M1 and M2 Macrophages Oracles of Health and Disease

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. Remarkably, 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-, 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 recommended 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 Lupus 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 persystem 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

AND RISE OF MACROPHAGES
attention was paid to the simpler cells (macrophages) that were mainly viewed as Servants,
performing tasks (such as killing pathogens and
cleaning up debris) under the direction of the
A. B.C. (Before Cells): Simple - Curative
Masters (T cells). It was a time of an Adaptive
Those who dont 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 problemcancer. 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 civilizations 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
Critical Reviews in Immunology
M1/M2 in Health and Disease
improve health requires a better understanding

of how immune responses work. It has also led
to recognition of the critical importance of Innate
Immunity and the primary cells that mediate it,
macrophagesthe simpler cells.
465
tocompatibility antigens so foreign antigens are

presented to T cells in the context of self.20,21
This sophistication allows T cells to develop specific responses, not only to exogenous pathogens,
but also self cells expressing alterations of self
because of viral infection or oncogenic transformation. One of these T cell responses causes B
III. MACROPHAGES
cells to produce antibody (although B recognition
of some microbial components is T cell indepenA. 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], alveorophages 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 macroof 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 doesnt respond
Volume 32, Number 2, 2012
466
inappropriately (preserve tolerance). To perform

these somewhat different daily functions, cells
in these various places have recognizable differences in the portfolios of genes and products they
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,
for the purpose of this review it is most useful to
view all these cells as macrophages.
Two other important cells of note will not
be covered separately here. Monocytes live in the
blood, and are important because they are the
precursors that supply the extravascular spaces
with new macrophages.24,25 At the same time,
these circulating cells have no known function, and
macrophages derived from monocytes in vitro do
not necessarily recapitulate in vivo macrophages.26
Myeloid-derived suppressor cells (MDSC) are
present in certain diseases (e.g., cancer), and are an
interesting lineage/group of cells which suppress
immune responses.6,27,28 MDSC includes macrophages, but they will not be covered as a separate
group of cells for the purpose of this review.
Mills
IV. M1 AND M2 MACROPHAGES

A. M1/M2: Fight or FixThe Arginine
Fork in the Road
As mentioned earlier, when macrophages are sampling and encounter something (be it a pathogen or
damaged tissue), their receptors trigger a decision
to Fight (kill it), or Fix (repair it). M1 and M2
biochemically defined molecules that macrophages
produce to perform these diametrically opposed
functions.1,2 Fight is mediated by macrophages
preferentially producing NO (Nitric Oxide) that
inhibits cell proliferation,31 while Fix is mediated by
macrophages producing Ornithine that promotes
proliferation and repair (through polyamines and
collagen).2,32-34
Both NO (and Citrulline) and Ornithine
(and Urea) result from the enzymatic cleavage of
terminal nitrogen linkages of Arginine in slightly
different ways:
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 (Ornithine) + 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 Road2 is difficult to envisage. That
cell basis), current evidence indicates that dendritic arginine metabolism is a central pathway in maccells do not perform any unique functions, or have rophages is indicated by the fact that the concenany 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 recepthis review.
tors).3-6 As will be seen, these different molecules
467
set in motion very different types of immune

responses. M2 is the default activity in resident
macrophages.1 And, TGF-b appears to be the most
important cytokine for maintaining M2 because
macrophages produced it endogenously, and it
strongly inhibits NO production.1,37 There are
several other macrophage cytokines (e.g., TNFa, IL-1b, IL-6) that have important physiologic
effects such as fever and joint pain.3 However,
the production of these inflammatory cytokines
does not directly correlate with NO or Ornithine
production (the very presence of macrophages at
a site is inflammatory). In this connection, M1
and M2 refer to two different opposing activities.1 It does not mean there are only two types
of macrophages, as will be discussed below. However, NO or Ornithine are the most characteristic
molecules of macrophage Fight or Fix responses.
This fact has made M1 and M2 very useful and
has largely replaced the older terms classically
and alternatively activated.
B. M1/M2: Masters, not Servants,
of T Cells
As discussed, M1 and M2 refer to NO or Ornithine
macrophage activity. However, the names M1 and
M2 were specifically chosen because of two other
sentinel findings: 1) M1 and M2 macrophage
activities exist without T or B cell influence; 2)
M1 and M2 macrophages stimulate Th1 and Th2
responses, respectively.1 The experiments that established M1/M2 and the sea change it helped bring
about in our understanding of immune responses
are discussed below.
1. The Dark Ages: The Th1/Th2
Paradigm.
As discussed earlier, the fascination with vaccines
and specificity allowed an Adaptive Dictator to
control immunology. In turn, it came to be believed
that differences in disease resistance between
individuals could be attributed solely to differences in T or B cell responses.38-40 In particular, it

was thought that one type of T cell response was
responsible for activating macrophages (Th1), and
another for stimulating antibody production (Th2).
The perceived importance of T cells and Th1/Th2
can be traced to differences in resistance between
C57Bl/6 and Balb/c mice to the Leishmania parasite.41 C57Bl/6 mice are more resistant and that
correlates with increased T cell production of IFN-
that activates macrophages to produce NO and kill
the parasite.42 In contrast, Leishmania infection in
Balb/c mice causes their T cells to produce more
IL-4 that stimulates antibody production, but
which does not kill the parasite. Actually, Th1 and
Th2 propensities are not disease specific because
we found that spleen cells of C57Bl/6 T mice
make more IFN-, and BALB/c spleen cells more
IL-4, in response to polyclonal stimulation also.1
Thus, according to the existing dogma, C57Bl/6
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
in T cell cytokine production between these mice
were not due to T cells alone.
2. The Age of Enlightenment:
Macrophage Renaissance
The first observation that cast doubt on the validity
of the Th1/Th2 paradigm was that isolated C57Bl/6
macrophages are more readily activated to produce
NO by IFN- or Lipopolysaccharide stimulation
than BALB/c macrophages.1,43,44 The results with
Lipopolysaccharide were particularly informative
because it demonstrated differences in macrophage
Toll receptor reactivity (TLR4), a T cell independent response. Therefore, these findings suggested
that differences in disease susceptibility between
individuals could result from inherent differences
in macrophage responsiveness. However, another
explanation could have been that C57Bl/6 and
BALB/c macrophages exhibited these differences
because they had already been primed in vivo in
468
a Th1- or Th2-dominant environment, respectively.

To eliminate the influence of T (or T and B) cells,
resident peritoneal macrophages were removed from
Nude and SCID C57Bl/6 and BALB/c mice, and
their M1 (NO) and M2 (Ornithine) activity was
examined. It was found that macrophages from
Nude or SCID C57Bl/6 mice exhibited M1dominant activity, while those from Nude or SCID
BALB/c mice exhibited M2 - dominant activity.
Indeed, it appeared that M1 and M2 differences
in these mice were greater than in their normal
counterparts. This was a surprising and important
result because it indicated that propensities for M1
or M2 dominant responses (and disease susceptibilities) can be independent of T cells.1
The foregoing results also brought up the very
intriguing possibility that differences in M1 and M2
responses between individuals could be responsible
for differences in T cell cytokine production. To test
this hypothesis, macrophages from SCID C57Bl/6
or BALB/c were again removed. Then, each was
mixed with macrophage-depleted CB6 (C57Bl/6 X
BALB/c F1 progeny) spleen cells (for histocompatibility). The polyclonal T cell activator, Con A, was
then added to stimulate T cell cytokine production.
It was found that SCID C57Bl/6 macrophages
(M1) stimulated CB6F1 spleen cells to produce
IFN-, while SCID BALB/c macrophages (M2)
stimulated TGF-b production (TGF-b is also
Th2-related; serum-free conditions did not allow
IL-4 production). Since the T cells were the same,
these results demonstrated that M1 and M2 macrophage- dominant responses stimulated T cells to
make Th1- or Th2-dominant cytokine responses.1
Results in other experimental systems had also
shown that antigen-presenting cells (whether called
macrophages or dendritic cells) can influence that
type of T cell response that occurs.45,46 However, the
experiments in SCID mice described above were
fundamentally different because they proved that
macrophages can drive T cell responses without
prior T cell influence. In turn, M1/M2 showed that
the Th1/Th2 paradigm was insufficient to explain
how immune responses work. As will be seen later,
if one looks at the design and function of Innate
Mills
Immunity, it makes sense that macrophages are the

central controlling element in immune responses.
3. M1/M2: Redefining Macrophage
Activation
Whereas I tried to inject some humor by describing an Adaptive Dictatorship, M1/M2 did help
change our concept of what macrophage activation
means that is worth explaining. Again, M1 and
M2 activities do not require T cell influence. This
fact is particularly important for understanding M2
macrophage activity. M2 (Ornithine production) is
the normal default program present in resident
macrophages. And, M2 macrophage activity that
is similar (or identical) to that of resident tissue
macrophages is found in sterile wounds and other
circumstances where there is no T cell component.3,36 In contrast, the concept of alternatively
activated macrophages arose mainly from the
observation that addition of the T cell cytokines
IL-4 or IL13 in vitro resulted in some changes
in macrophages.47-49 More recent results suggest
that alternatively activated macrophages are not a
distinct in vivo phenotype, but instead most closely
resemble resident macrophages (M2) in their gene
and protein expression.4,23 Thus, alternatively activated and M2 are not synonyms because M2 (and
M1) are T cell independent macrophage activities.
The older perception that macrophages depend
on T cell stimulation to be functional also lead
to the additional sub typing of M2 macrophages
into M2a, b and c based on in vitro stimulation
with: IL-4/13; Ig and Toll receptor agonists; and
IL-10 or TGF-b, and glucocorticoid hormones,
respectively.50 Like alternatively activated, macrophages treated in these or other ways in vitro
do have quantitatively different patterns of activity
(e.g., phagocytosis), gene expression and cell surface
markers.50 Macrophage M1 and M2 activity can
be elevated by T cell-derived cytokines. However,
M1/M2 most importantly means that macrophage
propensities precede and direct T cell responses,
not vice versa. Also, NO or Ornithine production is
469
the clearest and most functional way to categorize

opposing macrophage activities in Innate Immunity
(or in directing Adaptive Immunity).
C. M1/M2 and Macrophage Plasticity
M1 or M2 describes macrophage activity committed to Fighting or Fixing, or stimulating Th1 or
Th2 responses as described.1,2 Plasticity describes
macrophages as a continuum of phenotypes.51,52
The two ways of describing macrophages, while
seemingly at odds, are not. M1/M2 is useful because
it describes succinct macrophage functions that
influence the outcome of inflammation in polar
opposite ways. And, it is important to understand
that M1 and M2 specifically defines inflammatory
circumstances where NO or Ornithine production predominates.1 Predominate is the key word
because all inflammatory circumstances are clearly
a mix of M1 and M2 responses. One need not look
further than cultures of macrophages to know that
there is a mixture of different cells (Figure 1).1
Some have interpreted M1 and M2 as meaning
there are only two types of macrophages. Actually,
in the original description of M1 and M2, plasticity was the word used to allow for the possibility
that individual macrophages might produce varying
amounts of NO or Ornithine.1 It does appear, however, that all macrophages produce one or the other.
The important point then is that M1/M2 describes
polar opposite responses, and plasticity describes the
ability of macrophages to adapt incrementally (or
continuously) to achieve M1 or M2 predominate
activity, or varying mixtures of these responses.
It may be useful to think of M1 as a school
bus and M2 as a Ferrari; and plasticity as SUVs,
minivans and compact cars. Using this analogy,
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
above transport people, but only the school bus
and the Ferrari are ideal for these jobs. Similarly,
macrophages that produce NO or Ornithine are
ideally suited for certain circumstances. On the
other hand, there may be circumstances where

neither NO or Ornithine production is needed for
a desired macrophage function, like phagocytosis.
Going back to the analogy then, if one needed
to transport seven people across the country, an
intermediate type vehicle such as a minivan would
be ideal. Intermediate-type macrophages with
maximum phagocytic activity would likewise be
ideal, regardless of NO or Ornithine production
(if such cells exist).
Macrophages with intermediate activities
brings up a very important point relevant to
both M1/M2 and plasticity. In either case, it is
difficult to known from the study of populations
of cells what is going on at the single cell level.
For example, does an individual macrophage,
when maximally activated (e.g., IFN- and LPS),
fully turn on NO production and shut down the
other pathway, or is there plasticity in this and
the many other molecules macrophages produce.
Even the study of individual cells does not answer
this question because analysis at any point in time
is a snapshot. For example, if a macrophage is
committed to becoming pure M1, it can be caught
transitioning there from M2, and appear to be an
intermediate cell. Thus, by definition, there is
plasticity. That said, plasticity is not likely to be fully
bidirectional. In particular, once an M2 macrophage
(or population) becomes M1 (NO-producing), it
does not appear to revert back to M2. Although
not proven, this makes intuitive sense because NO
is toxic to macrophages also. Perhaps it depends
on how much NO is induced intracellularly. As
we learn more about the physiology of individual
macrophages perhaps new distinct phenotypes
will be discovered. In the meantime, M1/M2 and
plasticity both have important and different roles
in helping us understand macrophage functions.
D. Beyond Plasticity: Are Macrophages
Pluripotent?
Regardless of how one views or labels macrophage
activity, evidence keeps appearing to suggest that
470
Mills
FIGURE 1. Demonstration of the dramatic effects of serum on functions and morphology of macrophages.
TGF- 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

My own experience is that unstimulated macrophages, or, particularly, macrophages cultured
with TGF-b exhibit a fibroblast-like appearance
after a few days in vitro (Figure 1E). That these
macrophages may be transitioning into fibroblasts
(or some other cell) is supported by the fact that
their phagocytic activity is markedly reduced.1
Having a cell in most tissues of the body (like
macrophages) with pluripotent activity would
be an advantage in times of tissue damage (e.g.,
spinal cord injury) or tissue deterioration (e.g.,
Alzheimers). Therefore, although additional results
are needed to determine the potential pluripotency
of macrophages, I think this is an area worthy of
further investigation.
E. The Power and Pitfalls of In Vitro

Immunology in the Discovery of M1/M2.
The ability to analyze leukocytes in vitro has
unique advantages, but can also cause conflicting results and conclusions because conditions
dont resemble those occurring in situ or in vivo.
Appreciating these pitfalls can lead to a better
understanding of macrophages and other leukocytes. As an example, awareness of the composition of different culture medias played a key role
in elucidating the NO and Ornithine pathways
in macrophages. And, it also allowed experiments
to be performed showing that M1/M2 activities can control the type of T cell response as
discussed below.
471
The majority of immunologists still use fetal

bovine serum (or other serum supplements)
in culture media. Fetal serum is used to avoid
complications of heterophile and other antibodies present in adult serum. However, serum is
obtained by lysing platelets, which contain a lot
of TGF-b. And, TGF-b is the strongest inhibitor
of M1 activity (NO production) and promoter of
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
is sufficient to markedly inhibit macrophage NO
production.1,36 As shown in Figure 1, the effects
of TGF-b on macrophage morphology and functions are dramatic. And, the unknown presence of
this cytokine in vitro has lead to some erroneous
conclusions. For example, inhibition of NO production by TGF-b in serum resulted in the belief
that macrophages require two signals to become
activated (e.g., IFN- and LPS).58 The use of
serum-free media (actually contains albumin and
other necessary nutrients) allowed us to show that
either signal is sufficient.1
The use of serum (e.g., TGF-b) or other culture additives can lead to other types of pitfalls in
analyzing macrophage functions. For example, since
macrophages succumb to their own NO produced
in vitro,59 culture in serum-free (no TGF-b) media
elevates NO production, and decrease macrophage
viability after three days.59 Therefore, if one then
adds IFN-g or LPS to macrophages, one would
come to the incorrect conclusion that culture in
serum-free media decreased their ability to produce NO compared to serum-containing media.
However, if one had added live bacteria at the
beginning of culture, there would have been many
fewer bacteria three days later in serum-free media
compared to serum-containing media. Similar
misinterpretations have come from pre culturing
macrophages in IL-4 (that also decreases NO
production in macrophages). Because serum-free
media promoted M1, and serum supplementation
(TGF-b) promoted M2 activity, T cell responses
were also altered in important ways. Serum-free
media stimulated a Th1-dominant T cell response
(IFN- production), while serum stimulated a

Th2-dominant T cell response (IL-4 and TGFb
production). Recognition of this difference allowed
us to show that M1 and M2 dominant responses
can drive T cells toward Th1 and Th2 responses.1
Therefore, if a cell does not (or does) exhibit an
activity in vitro, it cannot necessarily be concluded
that the cell performs the same way in vivo.
F. Human M1 and M2 Macrophages
A particularly pertinent example of the tail wagging the dog (trying to make conclusions about in
vivo immunology from in vitro results) is continuing debates about whether human macrophages
display M1 or M2 characteristics.60-62 These debates
stem mainly from observations that human monocytes do not produce much NO or Ornithine.
But, compared to what? Mouse monocytes dont
produce much NO or Ornithine either! Moreover,
why would one expect monocytes (no known functions) to produce NO or Ornithine anyway? As
will be discussed later, civilization has decreased
infections (and wounds) in humans, so it seems
reasonable to postulate that human macrophages
produce somewhat less NO (or Ornithine) than
other species. But, there is abundance evidence
of the expression of both the iNOS and arginase
pathways in different human disease conditions.63-65
This makes sense because these two enzyme pathways are present in every other vertebrate (and
many invertebrate) species.15,33 Obviously, it is more
difficult to study macrophages in humans than in
rodents or other species. However, it is important to
measure NO and Ornithine production in human
macrophages because they are the best indicators
of Fight or Fix activities. As a side note here, one
might alternatively measure iNOS or arginase
enzyme activities, or the genes that code for these
enzymes as an indicator of M1/M2. It should
be kept in mind, however, that enzyme levels or
gene expression in general are indirect measures
of what really matters, the functional products of
macrophages, like NO or Ornithine. This fact is
472
sometimes forgotten when results with enzyme or

gene expression in macrophages are interpreted as
direct evidence of variations in functions.
V. M1/M2 MACROPHAGES IN NNATE
IMMUNITY: DESIGN, DANGER, AND
DIRECTION
Having identified the basic functions of macrophages, and what M1/M2 represents, it is useful to
describe how the design and the different responses
of Innate Immunity protect a host.
A. Design: Protect the Vascular System
Humans (and other vertebrates) have evolved
separate neural, endocrine, circulatory and respiratory organs connected by a vasculature system. The
importance of protecting these organs caused the
human Innate immune system to evolve a powerful
three-tier system of protection designed to keep
pathogens out of the vascular system.15,16
The first and most important tier is located
near surfaces exposed to the environment (skin,
lungs and the gut). Macrophages are the predominant cells in these areas.3 As macrophages sample
events, self or non-self material is recognized. Also,
epithelial cells secrete defensins at these surfaces.
In parallel with these events, the second tier of
protection begins with a natural fluid pressure
gradient that carries antigens and other materials
away from the local site into lymph channels and
into draining lymph nodes (or similar type collections of lymphocytes). Here, material is sampled by
macrophages again, and T and B cell proliferation
occurs if foreign antigens are detected. Finally, the
third tier of Innate protection comes from macrophages that reside in organs themselves (e.g.,
microglia). That the Innate System is designed to
keep pathogens out of the vasculature (and thus
the organs) was demonstrated by experiments
in the 1960s. Introduction of live organisms (or
antigens) intravenously rather than subcutaneously
Mills
was ineffective at generating immunity, and resulted

in death or tolerance, respectively.66 As mentioned
earlier then, by placing cowpox on the skin, Jenner inadvertently discovered much about how
Innate Immunity is designed. HIV is a clinically
relevant example of the importance of the route of
exposure because it is very poorly infective, unless
introduced directly into the blood. Of course, the
clinical term for what happens when the immune
system fails to keep pathogens out the vasculature
is sepsis, which is often fatal.
B. Danger: The Wake Up Call
Any breach of exposed surfaces (skin, lung, intestine) is potentially dangerous. Polly Matzinger
coined the fitting term Danger to describe the
critical nature of the events that occur immediately
after a breach.67 Speed and Overwhelming Force
are important in response to Danger, regardless of
the nature of threat.
1. Speed
Why must macrophages respond quickly? One
bacterium can become over 2 million in one day.
One white cell can become two in one day! So,
to overcome this overwhelming prokaryotic proliferative advantage, resident macrophages respond
within minutes to molecules such as prostaglandins
released from damaged cells in a skin wound. And,
inside of an hour, macrophages are phagocytosing
material and signaling other Innate cells to enter
the area from the vasculature.25
2. Overwhelming Force
Former US Defense Secretary, Colin Powell, used
this term to describe successful warfare, and it
aptly describes the goal of the initial Innate local
response. Because macrophages need time to
figure out if pathogens are present in the wound,
473
the first Biologic Priority of the initial Danger

response (416 hours) is simply to try to sterilize
the area.2,36,68 Recruited neutrophils, macrophages
(and sometimes Mast Cells) are the predominate
cells that do this through the secretion NO and
other oxygen radicals. Not surprisingly, there is collateral damage to normal tissue. The circumstances
in the lung, intestines, or in organs, are normally
somewhat different from a skin wound because the
tissue damage is microscopic, not macroscopic.
For example, macrophages in the lung are normally
encountering and phagocytosing inhaled material,
not damaged lung cells. Regardless of the location
of Danger, macrophages are critically required to
determine in what Direction the immune response
should proceed.
default (resident) M2 mode. Specifically, Ornithine, EGF, VEGF, and other growth factors are
produced that are necessary for repair and resolution of the wound.69 Also, wound signals such as
TGF-b and adenosine (from fibroblasts and other
cells) are important in maintaining M2 activity.69
In parallel, phagocytosis is important to clear and
digest damaged tissue and other debris. T cells
are not noticeably involved in wound healing.70,71
Therefore, Damage Danger provides an example
of the difference between M/1/M2 and Th1/Th2
because M2 macrophages heal the wound without
the need for alternative activation by T cells.47
C. Direction: What Type of Danger is

Present?
If pathogens are detected through PAMP, then a

different set of signals is received3-6,20 and resident
(and newly recruited) M2 macrophages become
M1 macrophages within a day of infection. Also,
IFN-g is produced by NK and other Innate cells
at this time to further elevate the M1 response.71
The intestine is somewhat different. Macrophages
there are routinely bathed in bacteria (and parasites
before civilization), but unknown unique elements
in that environment have evolved to routinely
suppress M1 activation to allow nutritionally beneficial symbiosis. Similarly, ingestion of food (or
other antigens) results in beneficial oral tolerance
compared to exposure at other sites.72 Outside
of a sterile operating room, most skin injuries
(or air breathed, food eaten) contain potential
pathogens. Therefore, it would seem that there
is daily Localized Danger, but M1 activation is
normally sufficient to contain pathogens without
T cell involvement.
Know when to hold em, know when to fold emKenny

Rogers (song lyric from The Gambler).
This lyric about poker playing colorfully describes

the critical nature of the decisions macrophages
make in determining what Direction to proceed
if there are pathogens present, if they can be contained, or if Adaptive Immunity is needed.
1. Damage Danger: False AlarmM2
Macrophage Repair
During the initial Danger phase, macrophages
are also determining what Direction to take based
on the nature of the threat. The extent of damage
is determined by receptors for DAMP (damage
associated molecular patterns). Concurrently, other
receptors recognize pathogens by PAMP (pathogen
associated molecular patterns). If no pathogens
are detected, the Biologic Priority switches from
sterilization to repair. Monocytes recruited from
the blood primarily handle this process.5 Because
there are no new Danger signals or pathogens
present, the recruited macrophages remain in the
2. Localized Danger: Real AlarmM1

Macrophage Containment
3. Definite Danger: M1/M2 Induction of

Adaptive Immunity
If the pathogen is not eliminated at the first tier of
defense within a few days, antigens or pathogens
474
enter the lymph channel and travel to the second

tier - the draining lymph nodes. There, macrophages (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
humor). This event results in clonal proliferation
of specific T cells.73 And, as mentioned earlier,
it is now clear that if the pathogen stimulates
an M1 dominant response, then macrophages
send signals to T cells (IL-12/23) to make a Th1
response (primarily IFN-g) that further elevates
the M1 response. M1 also seems to drive the more
recently discovered Th17 response associated with
autoimmunity.74 On the other hand, if macrophages
remain as M2 (because some pathogens subvert M1
responses, to be discussed), then a Th2 response
(e.g., IL-4/13, TGF-b, IL-10) occurs and results
in antibody production.75 And, the cytokines produced during Th1 or Th2 responses reciprocally
down regulate M2 and M1 macrophage responses
so one response (that may be optimally protective) can dominate.48 In this regard, only birds
and mammals make true antibody responses.(15)
Presumably, antibody responses evolved to better
handle pathogens by utilizing the circulation to
provide more effective systemic protection and/or
because antibody is more effective against certain
pathogens than macrophages. Also, antibody may
have also evolved to handle pathogens that can
subvert M1/Th1 responses (to be discussed).
The foregoing description of how Innate
Immunity functions was based on a host encountering a pathogen for the first time. If there is
preexisting T cell memory (a higher frequency
of specific T cells), or existing antibody from a
prior exposure, then Adaptive Immunity will play
a slightly different role. For example, preexisting
antibody might be sufficient to halt a pathogen
without requiring a secondary antibody response.
Nonetheless, Innate Immunity still functions as the
initial gatekeeper to identify threats and respond
accordingly. And, as will be seen later, M1/Th1
and M2/Th2 responses each have important and
different roles in preventing (or causing) many
modern diseases.
Mills
4. M1 Danger: Immunoregulation and

Damage by NO
Up until now, the protective functions of M1
responses (NO, antigen presentation) have been
described, but M1 also has important immunoregulatory functions.2 NO produced by what are often
called suppressor macrophages diffuses freely in
all directions, and can inhibit T cells, or whatever
other normal cells it encounters (recall collateral
damage in the early wound response).76,77 Therefore, NO creates a conundrum during immune
responses. For example, T cell production of IFN-g
(that elevates NO production in macrophages) is
required for protection against many major infections. However, it is clear in some circumstances
that the NO produced then inhibits the T cell
response in vivo.78
The suppressive effects of macrophage NO
production can cause different outcomes depending on the amount produced, the timing, and the
disease involved. For example, during a response
to a pathogen (like Leishmania, where macrophage
NO is required), if there is a lot of NO produced
early, then T cell activation and proliferation can
be strongly inhibited and the pathogen grows. If
sufficient NO is produced during T cell amplification, then the pathogen is eliminated. If surfeit
NO is produced during T cell amplification, the
T cell response can be partially inhibited and a
chronic infection may result. There are examples of
all three of these circumstances with pathogens.2 It
has not yet been determined if the inhibitory effect
of NO evolved to immunoregulate overzealous T
cell responses, or is just an unintended side effect.
Either way, there are other diseases (particularly
viruses), where only a T cell response (e.g., cytolytic)
is curative. In these circumstances, concomitant NO
production is clearly inhibitory what I termed
an effector deflector.2,78
Aside from these different immunoregulatory
effects, NO can also exacerbate certain diseases
directly because the cure is worse than the disease.
Specifically, excessive NO production can cause
more damaging effects in the lungs, intestines and

the brain than the pathogen itself. There are numerous examples of this phenomenon with bacteria
(Mycobacterium), parasites (Toxoplasma ghondi) and
viruses (HIV).2 Finally, another perplexing element
of the NO conundrum is the ability of M1 macrophages to stimulate a Th1 response, as described
earlier. It would seem that if an NO producing
macrophage presented antigen to a T cell, it would
kill it. Therefore, it seems like something is going on
during antigen presentation that remains unknown.
The foregoing results make it apparent that consideration of both the protective and destructive
effects of an M1 response need to be taken into
consideration when designing vaccines or other
therapeutic strategies to be discussed later.
VI. M1/M2 MACROPHAGES:
BIDIRECTIONAL COMMUNICATION
WITH NEURAL AND ENDOCRINE
SYSTEMS
A. Introduction
No man is an island John Donne (1624).
Just as we have seen that M1/M2 responses act in

concert with Th1/Th2 responses, it is also important to recognize that the immune system is not a
stand-alone organ. One only need be reminded of
how it feels to have the flu (or getting sick after
stressing out about a grant renewal!) to understand
that the immune system is interconnected with the
neural and endocrine systems. So, although it is not
my expertise, I thought a brief discussion of how
these systems influence each other is important.
B. Normal Stress and Beneficial M1/M2
Responses
The neural and endocrine systems help to maintain homeostatic balance by reacting to stress.79
The fundamental objective of a stress response
475
is likely to promote survival of the individual via

neurotransmitters and hormones that mobilize
energy from stores (e.g., glucose) to allow increased
physical and mental activity (e.g., running from a
dog), and increased immunologic activity (e.g., if
the dog bites your head).
Processive stressors are those that elicit the
fight-or-flight reaction from facets within the
hosts environment that are perceived by the
host as potential dangers, but do not cause harm
directly.80 When a host senses a threat, the pituitary gland responds involuntarily by releasing a
surge of ACTH, which acts on the adrenal glands
to release several stress hormones, including epinephrine and cortisol.
In contrast, systemic stressors are those that
actually pose a threat to homeostasis, such as
extreme pain, dehydration or injury. These stressors
require much less cognitive processing than processive stressors and often occur simultaneously with
processive stressors. Systemic stressors initially
activate the same type of neural and endocrine
responses as processive stressors.
As we saw earlier, the Innate Immune system
makes its own response to stress in the Danger
response. And, this response communicates with
the neural and endocrine systems in several ways.
For example, macrophages produce molecules such
as IL-1, IL-6, and TNF-a that cause the brain to
increase body temperature and create a feeling of
malaise. These effects are beneficial because fever
helps kill microorganisms, and malaise signals a
host to sequester itself (e.g., in the house) until
normal function is restored (dog bite heals). I used
the example of a dog bite in the head because it
illustrates that macrophages (in this case microglial
cells) are located in most organs, and form the third
tier in the Design of the Innate Immunity. In addition, microglial cells are in constant bidirectional
communication with astrocytes that is necessary
for normal brain function.81 Microglial cells, like
macrophages elsewhere, can make M1 responses to
deal with an infection (dog head bite), or an M2
response to heal a head injury (sterile dog head
bite). Thus, the neural, endocrine, and immune
476
systems have evolved important bidirectional

communication pathways that serve to optimize
host performance and survival under conditions
of processive or systemic stress.
C. Abnormal Stress and Detrimental M1/
M2 Responses
Unlike normal stress that is acute and beneficial,
chronic or severe stress degrades the neural, endocrine and immune network. In particular, prolonged
elevation of cortisol depresses Innate Immunity. One
example is life-long spouses. If one dies, the other
often dies shortly after, in part, because of depressed
immunity.82 Another important effect of chronic
stress is that it specifically decreases the M1/M2
ratio.83 Chronic inflammation also can be deleterious because byproducts of Innate Immunity, such as
glutamate, cause decreases in serotonin and dopamine that maintain a healthy mood. Schizophrenia,
depression, and other behavioral disorders can be
exacerbated by chronic inflammation.84 Although
macrophages are the primary link to the neural and
endocrine systems, if Innate Immunity stimulates
Adaptive Immunity, T cells produce similar molecules as macrophages that also degrade the neural
and endocrine systems.82 Civilization has markedly
affected the type of stress we experience, and has
contributed to changes in the balance of M1/M2
as described in the following section.
VII. M1/M2 MACROPHAGES IN
MODERN CIVILIZATION
As it was instructive to understand how Innate
Immunity works, it is useful to appreciate that
Civilization has changed Innate Immunity.
Civilization has roughly doubled humans lifespan.
Many modern diseases (cancer, atherosclerosis,
autoimmunity) are the result of these two changes.
Civilization (in evolutionary terms) is a recent
event. Therefore, the immune system has not had
much time to adapt. In addition, most modern
Mills
diseases occur later in life. Therefore, there is

little evolutionary pressure (breeding preferences)
for the immune system to adapt. Below are some
changes Progress (P) has brought in the input
that Innate Immunity receives that have changed
the balance of M1and M2.
A. Predation to Pets
Until fairly recently, the biggest threat to man
was being wounded or killed by tigers, or snakes
(predators), or other humans.85 Now, we only have
pets! Loss of predation/acute stress responses and
decreased infections have decreased M1 responses.
A useful comparison of early and modern man
is mice versus men. Mouse macrophages have a
stronger M1 component because they are exposed
to predators and more infectious agents.60,61
Net Result: Decreased M1/M2 ratio.
B. Pestilence to Purity
The major reason for the decrease in infectious
disease (and M1) in the last couple of centuries has
been increased sanitation. In addition, food sources
have been simplified and sanitized. Approximately
70% of the worlds food now comes from 6 grains
and one animal (cows), and more of the food consumed 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
vaccines have decreased infectious diseases resulting in decreased M1 responses. Because products
of M1 and M2 responses mutually inhibit each
other, M2 responses also have increased.
C. Paradise to Pressure
One might not think of earl mans existence as
paradise. However, industrialization, increased
population density and other factors have altered
477
the type of neuroendocrine stress we experience.

For example, housing and transportation have
decreased acute stress (e.g., being attacked while
sleeping or walking). In addition, crowding, jobs,
financial pressure, etc., have elevated chronic stress.
As discussed, both of these changes in the type
of stress experienced alter the M1/M2 balance.82
therapeutic potential for changing the M1/M2

balance in cancer.89,90 Also, the immunologic
circumstances that occur in cancer are applicable
to other diseases involving inappropriate M1/M2
balances. So, I will use cancer (and its relationship to a wound) as the central model to develop
some general ideas about how to develop effective
therapeutic strategies.
D. Petite to Pounds
A. Background to Cancer
The major increase in obesity in civilized countries

in the last century has caused excess circulating energy (glucose, fatty acids) that fools the
immune system into making chronic Danger
responses on vessel walls.86 Energy Imbalance and
Metabolic Syndrome are terms used to describe
this important new civilization-induced replacement for infection-induced M1 responses to be
discussed in the next section.
Net Result: Increased M1/M2 ratio
In summary, Civilization has markedly
decreased major infections, and brought other
changes to make modern human Innate Immunity
M2 dominant. M1 and M2 responses that evolved
to protect early man are now often inappropriate
in modern man because of damage they cause
during increased lifespan. Diseases associated with
inappropriate M1 and M2 responses, and how one
might alter them for better health is discussed in
the following section.
The history of diseases can be instructive. This

is particularly true in cancer. Cancer is with us
today because it occurs mostly later in life, and
therefore does not affect breeding; there is little
evolutionary pressure against cancer. Most other
modern diseases (e.g., atherosclerosis, allergy,
autoimmunity) are also with us for the same
reason. Cells populating tumors were observed
by Virchow in the 1800s. Around the turn of
the 20th century, the primary cells that populate
tumors came to be called macrophages. About
the same time, Dr. Coley had observed that
life-threatening infections in cancer patients
sometimes resulted in complete tumor regression.
So, he developed Coleys Toxin (a bacteria mix)
and used it to successfully cause complete cancer
regression in some patients.91 However, there
was unacceptable mortality and Coleys Toxin
has been mostly forgotten. Later on, during the
Adaptive Dictatorship, it came to be believed
in the 1960s and 70s that cancer develops now
and then, and the immune system specifically
recognizes and eliminates it.92 As appealing as
this notion was, a majority of cancers studied did
not express unique antigens. A telling blow to the
Immunosurveillance theory was the observation that T cell deficient mice (Nude) exhibited
nearly the same incidence of cancer as normal
mice.93,94 Additionally, it has long been observed
that invertebrates and other lower organism that
do not possess Adaptive Immunity do not have
a higher cancer incidence than humans.16 So,
what are macrophages doing in tumors, and what
VIII. M1/M2 BALANCES IN MODERN

DISEASES AND STRATEGIES FOR
HEALTH.
The causes and cures of modern diseases can be
complex. At the same time, many diseases exhibit
inappropriate M1/M2 balances that play a role in
disease pathologies. M1/M2 came about mainly
from my studies of cancer and wounds, and are
the conditions I know best from an immunologic perspective.2,87,88 And, there is considerable
478
mechanism had Coley (and others) been able

to stimulate that caused cancer regression? The
relationship between cancer and wound repair
provides salient insight.
B. Innate Immunity and Cancer: M2
Promoted Growth
As mentioned, macrophages are the primary leukocytes in tumor beds. M1 macrophage activity
can clearly kill tumor cells31,95 and the presence of
intratumor Fight M1 macrophages is very favorable for survival.96 However, evidence indicates
that intratumor macrophages are primarily M2
Fix dominant.6,97-99 What goes wrong? Cancer
and wounds may seem odd bedfellows, but are
intimately linked. Cancer has accurately been
called wounds that do not heal.100,101 Consider
back to the discussion of Damage Danger present in a wound. Initially, there is an M1 response
that serves to sterilize the area. However, if the
wound is sterile, an M2 -dominant response
occurs to provide Ornithine (polyamines, collagen), EGF, VEGF and other growth factors
that are required for proliferation, repair and
resolution. That this environment is conducive to
cancer is evidenced by the preferential appearance
of tumors at sites of wound repair,102-104 and is oft
called the Seed and Soil Hypothesis.105 Cancer is
analogous to a sterile wound in that there are no
(or insignificant) specific antigens, as mentioned.
Therefore, having M2 macrophages in a tumor is
not simply neutral, but can promote growth.106
That M2 macrophages are actively involved
in stimulating cancer growth is evidenced by
decreased growth rates in hosts depleted of
macrophages.107 In this regard, a tumor can only
grow to about one million cells before it requires
new blood vessel support (about the size of the
period at the end of this sentence). But cancers
dont normally produce angiogenic factors, and
so require M2 macrophages to provide it.108,109
Whereas a wound naturally heals, cancer keeps
proliferating and pushes normal tissue aside,
Mills
resulting in new Danger signals and signaling

more monocytes to enter from the circulation.
Returning to the wound analogy, why dont newly
recruited macrophages keep making the early M1
response that would kill cancer cells? Cancer cells
have evolved active strategies to subvert Innate
Immunity by preventing curative M1 responses
from developing. Cancer cells produce a variety
a factors such as TGF-b and prostaglandin E2
that inhibit M1 (NO) Fight activity, and keep
macrophages in M2 (Ornithine) Fix mode.110
Also, cancer cells stimulate metaloproteinases in
macrophages to aid in the breakdown of matrix
allowing continued encroachment into normal
tissue. Thus, cancer is mainly a disease that is
not recognized by T cells, and whose growth and
metastasis are aided by keeping macrophages in
M2- wound healing mode.
An unrelated, but interesting possible connection between cancer and M2 macrophages is the
influence of stress. As discussed earlier, chronic
stress is more of an element in modern life than
acute stress and chronic stress has been shown to
decrease the M1/M2 ratio.83 Although it is difficult
to establish cause and effect, there is a wealth of
evidence associating failure to handle stress (antisocial behavior, depression, etc.) with increased
cancer incidence and growth rates. Sometimes
this connection is referred to as the Melancholy
Personality in cancer.111
1. M2 Promotion of Infectious Diseases
Many pathogens, like cancer, have evolved
mechanisms to actively keep M2 macrophages
from transitioning to M1. For example, Mycobacterium, Leishmania and HIV all subvert
macrophage physiology for their own survival
and benefit.3,112,113Some do this by expressing an
antigen that fools macrophages into thinking they
have encountered self (preserving M2 activity), while others cause macrophages to secrete
cytokines (e.g., TGF-b or IL-10) that prevent
M1 activation.3,6
479
There are human cancers that do express

unique tumor antigens (e.g., Melanoma) and so
are foreign like infectious diseases.114 However,
as with infectious diseases, the same problem
remains. Cancer cells subvert M1 responses to
M2. And, because Innate Immunity is required
for T cell responses, the inhibition of M1 prevents
the development of an effective anti cancer Th1
response. This phenomenon was observed many
years ago in my laboratory using the P815 Mastocytoma in mice. P815 elicits a tumor-specific
cytolytic T cell and an activated macrophage
response in a host, but the responses are insufficient to cause tumor rejection.9,87 It can be seen
in Figure 2 that if a host is specifically preimmunized against the P815 tumor, and rechallenged with tumor, there is robust M1 activity
(NO) in the tumor bed and the tumor is rejected.
However, in a nave mouse, the P815 decreases
M1 and increases M2 activity allowing progressive tumor growth in much the same manner as
some pathogens.
3. Therapy of M2-Dominant Diseases
Once cancer appears, transiently boosting M1 (and/

or inhibiting M2) responses locally or systemically
seems to have great potential.90,119 Because allergy
is often localized and seasonal, there seems even
greater therapeutic potential for transiently increasing the M1/M2 ratio in the airways.120 Although
many autoimmune conditions seem to result from
overzealous M1 responses (e.g., MS, inflammatory
bowel disease, arthritis),3 SLE appears to mainly
result from excessive M2- mediated autoantibody
production, and could be a good candidate for
increasing the M1/M2 ratio also.121 Finally, as
discussed, certain infectious agents subvert M1
to M2 as recently discussed by others.3 There are
interesting new strategies to increase the M1/M2
ratio, including injected and inhaled adjuvants, as
well as probiotics and other materials that work
orally. Some of the most promising agents are Toll
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
480
a continuous cycle of M2 repair. And because

humans have limited capacity to regenerate tissue,
repair is imperfect (e.g., scarring), and lung function declines. In addition, continuing M2 repair
creates a fertile soil for the seed much like it
does in non-healing wounds.100 Sun-induced skin
damage resulting in skin cancers, or cancer of the
bowel from chronic inflammation, are two other
examples where chronic M1 oxidant damage are
implicated. Thus, M1 responses that were required
for survival in early man (and which are beneficial
against existent cancer) can result in accumulation
of damage over time and cause cancer.
1. M1 Responses and Autoimmunity
As in cancer, the accumulation of M1 Oxidant
damage (and associated M2 repair) seem to play
an important role in many autoimmune conditions
such as MS, inflammatory bowel disease, arthritis
and Alzheimers disease.3,127.128 This might seem at
odds with the fact that M1 responses overall are
less common in modern civilization, as discussed
Mills
earlier. However, as in cancer, accumulation of exposure because of increased longevity is important

because most autoimmune conditions develop over
many years. These conditions also resemble most
cancers because although there may sometimes be
a viral or microbial vector initially involved, once
established, they do not express classical foreign
antigens. Therefore, Innate, not Adaptive, Immunity is primarily responsible.3
2. M1 Responses and Atherosclerosis
In the last several years, it has come to be appreciated that macrophages are the primary leukocyte
component and main cause of atherosclerotic
plaques.86,129,130 And again, like cancer, the endothelial lining of vessels does not display classical
foreign antigens. So, why are macrophages causing reactions in the blood vessel walls resulting
in plaque formation? The answer seems to lie in
chronic excess energy availability, resulting from
what I called Pounds (obesity). Adipose tissue
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.
481
FIGURE 3. M1- or M2-dominant immune responses are associated with specific diseases.
rophages that produce TNF-a, NO and other

inflammatory mediators, and their production is
proportional to the amount of adipose tissue.86
Along with this inflammation, obesity causes
an inability of insulin to store glucose in fat
(insulin resistance), resulting in obesity-onset
diabetes (now called Type II). In turn, glucose
and fatty acids increase in the circulation. This
circumstance resembles what occurs in acute
stress (recall early man being chased by a tiger)
that shunts extra energy to the circulation to
properly deal with a threat. However, in obesity
this excess energy becomes chronic and fuels
M1 responses. We showed some years ago that
induction of diabetes in rats or mice results in
elevated M1 responses, and which was corrected
by insulin administration.131 Although it is not
altogether clear, the increased concentration of
glucose and fatty acids in the circulation seems
to be the stimuli that fools macrophages into
thinking there is Danger in the endothelial lining. Somewhat analogous to a smokers lung or
a non-healing wound, the continuing deposition

of fatty acids perpetuates a cycle of M1 damage
and M2 repair resulting in an enlarging plaque.
Thus, the problems associated with diabetes and
atherosclerosis are increasingly being viewed as
diseases of chronic M1 responses.132
3. Therapy of M1-Dominant Diseases
Unlike tumor growth where both decreased M1
and increased M2 activity can exacerbate disease,
diseases where M1 is contributory seem to result
primarily from chronic low -level M1 damage.
Because atherosclerosis is a disease of the vasculature and certain autoimmune conditions are localized to particular organs (e.g., inflammatory bowel
disease), potential exists to specifically decrease
M1 responses in the areas affected.125,133,134 How
one might devise particular therapeutic strategies
to simultaneously deal with M1 and M2related
conditions are discussed below.
482
D. Type, Territory, Timing and Tenacity of

M1/M2 Therapy
From the foregoing overview of M1/M2 imbalances in modern diseases, it is clear that no single
therapeutic strategy suits all conditions. At the
same time, diseases appear at different places in
the body and at different times in life that opens
up windows of opportunity for developing targeted
immunologic interventions. However, success will
depend on taking into account the Type, Territory
(e.g., systemic or local), Timing and Tenacity of
the therapy.
As an example, it is widely believed that
decreasing oxidant damage (e.g., M1) through
nutrition and lifestyle changes is a successful Type
of strategy. This seems logical in so far as chronic
cellular damage results in aging and some cancers.
Also, atherosclerosis would benefit from decreased
M1 responses. The Territory to be covered would
be systemic because damaging M1 responses
are. As for Timing, this strategy makes sense in
adults, after childhood diseases have waned, and
when M1-mediated atherosclerosis appears. As
for Tenacity, it is worth considering that M1 is
already decreased, so a mild lifetime decrease in
M1 might be appropriate. Decreasing M1 over
a long period does again bring up the TwoEdged Sword nature of immune responses. That
is, to the degree that Innate Immunity routinely
eliminates cancer, decreasing M1 could result in
greater incidences.
By considering the Type, Territory, Timing,
or Tenacity, more than one intervention could be
employed in the same individual for another disease. For example, increasing the M1/M2 ratio is
a different Type of strategy than discussed above
that would be beneficial against growing cancer
or allergies. However, the Territory treated could
be limited in the case of localized cancer. Allergy
is an even better example because the Territory
is most often localized in the airways. Even if
increasing the M1/M2 ratio needed to be systemic,
the Timing of cancer treatment could be brief, or
Mills
seasonal with allergy. The Tenacity in the case of

cancer might have to be high because of previous
observations that regression only occurs if there
is a substantial infection.91
Therefore, as one looks ahead to therapeutic
strategies it is useful to view immune responses not
as Two-Edged Swords, but different swords that
can be wielded in different ways and at different
times for better health.
IX. SUMMARY AND PERSPECTIVE
It is apparent that most diseases plaguing modern civilization result from inappropriate Innate
immune responses, be it Type, Territory, Timing,
or Tenacity. Most of these responses distill down to
overactive or underactive M1 Fight or M2 Fix
responses. It is also helpful to view todays human
M1 and M2 responses as vestiges of a dirtier
time when they were necessary for survival (early
in lifeto reach breeding age). In turn, in our
increasingly germ-free and long-lived societies it
may be useful to somewhat decrease overall immunity (M1 and M2), towards what could be termed
immunocivilization or M0. After all, we seem
to be doing fine with decreased M1, and the level
of M2 doesnt seem beneficial (and contributes to
cancer and allergy, etc.). Given the central role of
the immune system in modern disease pathologies,
it is now important to include routine immunologic testing of individuals to assess who might
benefit from an immunologic intervention before
disease onset. In this regard, it is apparent that M1
and Th1 (or M2 and Th2) responses most often
work in concert to create characteristic immune
responses. Therefore, I think it would be useful
in many circumstances to adapt a simpler, more
inclusive terminology such as Immune Type 1, 2,
or 0 (IT1, IT2, IT0) to describe an individuals
immunologic status.
In conclusion, viewing Innate Immunity
mainly from an M1/M2 perspective has limitations
as I said at the outset. However, it has become
abundantly clear in the last few years that Innate
483
Immunity controls both inflammatory and specific

responses, and M1 and M2 activities are the essence
of this control. Simple.
ACKNOWLEDGMENTS
I am grateful for the associations I have had
over the years that have allowed me to look at
the immune system from different angles. As my
postdoctoral advisor, Robert North of the Trudeau
Institute introduced me to macrophages (where,
with his help, macrophage activation had been
discovered).22,135 At Brown University, my association with Michael Caldwell and Jorge Albina
opened my eyes to the importance of metabolism,
specifically arginine, in macrophage physiology. I
am grateful to my friend, John Hibbs, in Utah
who discovered Nitric Oxide that helped me
develop M1 and M2 macrophages. Finally, I am
most fortunate to have had superior students and
employees to supply fresh ideas, and my many
friends and my children that make life rich and
whole. I dedicate this article to two such friends
who died before their time this year: Dr. Bob Stout
(a fellow mac guy 51,52), and Dr. George Ebert
(my life-long friend), who died suddenly while
attending his mothers funeral.
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M1 and M2 Macrophages Oracles of Health and Disease

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

M1 and M2 Macrophages: Oracles of

I. SCOPE AND GOAL

of clinical applications. Others have expertly

II. HISTORY: VACCINES AND THE FALL

M1/M2 in Health and Disease

improve health requires a better understanding

tocompatibility antigens so foreign antigens are

inappropriately (preserve tolerance). To perform

IV. M1 AND M2 MACROPHAGES

M1/M2 in Health and Disease

set in motion very different types of immune

individuals could be attributed solely to differences in T or B cell responses.38-40 In particular, it

a Th1- or Th2-dominant environment, respectively.

Immunity, it makes sense that macrophages are the

M1/M2 in Health and Disease

the clearest and most functional way to categorize

other hand, there may be circumstances where

macrophages can transform into other cells.53-57

E. The Power and Pitfalls of In Vitro

M1/M2 in Health and Disease

The majority of immunologists still use fetal

(IFN- production), while serum stimulated a

sometimes forgotten when results with enzyme or

was ineffective at generating immunity, and resulted

M1/M2 in Health and Disease

the first Biologic Priority of the initial Danger

C. Direction: What Type of Danger is

If pathogens are detected through PAMP, then a

Know when to hold em, know when to fold emKenny

This lyric about poker playing colorfully describes

2. Localized Danger: Real AlarmM1

3. Definite Danger: M1/M2 Induction of

enter the lymph channel and travel to the second

4. M1 Danger: Immunoregulation and

M1/M2 in Health and Disease

more damaging effects in the lungs, intestines and

Just as we have seen that M1/M2 responses act in

is likely to promote survival of the individual via

systems have evolved important bidirectional

diseases occur later in life. Therefore, there is

M1/M2 in Health and Disease

the type of neuroendocrine stress we experience.

therapeutic potential for changing the M1/M2

The major increase in obesity in civilized countries

The history of diseases can be instructive. This

VIII. M1/M2 BALANCES IN MODERN

mechanism had Coley (and others) been able

resulting in new Danger signals and signaling

M1/M2 in Health and Disease

There are human cancers that do express

3. Therapy of M2-Dominant Diseases

Once cancer appears, transiently boosting M1 (and/

a continuous cycle of M2 repair. And because

earlier. However, as in cancer, accumulation of exposure because of increased longevity is important

M1/M2 in Health and Disease

rophages that produce TNF-a, NO and other

a non-healing wound, the continuing deposition

D. Type, Territory, Timing and Tenacity of

seasonal with allergy. The Tenacity in the case of

M1/M2 in Health and Disease

Immunity controls both inflammatory and specific

lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes. J Exp Med.