Innate Immunity to
protect exposed surfaces like the skin and lungs, C. Paradise (Specificity) Lost: Innate
and other characteristics of Innate Immunity, Immunity Found
are critical to survival. However, scientists first
focused on the “Holy Grail” of immunology— Dreams of a cancer “vaccine” have faded because
“Specificity”. the majority of cancer cells do not express antigens
that can be made to be immunogenic. Also, most
of modern civilization’s other important diseases
B. A.D. (Adaptive Dictatorship): (e.g., atherosclerosis, autoimmunity) also do not
Macrophages as Servants express classical “foreign” antigens. Does this mean
that the immune system will continue mainly
The ability to simply isolate a causative agent (e.g., as a weapon against exogenous pathogens (i.e.,
smallpox or polio), inject it (or something similar), bacteria, parasites, viruses)? The good news is no.
and have the body take care of the rest with specific The immune system does play an important role
immunity was a remarkable scientific advance. In in most modern diseases, just in a different way
turn, immunologists naturally focused on figuring than against pathogens. Additionally, some current
out what constituted specificity to try and cure problem pathogens (or vaccines made from them)
other diseases. In some cases, T cells were found elicit a response, but it is ineffective. For example,
to be critical for protection (e.g., Tuberculosis); HIV naturally elicits an antibody response, but
in others, B cells/antibody were found to be most a cytolytic T cell response seems required for
effective (e.g., Diphtheria). During this quest, scant immunity.12 Therefore, understanding how to

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M1/M2 in Health and Disease 465

improve health requires a better understanding tocompatibility antigens so foreign antigens are
of how immune responses work. It has also led presented to T cells in the context of self.20,21
to recognition of the critical importance of Innate This sophistication allows T cells to develop spe-
Immunity and the primary cells that mediate it, cific responses, not only to exogenous pathogens,
macrophages—the “simpler” cells. but also “self ” cells expressing alterations of self
because of viral infection or oncogenic transfor-
mation. One of these T cell responses causes B
III. MACROPHAGES cells to produce antibody (although B recognition
of some microbial components is T cell indepen-
A. Basic Definition dent). Another T cell response (discovered at the
Trudeau Institute by Mackaness and colleagues)
A macrophage is a cell that “samples” the environ- causes further amplification of the macrophage
ment, often by engulfing (phagocytosing) mate- response (termed macrophage “activation) that is
rial. So, Metchnikov picked a good name in “big necessary to kill many intracellular pathogens.3,22
eater”.13 Metchnikov had other pioneering ideas Thus, throughout evolution, macrophages (or
about immunity and health as well.14 “Sampling” macrophage —like cells) perform critical functions
is a primordial function (amoebas do it) that origi- that are directly (Innate Response) or indirectly
nally served to help simple organisms find food, and (Adaptive Response) necessary for survival.
to avoid using other like members of the species
as food (preservation of the species)!15,16 Later on
in evolution, sampling became more sophisticated, B. Macrophages Living in Different
mainly through the use of Toll receptors17,18 that Locations
recognize pathogens by what are called Pathogen
Associated Molecular Patterns (PAMP). More Depending on where they reside in the body,
recently, Damage Associated Molecular Patterns macrophage-like cells have different names (e.g.,
(DAMP) have been identified that also allow mac- microglial cells [brain], Kuppfer cells [liver], alveo-
rophages to detect damaged or effete self-tissue.19 lar macrophages [lung], peritoneal macrophages
Once a human macrophage recognizes what [peritoneum], foam cells [blood vessel plaques],
it is dealing with, it selects a response appropriate Langherhan cells [skin]). The presence of these
for the circumstance. Macrophages have a battery macrophage-like cells in different places is essential,
of responses available, many of which are present not only for defense, but also for fetal development
far back in evolution (e.g., digestion, NO and and adult life (In contrast, T and B cells are not
oxygen radical production, factors that promote required for life under germ-free conditions).15
proliferation and angiogenesis or matrix deposi- There are no macrophage “knock out” mice. At the
tion).3,15 Essentially though, a macrophage does same time, the functions of these cells in different
one of two things. It either decides to “Fight” by locations all depend on sampling the environment
turning on a killing program (e.g., a pathogen), or for self/non-self like “classical” macrophages. For
“Fix” using a repair program (e.g., wound healing). example, resident tissue macrophages in the skin
If the material is “foreign”, human macrophages often encounter tissue damage signals (wounds)
(also other mammals and birds) have evolved a and make a Fight response. Alveolar macrophages
second, more sophisticated, system to commu- more commonly encounter inhaled material that
nicate with T cells that goes beyond recognition they engulf to Fix the problem. Intestinal macro-
of non-self to recognition of subtle variations of phages encounter ingested material (and resident
self. To achieve this, macrophages (and dendritic bacteria, etc.), and sampling in this environment
cells and B cells) uniquely possess Class II his- helps Fix the immune system so it doesn’t respond

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466 Mills

inappropriately (preserve tolerance). To perform IV. M1 AND M2 MACROPHAGES


these somewhat different daily functions, cells
in these various places have recognizable differ- A. M1/M2: Fight or Fix—The Arginine
ences in the portfolios of genes and products they Fork in the Road
express.4,5,23 However, all these cells make the same
Innate Fight or Fix decisions (and all employ
similar intracellular processes to do so). Therefore, As mentioned earlier, when macrophages are sam-
for the purpose of this review it is most useful to pling and encounter something (be it a pathogen or
view all these cells as macrophages. damaged tissue), their receptors trigger a decision
Two other important cells of note will not to Fight (kill it), or Fix (repair it). M1 and M2
be covered separately here. Monocytes live in the biochemically defined molecules that macrophages
blood, and are important because they are the produce to perform these diametrically opposed
precursors that supply the extravascular spaces functions.1,2 Fight is mediated by macrophages
with new macrophages.24,25 At the same time, preferentially producing NO (Nitric Oxide) that
these circulating cells have no known function, and inhibits cell proliferation,31 while Fix is mediated by
“macrophages” derived from monocytes in vitro do macrophages producing Ornithine that promotes
not necessarily recapitulate in vivo macrophages.26 proliferation and repair (through polyamines and
Myeloid-derived suppressor cells (MDSC) are collagen).2,32-34
present in certain diseases (e.g., cancer), and are an Both NO (and Citrulline) and Ornithine
interesting lineage/group of cells which suppress (and Urea) result from the enzymatic cleavage of
immune responses.6,27,28 MDSC includes macro- terminal nitrogen linkages of Arginine in slightly
phages, but they will not be covered as a separate different ways:
group of cells for the purpose of this review. Arginine: O2CCHNH3 – C3H6 – NH – C
– (NH)2
iNOS: O2CCHNH3 – C3H6 – NH – C - NH
C. Macrophages Wearing (Citrulline) + NO (Nitric Oxide)
Different Outfits Arginase: O2CCHNH3 – C3H6 – NH (Orni-
thine) + C – (NH)2 (Urea)
Dendritic cells are unusual looking cells compared Importantly, NO or Ornithine production is
to macrophages. But, evidence suggests that they favored in a macrophage because intermediates
arise from the same precursor as macrophages in in each enzyme pathway inhibit the opposing
bone marrow.29 Despite their distinctive morphol- pathway.35 A more elegantly simple mechanism for
ogy, location in T cell areas of lymph nodes, and making a Fight or Fix response than this Arginine
enhanced antigen-presenting capacity (on a cell per “Fork in the Road”2 is difficult to envisage. That
cell basis), current evidence indicates that dendritic arginine metabolism is a central pathway in mac-
cells do not perform any unique functions, or have rophages is indicated by the fact that the concen-
any unique markers, compared to macrophages.29 tration of this amino acid at sites of inflammation
Like macrophages, they sample the environment, often declines to undetectable levels.36
make innate responses, and present antigens. Often, There are other changes in macrophages that
dendritic cells and macrophages are separated in typically occur in concert with NO production
articles and in immunologists’ minds that can be (e.g., increased Class II expression and production
counterproductive. So, I will leave that debate to of IL-12/23 and IL-8) or Ornithine production
others30 and consider dendritic cells as a special- (e.g., increased TGF-b, IL-10, IFN a/b, chitinases,
ized subset of macrophages for the purpose of matrix metaloproteinases, and scavenger recep-
this review. tors).3-6 As will be seen, these different molecules

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M1/M2 in Health and Disease 467

set in motion very different types of immune individuals could be attributed solely to differ-
responses. M2 is the “default” activity in resident ences in T or B cell responses.38-40 In particular, it
macrophages.1 And, TGF-b appears to be the most was thought that one type of T cell response was
important cytokine for maintaining M2 because responsible for activating macrophages (Th1), and
macrophages produced it endogenously, and it another for stimulating antibody production (Th2).
strongly inhibits NO production.1,37 There are The perceived importance of T cells and Th1/Th2
several other macrophage cytokines (e.g., TNF- can be traced to differences in resistance between
a, IL-1b, IL-6) that have important physiologic C57Bl/6 and Balb/c mice to the Leishmania para-
effects such as fever and joint pain.3 However, site.41 C57Bl/6 mice are more resistant and that
the production of these “inflammatory” cytokines correlates with increased T cell production of IFN-γ
does not directly correlate with NO or Ornithine that activates macrophages to produce NO and kill
production (the very presence of macrophages at the parasite.42 In contrast, Leishmania infection in
a site is “inflammatory”). In this connection, M1 Balb/c mice causes their T cells to produce more
and M2 refer to two different opposing activi- IL-4 that stimulates antibody production, but
ties.1 It does not mean there are only two types which does not kill the parasite. Actually, Th1 and
of macrophages, as will be discussed below. How- Th2 propensities are not disease specific because
ever, NO or Ornithine are the most characteristic we found that spleen cells of C57Bl/6 T mice
molecules of macrophage Fight or Fix responses. make more IFN-γ, and BALB/c spleen cells more
This fact has made M1 and M2 very useful and IL-4, in response to polyclonal stimulation also.1
has largely replaced the older terms “classically” Thus, according to the existing dogma, C57Bl/6
and “alternatively” activated. mice were “Th1” and BALB/c “Th2”-type mice.
However, as will be seen, other experiments in
the lab were beginning to suggest that differences
B. M1/M2: Masters, not Servants, in T cell cytokine production between these mice
of T Cells were not due to T cells alone.

As discussed, M1 and M2 refer to NO or Ornithine


macrophage activity. However, the names M1 and 2. The Age of Enlightenment:
M2 were specifically chosen because of two other Macrophage Renaissance
sentinel findings: 1) M1 and M2 macrophage
activities exist without T or B cell influence; 2) The first observation that cast doubt on the validity
M1 and M2 macrophages stimulate Th1 and Th2 of the Th1/Th2 paradigm was that isolated C57Bl/6
responses, respectively.1 The experiments that estab- macrophages are more readily activated to produce
lished M1/M2 and the sea change it helped bring NO by IFN-γ or Lipopolysaccharide stimulation
about in our understanding of immune responses than BALB/c macrophages.1,43,44 The results with
are discussed below. Lipopolysaccharide were particularly informative
because it demonstrated differences in macrophage
Toll receptor reactivity (TLR4), a T cell indepen-
1. The “Dark” Ages: The Th1/Th2 dent response. Therefore, these findings suggested
Paradigm. that differences in disease susceptibility between
individuals could result from inherent differences
As discussed earlier, the fascination with vaccines in macrophage responsiveness. However, another
and specificity allowed an “Adaptive Dictator” to explanation could have been that C57Bl/6 and
control immunology. In turn, it came to be believed BALB/c macrophages exhibited these differences
that differences in disease resistance between because they had already been “primed” in vivo in

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468 Mills

a Th1- or Th2-dominant environment, respectively. Immunity, it makes sense that macrophages are the
To eliminate the influence of T (or T and B) cells, central controlling element in immune responses.
resident peritoneal macrophages were removed from
Nude and SCID C57Bl/6 and BALB/c mice, and
their M1 (NO) and M2 (Ornithine) activity was 3. M1/M2: Redefining Macrophage
examined. It was found that macrophages from “Activation”
Nude or SCID C57Bl/6 mice exhibited M1-
dominant activity, while those from Nude or SCID Whereas I tried to inject some humor by describ-
BALB/c mice exhibited M2 - dominant activity. ing an “Adaptive Dictatorship,” M1/M2 did help
Indeed, it appeared that M1 and M2 differences change our concept of what macrophage “activation”
in these mice were greater than in their normal means that is worth explaining. Again, M1 and
counterparts. This was a surprising and important M2 activities do not require T cell influence. This
result because it indicated that propensities for M1 fact is particularly important for understanding M2
or M2 dominant responses (and disease suscepti- macrophage activity. M2 (Ornithine production) is
bilities) can be independent of T cells.1 the normal “default” program present in resident
The foregoing results also brought up the very macrophages. And, M2 macrophage activity that
intriguing possibility that differences in M1 and M2 is similar (or identical) to that of resident tissue
responses between individuals could be responsible macrophages is found in sterile wounds and other
for differences in T cell cytokine production. To test circumstances where there is no T cell compo-
this hypothesis, macrophages from SCID C57Bl/6 nent.3,36 In contrast, the concept of “alternatively
or BALB/c were again removed. Then, each was activated” macrophages arose mainly from the
mixed with macrophage-depleted CB6 (C57Bl/6 X observation that addition of the T cell cytokines
BALB/c F1 progeny) spleen cells (for histocompat- IL-4 or IL13 in vitro resulted in some changes
ibility). The polyclonal T cell activator, Con A, was in macrophages.47-49 More recent results suggest
then added to stimulate T cell cytokine production. that “alternatively activated” macrophages are not a
It was found that SCID C57Bl/6 macrophages distinct in vivo phenotype, but instead most closely
(M1) stimulated CB6F1 spleen cells to produce resemble resident macrophages (M2) in their gene
IFN-γ, while SCID BALB/c macrophages (M2) and protein expression.4,23 Thus, “alternatively acti-
stimulated TGF-b production (TGF-b is also vated” and M2 are not synonyms because M2 (and
Th2-related; serum-free conditions did not allow M1) are T cell independent macrophage activities.
IL-4 production). Since the T cells were the same, The older perception that macrophages depend
these results demonstrated that M1 and M2 mac- on T cell stimulation to be functional also lead
rophage- dominant responses stimulated T cells to to the additional sub typing of M2 macrophages
make Th1- or Th2-dominant cytokine responses.1 into M2a, b and c based on in vitro stimulation
Results in other experimental systems had also with: IL-4/13; Ig and Toll receptor agonists; and
shown that “antigen-presenting cells” (whether called IL-10 or TGF-b, and glucocorticoid hormones,
macrophages or dendritic cells) can influence that respectively.50 Like “alternatively activated”, mac-
type of T cell response that occurs.45,46 However, the rophages treated in these or other ways in vitro
experiments in SCID mice described above were do have quantitatively different patterns of activity
fundamentally different because they proved that (e.g., phagocytosis), gene expression and cell surface
macrophages can drive T cell responses without markers.50 Macrophage M1 and M2 activity can
prior T cell influence. In turn, M1/M2 showed that be elevated by T cell-derived cytokines. However,
the Th1/Th2 paradigm was insufficient to explain M1/M2 most importantly means that macrophage
how immune responses work. As will be seen later, propensities precede and direct T cell responses,
if one looks at the design and function of Innate not vice versa. Also, NO or Ornithine production is

Critical Reviews™ in Immunology


M1/M2 in Health and Disease 469

the clearest and most functional way to categorize other hand, there may be circumstances where
opposing macrophage activities in Innate Immunity neither NO or Ornithine production is needed for
(or in directing Adaptive Immunity). a desired macrophage function, like phagocytosis.
Going back to the analogy then, if one needed
to transport seven people across the country, an
C. M1/M2 and Macrophage “Plasticity” intermediate type vehicle such as a minivan would
be ideal. Intermediate-type macrophages with
M1 or M2 describes macrophage activity commit- maximum phagocytic activity would likewise be
ted to Fighting or Fixing, or stimulating Th1 or ideal, regardless of NO or Ornithine production
Th2 responses as described.1,2 Plasticity describes (if such cells exist).
macrophages as a continuum of phenotypes.51,52 Macrophages with intermediate activities
The two ways of describing macrophages, while brings up a very important point relevant to
seemingly at odds, are not. M1/M2 is useful because both M1/M2 and plasticity. In either case, it is
it describes succinct macrophage functions that difficult to known from the study of populations
influence the outcome of inflammation in polar of cells what is going on at the single cell level.
opposite ways. And, it is important to understand For example, does an individual macrophage,
that M1 and M2 specifically defines inflammatory when maximally activated (e.g., IFN-γ and LPS),
circumstances where NO or Ornithine produc- fully turn on NO production and shut down the
tion predominates.1 Predominate is the key word other pathway, or is there “plasticity” in this and
because all inflammatory circumstances are clearly the many other molecules macrophages produce.
a mix of M1 and M2 responses. One need not look Even the study of individual cells does not answer
further than cultures of macrophages to know that this question because analysis at any point in time
there is a mixture of different cells (Figure 1).1 is a “snapshot.” For example, if a macrophage is
Some have interpreted M1 and M2 as meaning committed to becoming pure M1, it can be caught
there are only two types of macrophages. Actually, transitioning there from M2, and appear to be an
in the original description of M1 and M2, “plastic- “intermediate” cell. Thus, by definition, there is
ity” was the word used to allow for the possibility plasticity. That said, plasticity is not likely to be fully
that individual macrophages might produce varying bidirectional. In particular, once an M2 macrophage
amounts of NO or Ornithine.1 It does appear, how- (or population) becomes M1 (NO-producing), it
ever, that all macrophages produce one or the other. does not appear to revert back to M2. Although
The important point then is that M1/M2 describes not proven, this makes intuitive sense because NO
polar opposite responses, and plasticity describes the is toxic to macrophages also. Perhaps it depends
ability of macrophages to adapt incrementally (or on how much NO is induced intracellularly. As
continuously) to achieve M1 or M2 predominate we learn more about the physiology of individual
activity, or varying mixtures of these responses. macrophages perhaps new distinct phenotypes
It may be useful to think of M1 as a school will be discovered. In the meantime, M1/M2 and
bus and M2 as a Ferrari; and plasticity as SUVs, plasticity both have important and different roles
minivans and compact cars. Using this analogy, in helping us understand macrophage functions.
if you needed to transport 40 people across the
country, and two other people across the country
quickly, what would you do? All the vehicles D. Beyond Plasticity: Are Macrophages
above transport people, but only the school bus Pluripotent?
and the Ferrari are ideal for these jobs. Similarly,
macrophages that produce NO or Ornithine are Regardless of how one views or labels macrophage
ideally suited for certain circumstances. On the activity, evidence keeps appearing to suggest that

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470 Mills

FIGURE 1. Demonstration of the dramatic effects of serum on functions and morphology of macrophages.
TGF-β appears primarily responsible. Reprinted by permission, J. of Immunology (Mills, C.D., K. Kincaid, J. A.
Alt, M. J. Heilman, A. H. Hill. 2000. M-1/M-2 Macrophages and the Th1/Th2 Paradigm. J. Immunol. 164:6166.)

macrophages can transform into other cells.53-57 E. The Power and Pitfalls of In Vitro
My own experience is that unstimulated mac- Immunology in the Discovery of M1/M2.
rophages, or, particularly, macrophages cultured
with TGF-b exhibit a fibroblast-like appearance The ability to analyze leukocytes in vitro has
after a few days in vitro (Figure 1E). That these unique advantages, but can also cause conflict-
macrophages may be transitioning into fibroblasts ing results and conclusions because conditions
(or some other cell) is supported by the fact that don’t resemble those occurring in situ or in vivo.
their phagocytic activity is markedly reduced.1 Appreciating these pitfalls can lead to a better
Having a cell in most tissues of the body (like understanding of macrophages and other leuko-
macrophages) with pluripotent activity would cytes. As an example, awareness of the composi-
be an advantage in times of tissue damage (e.g., tion of different culture medias played a key role
spinal cord injury) or tissue deterioration (e.g., in elucidating the NO and Ornithine pathways
Alzheimer’s). Therefore, although additional results in macrophages. And, it also allowed experiments
are needed to determine the potential pluripotency to be performed showing that M1/M2 activi-
of macrophages, I think this is an area worthy of ties can control the type of T cell response as
further investigation. discussed below.

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M1/M2 in Health and Disease 471

The majority of immunologists still use fetal (IFN- production), while serum stimulated a
bovine serum (or other serum supplements) Th2-dominant T cell response (IL-4 and TGF–b
in culture media. Fetal serum is used to avoid production). Recognition of this difference allowed
complications of heterophile and other antibod- us to show that M1 and M2 dominant responses
ies present in adult serum. However, serum is can drive T cells toward Th1 and Th2 responses.1
obtained by lysing platelets, which contain a lot Therefore, if a cell does not (or does) exhibit an
of TGF-b. And, TGF-b is the strongest inhibitor activity in vitro, it cannot necessarily be concluded
of M1 activity (NO production) and promoter of that the cell performs the same way in vivo.
M2 activity (Ornithine production).2,35,37 Serum
contains over 500 pg/ml TGF-b that is similar
to the concentration found in wounds, and which F. Human M1 and M2 Macrophages
is sufficient to markedly inhibit macrophage NO
production.1,36 As shown in Figure 1, the effects A particularly pertinent example of “the tail wag-
of TGF-b on macrophage morphology and func- ging the dog” (trying to make conclusions about in
tions are dramatic. And, the unknown presence of vivo immunology from in vitro results) is continu-
this cytokine in vitro has lead to some erroneous ing debates about whether human macrophages
conclusions. For example, inhibition of NO pro- display M1 or M2 characteristics.60-62 These debates
duction by TGF-b in serum resulted in the belief stem mainly from observations that human mono-
that macrophages require two signals to become cytes do not produce much NO or Ornithine.
“activated” (e.g., IFN-γ and LPS).58 The use of But, compared to what? Mouse monocytes don’t
serum-free media (actually contains albumin and produce much NO or Ornithine either! Moreover,
other necessary nutrients) allowed us to show that why would one expect monocytes (no known func-
either signal is sufficient.1 tions) to produce NO or Ornithine anyway? As
The use of serum (e.g., TGF-b) or other cul- will be discussed later, civilization has decreased
ture additives can lead to other types of pitfalls in infections (and wounds) in humans, so it seems
analyzing macrophage functions. For example, since reasonable to postulate that human macrophages
macrophages succumb to their own NO produced produce somewhat less NO (or Ornithine) than
in vitro,59 culture in serum-free (no TGF-b) media other species. But, there is abundance evidence
elevates NO production, and decrease macrophage of the expression of both the iNOS and arginase
viability after three days.59 Therefore, if one then pathways in different human disease conditions.63-65
adds IFN-g or LPS to macrophages, one would This makes sense because these two enzyme path-
come to the incorrect conclusion that culture in ways are present in every other vertebrate (and
serum-free media decreased their ability to pro- many invertebrate) species.15,33 Obviously, it is more
duce NO compared to serum-containing media. difficult to study macrophages in humans than in
However, if one had added live bacteria at the rodents or other species. However, it is important to
beginning of culture, there would have been many measure NO and Ornithine production in human
fewer bacteria three days later in serum-free media macrophages because they are the best indicators
compared to serum-containing media. Similar of Fight or Fix activities. As a side note here, one
misinterpretations have come from pre culturing might alternatively measure iNOS or arginase
macrophages in IL-4 (that also decreases NO enzyme activities, or the genes that code for these
production in macrophages). Because serum-free enzymes as an indicator of M1/M2. It should
media promoted M1, and serum supplementation be kept in mind, however, that enzyme levels or
(TGF-b) promoted M2 activity, T cell responses gene expression in general are indirect measures
were also altered in important ways. Serum-free of what really matters, the functional products of
media stimulated a Th1-dominant T cell response macrophages, like NO or Ornithine. This fact is

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472 Mills

sometimes forgotten when results with enzyme or was ineffective at generating immunity, and resulted
gene expression in macrophages are interpreted as in death or tolerance, respectively.66 As mentioned
direct evidence of variations in functions. earlier then, by placing cowpox on the skin, Jen-
ner inadvertently discovered much about how
Innate Immunity is designed. HIV is a clinically
V. M1/M2 MACROPHAGES IN NNATE relevant example of the importance of the route of
IMMUNITY: DESIGN, DANGER, AND exposure because it is very poorly infective, unless
DIRECTION introduced directly into the blood. Of course, the
clinical term for what happens when the immune
Having identified the basic functions of macro- system fails to keep pathogens out the vasculature
phages, and what M1/M2 represents, it is useful to is sepsis, which is often fatal.
describe how the design and the different responses
of Innate Immunity protect a host.
B. “Danger”: The Wake Up Call

A. Design: Protect the Vascular System Any breach of exposed surfaces (skin, lung, intes-
tine) is potentially dangerous. Polly Matzinger
Humans (and other vertebrates) have evolved coined the fitting term “Danger” to describe the
separate neural, endocrine, circulatory and respira- critical nature of the events that occur immediately
tory organs connected by a vasculature system. The after a breach.67 Speed and “Overwhelming Force”
importance of protecting these organs caused the are important in response to Danger, regardless of
human Innate immune system to evolve a powerful the nature of threat.
three-tier system of protection designed to keep
pathogens out of the vascular system.15,16
The first and most important tier is located 1. Speed
near surfaces exposed to the environment (skin,
lungs and the gut). Macrophages are the predomi- Why must macrophages respond quickly? One
nant cells in these areas.3 As macrophages sample bacterium can become over 2 million in one day.
events, self or non-self material is recognized. Also, One “white” cell can become two in one day! So,
epithelial cells secrete defensins at these surfaces. to overcome this overwhelming prokaryotic pro-
In parallel with these events, the second tier of liferative advantage, resident macrophages respond
protection begins with a natural fluid pressure within minutes to molecules such as prostaglandins
gradient that carries antigens and other materials released from damaged cells in a skin wound. And,
away from the local site into lymph channels and inside of an hour, macrophages are phagocytosing
into draining lymph nodes (or similar type collec- material and signaling other Innate cells to enter
tions of lymphocytes). Here, material is sampled by the area from the vasculature.25
macrophages again, and T and B cell proliferation
occurs if foreign antigens are detected. Finally, the
third tier of Innate protection comes from mac- 2. Overwhelming Force
rophages that reside in organs themselves (e.g.,
microglia). That the Innate System is designed to Former US Defense Secretary, Colin Powell, used
keep pathogens out of the vasculature (and thus this term to describe successful warfare, and it
the organs) was demonstrated by experiments aptly describes the goal of the initial Innate local
in the 1960s. Introduction of live organisms (or response. Because macrophages need time to
antigens) intravenously rather than subcutaneously figure out if pathogens are present in the wound,

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M1/M2 in Health and Disease 473

the first Biologic Priority of the initial Danger default (resident) M2 mode. Specifically, Orni-
response (4–16 hours) is simply to try to sterilize thine, EGF, VEGF, and other growth factors are
the area.2,36,68 Recruited neutrophils, macrophages produced that are necessary for repair and resolu-
(and sometimes Mast Cells) are the predominate tion of the wound.69 Also, wound signals such as
cells that do this through the secretion NO and TGF-b and adenosine (from fibroblasts and other
other oxygen radicals. Not surprisingly, there is col- cells) are important in maintaining M2 activity.69
lateral damage to normal tissue. The circumstances In parallel, phagocytosis is important to clear and
in the lung, intestines, or in organs, are normally digest damaged tissue and other debris. T cells
somewhat different from a skin wound because the are not noticeably involved in wound healing.70,71
tissue “damage” is microscopic, not macroscopic. Therefore, Damage Danger provides an example
For example, macrophages in the lung are normally of the difference between M/1/M2 and Th1/Th2
encountering and phagocytosing inhaled material, because M2 macrophages heal the wound without
not damaged lung cells. Regardless of the location the need for “alternative activation” by T cells.47
of Danger, macrophages are critically required to
determine in what Direction the immune response
should proceed. 2. Localized Danger: Real Alarm—M1
Macrophage Containment

C. Direction: What Type of Danger is If pathogens are detected through PAMP, then a
Present? different set of signals is received3-6,20 and resident
(and newly recruited) M2 macrophages become
“Know when to hold ’em, know when to fold ’em”—Kenny
M1 macrophages within a day of infection. Also,
Rogers (song lyric from “The Gambler”).
IFN-g is produced by NK and other Innate cells
This lyric about poker playing colorfully describes at this time to further elevate the M1 response.71
the critical nature of the decisions macrophages The intestine is somewhat different. Macrophages
make in determining what Direction to proceed there are routinely bathed in bacteria (and parasites
if there are pathogens present, if they can be con- before civilization), but unknown unique elements
tained, or if Adaptive Immunity is needed. in that environment have evolved to routinely
suppress M1 activation to allow nutritionally ben-
eficial symbiosis. Similarly, ingestion of food (or
1. Damage Danger: False Alarm—M2 other antigens) results in beneficial “oral tolerance”
Macrophage Repair compared to exposure at other sites.72 Outside
of a sterile operating room, most skin injuries
During the initial “Danger” phase, macrophages (or air breathed, food eaten) contain potential
are also determining what Direction to take based pathogens. Therefore, it would seem that there
on the nature of the threat. The extent of damage is daily Localized Danger, but M1 activation is
is determined by receptors for DAMP (damage normally sufficient to contain pathogens without
associated molecular patterns). Concurrently, other T cell involvement.
receptors recognize pathogens by PAMP (pathogen
associated molecular patterns). If no pathogens
are detected, the Biologic Priority switches from 3. Definite Danger: M1/M2 Induction of
sterilization to repair. Monocytes recruited from Adaptive Immunity
the blood primarily handle this process.5 Because
there are no new “Danger” signals or pathogens If the pathogen is not eliminated at the first tier of
present, the recruited macrophages remain in the defense within a few days, antigens or pathogens

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474 Mills

enter the lymph channel and travel to the second 4. M1 Danger: Immunoregulation and
tier - the draining lymph nodes. There, macro- Damage by NO
phages (and/or dendritic cells) present antigens and
Class II antigens together because T cells are too
“stupid” to do it on their own (Innate Immunity Up until now, the protective functions of M1
humor). This event results in clonal proliferation responses (NO, antigen presentation) have been
of specific T cells.73 And, as mentioned earlier, described, but M1 also has important immunoregu-
it is now clear that if the pathogen stimulates latory functions.2 NO produced by what are often
an M1 dominant response, then macrophages called “suppressor” macrophages diffuses freely in
send signals to T cells (IL-12/23) to make a Th1 all directions, and can inhibit T cells, or whatever
response (primarily IFN-g) that further elevates other normal cells it encounters (recall collateral
the M1 response. M1 also seems to drive the more damage in the early wound response).76,77 There-
recently discovered Th17 response associated with fore, NO creates a conundrum during immune
autoimmunity.74 On the other hand, if macrophages responses. For example, T cell production of IFN-g
remain as M2 (because some pathogens subvert M1 (that elevates NO production in macrophages) is
responses, to be discussed), then a Th2 response required for protection against many major infec-
(e.g., IL-4/13, TGF-b, IL-10) occurs and results tions. However, it is clear in some circumstances
in antibody production.75 And, the cytokines pro- that the NO produced then inhibits the T cell
duced during Th1 or Th2 responses reciprocally response in vivo.78
down regulate M2 and M1 macrophage responses The “suppressive” effects of macrophage NO
so one response (that may be optimally protec- production can cause different outcomes depend-
tive) can dominate.48 In this regard, only birds ing on the amount produced, the timing, and the
and mammals make true antibody responses.(15) disease involved. For example, during a response
Presumably, antibody responses evolved to better to a pathogen (like Leishmania, where macrophage
handle pathogens by utilizing the circulation to NO is required), if there is a lot of NO produced
provide more effective systemic protection and/or early, then T cell activation and proliferation can
because antibody is more effective against certain be strongly inhibited and the pathogen grows. If
pathogens than macrophages. Also, antibody may sufficient NO is produced during T cell amplifi-
have also evolved to handle pathogens that can cation, then the pathogen is eliminated. If surfeit
subvert M1/Th1 responses (to be discussed). NO is produced during T cell amplification, the
The foregoing description of how Innate T cell response can be partially inhibited and a
Immunity functions was based on a host encoun- chronic infection may result. There are examples of
tering a pathogen for the first time. If there is all three of these circumstances with pathogens.2 It
preexisting T cell memory (a higher frequency has not yet been determined if the inhibitory effect
of specific T cells), or existing antibody from a of NO evolved to immunoregulate overzealous T
prior exposure, then Adaptive Immunity will play cell responses, or is just an unintended side effect.
a slightly different role. For example, preexisting Either way, there are other diseases (particularly
antibody might be sufficient to halt a pathogen viruses), where only a T cell response (e.g., cytolytic)
without requiring a secondary antibody response. is curative. In these circumstances, concomitant NO
Nonetheless, Innate Immunity still functions as the production is clearly inhibitory – what I termed
initial gatekeeper to identify threats and respond an “effector deflector.”2,78
accordingly. And, as will be seen later, M1/Th1 Aside from these different immunoregulatory
and M2/Th2 responses each have important and effects, NO can also exacerbate certain diseases
different roles in preventing (or causing) many directly because the “cure is worse than the disease.”
modern diseases. Specifically, excessive NO production can cause

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M1/M2 in Health and Disease 475

more damaging effects in the lungs, intestines and is likely to promote survival of the individual via
the brain than the pathogen itself. There are numer- neurotransmitters and hormones that mobilize
ous examples of this phenomenon with bacteria energy from stores (e.g., glucose) to allow increased
(Mycobacterium), parasites (Toxoplasma ghondi) and physical and mental activity (e.g., running from a
viruses (HIV).2 Finally, another perplexing element dog), and increased immunologic activity (e.g., if
of the NO conundrum is the ability of M1 mac- the dog bites your head).
rophages to stimulate a Th1 response, as described Processive stressors are those that elicit the
earlier. It would seem that if an NO – producing “fight-or-flight” reaction from facets within the
macrophage presented antigen to a T cell, it would host’s environment that are perceived by the
kill it. Therefore, it seems like something is going on host as potential dangers, but do not cause harm
during antigen presentation that remains unknown. directly.80 When a host senses a threat, the pitu-
The foregoing results make it apparent that con- itary gland responds involuntarily by releasing a
sideration of both the protective and destructive surge of ACTH, which acts on the adrenal glands
effects of an M1 response need to be taken into to release several stress hormones, including epi-
consideration when designing vaccines or other nephrine and cortisol.
therapeutic strategies to be discussed later. In contrast, systemic stressors are those that
actually pose a threat to homeostasis, such as
extreme pain, dehydration or injury. These stressors
VI. M1/M2 MACROPHAGES: require much less cognitive processing than proces-
BIDIRECTIONAL COMMUNICATION sive stressors and often occur simultaneously with
WITH NEURAL AND ENDOCRINE processive stressors. Systemic stressors initially
SYSTEMS activate the same type of neural and endocrine
responses as processive stressors.
A. Introduction As we saw earlier, the Innate Immune system
makes its own response to “stress” in the “Danger”
“No man is an island” —John Donne (1624). response. And, this response communicates with
the neural and endocrine systems in several ways.
Just as we have seen that M1/M2 responses act in For example, macrophages produce molecules such
concert with Th1/Th2 responses, it is also impor- as IL-1, IL-6, and TNF-a that cause the brain to
tant to recognize that the immune system is not a increase body temperature and create a feeling of
stand-alone organ. One only need be reminded of malaise. These effects are beneficial because fever
how it feels to have the flu (or getting sick after helps kill microorganisms, and malaise signals a
stressing out about a grant renewal!) to understand host to sequester itself (e.g., in the house) until
that the immune system is interconnected with the normal function is restored (dog bite heals). I used
neural and endocrine systems. So, although it is not the example of a dog bite in the head because it
my expertise, I thought a brief discussion of how illustrates that macrophages (in this case microglial
these systems influence each other is important. cells) are located in most organs, and form the third
tier in the Design of the Innate Immunity. In addi-
tion, microglial cells are in constant bidirectional
B. Normal Stress and Beneficial M1/M2 communication with astrocytes that is necessary
Responses for normal brain function.81 Microglial cells, like
macrophages elsewhere, can make M1 responses to
The neural and endocrine systems help to main- deal with an infection (dog head bite), or an M2
tain homeostatic balance by reacting to stress.79 response to heal a head injury (sterile dog head
The fundamental objective of a stress response bite). Thus, the neural, endocrine, and immune

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476 Mills

systems have evolved important bidirectional diseases occur later in life. Therefore, there is
communication pathways that serve to optimize little evolutionary pressure (breeding preferences)
host performance and survival under conditions for the immune system to adapt. Below are some
of processive or systemic stress. changes “Progress” (P) has brought in the input
that Innate Immunity receives that have changed
the balance of M1and M2.
C. Abnormal Stress and Detrimental M1/
M2 Responses
A. Predation to Pets
Unlike “normal” stress that is acute and beneficial,
chronic or severe stress degrades the neural, endo- Until fairly recently, the biggest threat to man
crine and immune network. In particular, prolonged was being wounded or killed by tigers, or snakes
elevation of cortisol depresses Innate Immunity. One (predators), or other humans.85 Now, we only have
example is life-long spouses. If one dies, the other pets! Loss of predation/acute stress responses and
often dies shortly after, in part, because of depressed decreased infections have decreased M1 responses.
immunity.82 Another important effect of chronic A useful comparison of early and modern man
stress is that it specifically decreases the M1/M2 is mice versus men. Mouse macrophages have a
ratio.83 Chronic inflammation also can be deleteri- stronger M1 component because they are exposed
ous because byproducts of Innate Immunity, such as to predators and more infectious agents.60,61
glutamate, cause decreases in serotonin and dopa- Net Result: Decreased M1/M2 ratio.
mine that maintain a healthy mood. Schizophrenia,
depression, and other behavioral disorders can be
exacerbated by chronic inflammation.84 Although B. Pestilence to Purity
macrophages are the primary link to the neural and
endocrine systems, if Innate Immunity stimulates The major reason for the decrease in infectious
Adaptive Immunity, T cells produce similar mol- disease (and M1) in the last couple of centuries has
ecules as macrophages that also degrade the neural been increased sanitation. In addition, food sources
and endocrine systems.82 “Civilization” has markedly have been simplified and sanitized. Approximately
affected the type of stress we experience, and has 70% of the world’s food now comes from 6 grains
contributed to changes in the balance of M1/M2 and one animal (cows), and more of the food con-
as described in the following section. sumed is sterile. This has reduced the variety and
quantity of the intestinal floral which also decreases
M1 responses.85 In a similar manner, antibiotics and
VII. M1/M2 MACROPHAGES IN vaccines have decreased infectious diseases result-
MODERN CIVILIZATION ing in decreased M1 responses. Because products
of M1 and M2 responses mutually inhibit each
As it was instructive to understand how Innate other, M2 responses also have increased.
Immunity works, it is useful to appreciate that Net Result: Decreased M1/M2 ratio.
“Civilization” has changed Innate Immunity.
Civilization has roughly doubled humans’ lifespan.
Many modern diseases (cancer, atherosclerosis, C. Paradise to Pressure
autoimmunity) are the result of these two changes.
Civilization (in evolutionary terms) is a recent One might not think of earl man’s existence as
event. Therefore, the immune system has not had paradise. However, industrialization, increased
much time to adapt. In addition, most modern population density and other factors have altered

Critical Reviews™ in Immunology


M1/M2 in Health and Disease 477

the type of neuroendocrine stress we experience. therapeutic potential for changing the M1/M2
For example, housing and transportation have balance in cancer.89,90 Also, the immunologic
decreased acute stress (e.g., being attacked while circumstances that occur in cancer are applicable
sleeping or walking). In addition, crowding, jobs, to other diseases involving inappropriate M1/M2
financial pressure, etc., have elevated chronic stress. balances. So, I will use cancer (and its relation-
As discussed, both of these changes in the type ship to a wound) as the central model to develop
of stress experienced alter the M1/M2 balance.82 some general ideas about how to develop effective
Net Result: Decreased M1/M2 ratio. therapeutic strategies.

D. Petite to Pounds A. Background to Cancer

The major increase in obesity in civilized countries The history of diseases can be instructive. This
in the last century has caused excess circulat- is particularly true in cancer. Cancer is with us
ing “energy” (glucose, fatty acids) that fools the today because it occurs mostly later in life, and
immune system into making chronic “Danger” therefore does not affect breeding; there is little
responses on vessel walls.86 Energy Imbalance and evolutionary pressure against cancer. Most other
Metabolic Syndrome are terms used to describe modern diseases (e.g., atherosclerosis, allergy,
this important new civilization-induced replace- autoimmunity) are also with us for the same
ment for infection-induced M1 responses to be reason. Cells populating tumors were observed
discussed in the next section. by Virchow in the 1800s. Around the turn of
Net Result: Increased M1/M2 ratio the 20th century, the primary cells that populate
In summary, “Civilization” has markedly tumors came to be called macrophages. About
decreased major infections, and brought other the same time, Dr. Coley had observed that
changes to make modern human Innate Immunity life-threatening infections in cancer patients
M2 dominant. M1 and M2 responses that evolved sometimes resulted in complete tumor regression.
to protect early man are now often inappropriate So, he developed “Coley’s Toxin” (a bacteria mix)
in modern man because of damage they cause and used it to successfully cause complete cancer
during increased lifespan. Diseases associated with regression in some patients.91 However, there
inappropriate M1 and M2 responses, and how one was unacceptable mortality and Coley’s Toxin
might alter them for better health is discussed in has been mostly forgotten. Later on, during the
the following section. “Adaptive Dictatorship,” it came to be believed
in the 1960s and ’70s that cancer develops now
and then, and the immune system specifically
VIII. M1/M2 BALANCES IN MODERN recognizes and eliminates it.92 As appealing as
DISEASES AND STRATEGIES FOR this notion was, a majority of cancers studied did
HEALTH. not express unique antigens. A telling blow to the
“Immunosurveillance” theory was the observa-
The causes and cures of modern diseases can be tion that T cell deficient mice (Nude) exhibited
complex. At the same time, many diseases exhibit nearly the same incidence of cancer as normal
inappropriate M1/M2 balances that play a role in mice.93,94 Additionally, it has long been observed
disease pathologies. M1/M2 came about mainly that invertebrates and other lower organism that
from my studies of cancer and wounds, and are do not possess Adaptive Immunity do not have
the conditions I know best from an immuno- a higher cancer incidence than humans.16 So,
logic perspective.2,87,88 And, there is considerable what are macrophages doing in tumors, and what

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478 Mills

mechanism had Coley (and others) been able resulting in new “Danger” signals and signaling
to stimulate that caused cancer regression? The more monocytes to enter from the circulation.
relationship between cancer and wound repair Returning to the wound analogy, why don’t newly
provides salient insight. recruited macrophages keep making the early M1
response that would kill cancer cells? Cancer cells
have evolved active strategies to subvert Innate
B. Innate Immunity and Cancer: M2— Immunity by preventing curative M1 responses
Promoted Growth from developing. Cancer cells produce a variety
a factors such as TGF-b and prostaglandin E2
As mentioned, macrophages are the primary leu- that inhibit M1 (NO) “Fight” activity, and keep
kocytes in tumor beds. M1 macrophage activity macrophages in M2 (Ornithine) “Fix” mode.110
can clearly kill tumor cells31,95 and the presence of Also, cancer cells stimulate metaloproteinases in
intratumor “Fight” M1 macrophages is very favor- macrophages to aid in the breakdown of matrix
able for survival.96 However, evidence indicates allowing continued encroachment into normal
that intratumor macrophages are primarily M2 tissue. Thus, cancer is mainly a disease that is
“Fix” dominant.6,97-99 What goes wrong? Cancer not recognized by T cells, and whose growth and
and wounds may seem odd bedfellows, but are metastasis are aided by keeping macrophages in
intimately linked. Cancer has accurately been M2- wound healing mode.
called “wounds that do not heal.”100,101 Consider An unrelated, but interesting possible connec-
back to the discussion of “Damage Danger” pres- tion between cancer and M2 macrophages is the
ent in a wound. Initially, there is an M1 response influence of “stress”. As discussed earlier, chronic
that serves to sterilize the area. However, if the stress is more of an element in modern life than
wound is sterile, an M2 -dominant response acute stress and chronic stress has been shown to
occurs to provide Ornithine (polyamines, col- decrease the M1/M2 ratio.83 Although it is difficult
lagen), EGF, VEGF and other growth factors to establish cause and effect, there is a wealth of
that are required for proliferation, repair and evidence associating failure to handle stress (anti-
resolution. That this environment is conducive to social behavior, depression, etc.) with increased
cancer is evidenced by the preferential appearance cancer incidence and growth rates. Sometimes
of tumors at sites of wound repair,102-104 and is oft this connection is referred to as the “Melancholy
called the Seed and Soil Hypothesis.105 Cancer is Personality” in cancer.111
analogous to a sterile wound in that there are no
(or insignificant) specific antigens, as mentioned.
Therefore, having M2 macrophages in a tumor is 1. M2 Promotion of Infectious Diseases
not simply neutral, but can promote growth.106
That M2 macrophages are actively involved Many pathogens, like cancer, have evolved
in stimulating cancer growth is evidenced by mechanisms to actively keep M2 macrophages
decreased growth rates in hosts depleted of from transitioning to M1. For example, Myco-
macrophages.107 In this regard, a tumor can only bacterium, Leishmania and HIV all subvert
grow to about one million cells before it requires macrophage physiology for their own survival
new blood vessel support (about the size of the and benefit.3,112,113Some do this by expressing an
period at the end of this sentence). But cancers antigen that fools macrophages into thinking they
don’t normally produce angiogenic factors, and have encountered “self ” (preserving M2 activ-
so require M2 macrophages to provide it.108,109 ity), while others cause macrophages to secrete
Whereas a wound naturally heals, cancer keeps cytokines (e.g., TGF-b or IL-10) that prevent
proliferating and pushes normal tissue aside, M1 activation.3,6

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M1/M2 in Health and Disease 479

There are human cancers that do express 3. Therapy of M2-Dominant Diseases


unique tumor antigens (e.g., Melanoma) and so
are “foreign” like infectious diseases.114 However,
as with infectious diseases, the same problem Once cancer appears, transiently boosting M1 (and/
remains. Cancer cells subvert M1 responses to or inhibiting M2) responses locally or systemically
M2. And, because Innate Immunity is required seems to have great potential.90,119 Because allergy
for T cell responses, the inhibition of M1 prevents is often localized and seasonal, there seems even
the development of an effective anti cancer Th1 greater therapeutic potential for transiently increas-
response. This phenomenon was observed many ing the M1/M2 ratio in the airways.120 Although
years ago in my laboratory using the P815 Mas- many autoimmune conditions seem to result from
tocytoma in mice. P815 elicits a tumor-specific overzealous M1 responses (e.g., MS, inflammatory
cytolytic T cell and an activated macrophage bowel disease, arthritis),3 SLE appears to mainly
response in a host, but the responses are insuf- result from excessive M2- mediated autoantibody
ficient to cause tumor rejection.9,87 It can be seenproduction, and could be a good candidate for
in Figure 2 that if a host is specifically preim- increasing the M1/M2 ratio also.121 Finally, as
munized against the P815 tumor, and rechal- discussed, certain infectious agents subvert M1
lenged with tumor, there is robust M1 activity to M2 as recently discussed by others.3 There are
(NO) in the tumor bed and the tumor is rejected. interesting new strategies to increase the M1/M2
However, in a naïve mouse, the P815 decreases ratio, including injected and inhaled adjuvants, as
M1 and increases M2 activity allowing progres- well as “probiotics” and other materials that work
sive tumor growth in much the same manner as orally. Some of the most promising agents are Toll
some pathogens. receptor agonists. However, it is beyond the scope
of this review so readers are referred elsewhere for
that information.119,120,122-125
2. M2 Promotion of Allergy Increasing the M1/M2 ratio in cancer seems
logical. However, the vexing “Two-Edged Sword”
Cancer may seem unrelated to allergy. However, nature of immune responses comes into play in
consider both conditions in light of civilization- cancer and other diseases as discussed below.
induced M2- dominant immune systems. Allergy
and Asthma are clearly increasing because of
increased M2/Th2 responses, and/or decreased C. M1 Oxidants and Cancer:
M1/Th1 responses.3,115,116 Decreased infections Accumulation of Damage
combined with increased hygiene have lead to the
popular “Hygiene Hypothesis” to explain increased For cancer to appear, a normal cell must lose growth
allergy.117 For example, people raised on a farm control —become immortal. And, unlike a growing
(dirtier) environment have lower incidences of tumor, it appears that overactive M1 responses over
allergy.118 Although somewhat different, cancer the course of a lifetime may not only not routinely
actively promotes M2 responses for its growth. eliminate new cancer, but can sometimes cause it.
And, recall that the only well documented cases That is, expression of NO and other oxygen radicals
of cancer regression occurred when hosts had mas- can be carcinogenic/mutagenic.126 Lung cancer
sive infections.91 Looked at in this light, allergy seems the best example. Continued inhalation of
and cancer are both conditions exacerbated by tar and other materials causes resident alveolar
a decreased M1/M2 ratio. A summary of some M2 macrophages to chronically sense “Danger”
diseases where an M1/M2 imbalance is important resulting in an M1 response with attendant local
is shown in Figure 3. damage. In line with this “wounding” there is

Volume 32, Number 2, 2012


480 Mills

a continuous cycle of M2 repair. And because earlier. However, as in cancer, accumulation of expo-
humans have limited capacity to regenerate tissue, sure because of increased longevity is important
repair is imperfect (e.g., scarring), and lung func- because most autoimmune conditions develop over
tion declines. In addition, continuing M2 repair many years. These conditions also resemble most
creates a fertile “soil for the seed” much like it cancers because although there may sometimes be
does in non-healing wounds.100 Sun-induced skin a viral or microbial vector initially involved, once
damage resulting in skin cancers, or cancer of the established, they do not express classical “foreign”
bowel from chronic inflammation, are two other antigens. Therefore, Innate, not Adaptive, Immu-
examples where chronic M1 oxidant damage are nity is primarily responsible.3
implicated. Thus, M1 responses that were required
for survival in early man (and which are beneficial
against existent cancer) can result in accumulation 2. M1 Responses and Atherosclerosis
of damage over time and cause cancer.
In the last several years, it has come to be appreci-
ated that macrophages are the primary leukocyte
1. M1 Responses and Autoimmunity component and main cause of atherosclerotic
plaques.86,129,130 And again, like cancer, the endo-
As in cancer, the accumulation of M1 Oxidant thelial lining of vessels does not display classical
damage (and associated M2 repair) seem to play “foreign” antigens. So, why are macrophages caus-
an important role in many autoimmune conditions ing reactions in the blood vessel walls resulting
such as MS, inflammatory bowel disease, arthritis in plaque formation? The answer seems to lie in
and Alzheimer’s disease.3,127.128 This might seem at chronic excess energy availability, resulting from
odds with the fact that M1 responses overall are what I called “Pounds” (obesity). Adipose tissue
less common in modern civilization, as discussed somehow resembles a wound and attracts mac-

FIGURE 2. Evidence that intratumor macrophage arginine to NO or Ornithine is responsible for rejection or
progression of cancer. Reprinted by permission, J. of Immunology Mills, C.D., J.D. Shearer, R. Evans, M.D.
Caldwell. 1992. Macrophage arginine metabolism and the inhibition or stimulation of cancer. J. Immunol.
149:2709.

Critical Reviews™ in Immunology


M1/M2 in Health and Disease 481

FIGURE 3. M1- or M2-dominant immune responses are associated with specific diseases.

rophages that produce TNF-a, NO and other a non-healing wound, the continuing deposition
inflammatory mediators, and their production is of fatty acids perpetuates a cycle of M1 damage
proportional to the amount of adipose tissue.86 and M2 repair resulting in an enlarging plaque.
Along with this inflammation, obesity causes Thus, the problems associated with diabetes and
an inability of insulin to store glucose in fat atherosclerosis are increasingly being viewed as
(insulin resistance), resulting in obesity-onset diseases of chronic M1 responses.132
diabetes (now called Type II). In turn, glucose
and fatty acids increase in the circulation. This
circumstance resembles what occurs in acute 3. Therapy of M1-Dominant Diseases
stress (recall early man being chased by a tiger)
that shunts extra energy to the circulation to Unlike tumor growth where both decreased M1
properly deal with a threat. However, in obesity and increased M2 activity can exacerbate disease,
this excess energy becomes chronic and fuels diseases where M1 is contributory seem to result
M1 responses. We showed some years ago that primarily from chronic low -level M1 damage.
induction of diabetes in rats or mice results in Because atherosclerosis is a disease of the vascula-
elevated M1 responses, and which was corrected ture and certain autoimmune conditions are local-
by insulin administration.131 Although it is not ized to particular organs (e.g., inflammatory bowel
altogether clear, the increased concentration of disease), potential exists to specifically decrease
glucose and fatty acids in the circulation seems M1 responses in the areas affected.125,133,134 How
to be the stimuli that fools macrophages into one might devise particular therapeutic strategies
thinking there is “Danger” in the endothelial lin- to simultaneously deal with M1 and M2—related
ing. Somewhat analogous to a smoker’s lung or conditions are discussed below.

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482 Mills

D. Type, Territory, Timing and Tenacity of seasonal with allergy. The Tenacity in the case of
M1/M2 Therapy cancer might have to be high because of previous
observations that regression only occurs if there
is a substantial infection.91
From the foregoing overview of M1/M2 imbal- Therefore, as one looks ahead to therapeutic
ances in modern diseases, it is clear that no single strategies it is useful to view immune responses not
therapeutic strategy suits all conditions. At the as Two-Edged Swords, but different swords that
same time, diseases appear at different places in can be wielded in different ways and at different
the body and at different times in life that opens times for better health.
up windows of opportunity for developing targeted
immunologic interventions. However, success will
depend on taking into account the Type, Territory IX. SUMMARY AND PERSPECTIVE
(e.g., systemic or local), Timing and Tenacity of
the therapy. It is apparent that most diseases plaguing mod-
As an example, it is widely believed that ern civilization result from inappropriate Innate
decreasing oxidant damage (e.g., M1) through immune responses, be it Type, Territory, Timing,
nutrition and lifestyle changes is a successful Type or Tenacity. Most of these responses distill down to
of strategy. This seems logical in so far as chronic overactive or underactive M1 “Fight” or M2 “Fix”
cellular damage results in aging and some cancers. responses. It is also helpful to view today’s human
Also, atherosclerosis would benefit from decreased M1 and M2 responses as vestiges of a “dirtier”
M1 responses. The Territory to be covered would time when they were necessary for survival (early
be systemic because damaging M1 responses in life—to reach breeding age). In turn, in our
are. As for Timing, this strategy makes sense in increasingly “germ-free” and long-lived societies it
adults, after childhood diseases have waned, and may be useful to somewhat decrease overall immu-
when M1-mediated atherosclerosis appears. As nity (M1 and M2), towards what could be termed
for Tenacity, it is worth considering that M1 is “immunocivilization” or “M0”. After all, we seem
already decreased, so a mild lifetime decrease in to be doing fine with decreased M1, and the level
M1 might be appropriate. Decreasing M1 over of M2 doesn’t seem beneficial (and contributes to
a long period does again bring up the “Two- cancer and allergy, etc.). Given the central role of
Edged Sword” nature of immune responses. That the immune system in modern disease pathologies,
is, to the degree that Innate Immunity routinely it is now important to include routine immuno-
eliminates cancer, decreasing M1 could result in logic testing of individuals to assess who might
greater incidences. benefit from an immunologic intervention before
By considering the Type, Territory, Timing, disease onset. In this regard, it is apparent that M1
or Tenacity, more than one intervention could be and Th1 (or M2 and Th2) responses most often
employed in the same individual for another dis- work in concert to create characteristic immune
ease. For example, increasing the M1/M2 ratio is responses. Therefore, I think it would be useful
a different Type of strategy than discussed above in many circumstances to adapt a simpler, more
that would be beneficial against growing cancer inclusive terminology such as Immune Type 1, 2,
or allergies. However, the Territory treated could or 0 (IT1, IT2, IT0) to describe an individual’s
be limited in the case of localized cancer. Allergy immunologic status.
is an even better example because the Territory In conclusion, viewing Innate Immunity
is most often localized in the airways. Even if mainly from an M1/M2 perspective has limitations
increasing the M1/M2 ratio needed to be systemic, as I said at the outset. However, it has become
the Timing of cancer treatment could be brief, or abundantly clear in the last few years that Innate

Critical Reviews™ in Immunology


M1/M2 in Health and Disease 483

Immunity controls both inflammatory and specific Journ. 2011;11:2,391–402.


responses, and M1 and M2 activities are the essence 6. Gabrilovich DI, Ostrand-Rosenberg S, Bronte
of this control. Simple. V. Coordinated regulation of myeloid cells by
tumours. Nat Rev Immunol. 2012;12:253–68.
7. Morgan AJ, Poland GA. The Jenner Society
ACKNOWLEDGMENTS and the Edward Jenner Museum: tributes to a
physician-scientist. Vaccine. 2011;30:152–4.
I am grateful for the associations I have had 8. Mills, CD, North RJ, Dye ES. Mechanisms of
over the years that have allowed me to look at anti-tumor action of Corynebacterium parvum.
the immune system from different angles. As my II. Potentiated cytolytic T cell response and
postdoctoral advisor, Robert North of the Trudeau its tumor-induced suppression. J Exp Med.
1981;154:621–30.
Institute introduced me to macrophages (where,
with his help, macrophage “activation” had been 9. Mills CD, North RJ. Expression of passively
transferred immunity against an established tumor
discovered).22,135 At Brown University, my asso-
depends on generation of cytolytic T cells in the
ciation with Michael Caldwell and Jorge Albina recipient. Inhibition by suppressor T cells. J Exp
opened my eyes to the importance of metabolism, Med. 1983;157:1448–60.
specifically arginine, in macrophage physiology. I 10. Chaudhuri D, Suriano R, Mittelman A, Tiwari
am grateful to my friend, John Hibbs, in Utah RK. Targeting the immune system in cancer. Curr
who discovered Nitric Oxide that helped me Pharm Biotechnol. 2009;10:166–84.
develop M1 and M2 macrophages. Finally, I am 11. Scott AM, Wolchok JD, Old LJ. Antibody therapy
most fortunate to have had superior students and of cancer. Nat Rev Cancer. 2012;12:278–87.
employees to supply fresh ideas, and my many 12. Soghoian DZ, Streeck H. Cytolytic CD4(+) T
friends and my children that make life rich and cells in viral immunity. Expert Rev Vaccines.
whole. I dedicate this article to two such friends 2010;12:1453–63.
who died before their time this year: Dr. Bob Stout 13. Metchnikoff E. Immunity in Infective Diseases.
(a fellow “mac” guy 51,52), and Dr. George Ebert Cambridge University Press, New York 1905.
(my life-long friend), who died suddenly while 14. Cooper EL. eCAM: Darwin and Metchnikoff.
attending his mother’s funeral. Evid Based Complement Alternat. Med. 2009;
6:421–2.
15. Dzik JM. The ancestry and cumulative evolution
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