M-1/M-2 Macrophages and the Th1/Th2 Paradigm1

Charles D. Mills,2 Kristi Kincaid, Jennifer M. Alt, Michelle J. Heilman, and Annette M. Hill
Evidence is provided that macrophages can make M-1 or M-2 responses. The concept of M-1/M-2 fomented from observations that
macrophages from prototypical Th1 strains (C57BL/6, B10D2) are more easily activated to produce NO with either IFN-␥ or LPS
than macrophages from Th2 strains (BALB/c, DBA/2). In marked contrast, LPS stimulates Th2, but not Th1, macrophages to
increase arginine metabolism to ornithine. Thus, M-1/M-2 does not simply describe activated or unactivated macrophages, but
cells expressing distinct metabolic programs. Because NO inhibits cell division, while ornithine can stimulate cell division (via
polyamines), these results also indicate that M-1 and M-2 responses can influence inflammatory reactions in opposite ways.
Macrophage TGF-␤1, which inhibits inducible NO synthase and stimulates arginase, appears to play an important role in reg-
ulating the balance between M-1 and M-2. M-1/M-2 phenotypes are independent of T or B lymphocytes because C57BL/6 and
BALB/c NUDE or SCID macrophages also exhibit M-1/M-2. Indeed, M-1/M-2 proclivities are magnified in NUDE and SCID mice.
Finally, C57BL/6 SCID macrophages cause CB6F1 lymphocytes to increase IFN-␥ production, while BALB/c SCID macrophages
increase TGF-␤ production. Together, the results indicate that M-1- or M-2-dominant macrophage responses can influence
whether Th1/Th2 or other types of inflammatory responses occur. The Journal of Immunology, 2000, 164: 6166 – 6173.

M acrophages are essential for host defense (1–7). In cytokines that activate or inhibit macrophages, other reports have
primitive organisms, macrophages are the host de- shown that macrophages from the Th1 strains are more easily ac-
fense, in that they are responsible for everything from tivated than those from Th2 strains (17–21). Thus, the ability to
recognition to engulfment to destruction of threats (8). During phy- generate a Th1- or Th2-type response does not wholly depend on
logeny, lymphocytes take on progressively more of these macro- T lymphocytes. That macrophages themselves may determine im-
phage duties using their superior recognition structures. The shift munologic outcomes is suggested by results such as those showing
in the importance from innate to adaptive immune responses in that Leishmania infection of macrophages can increase their ability
evolution (9) is illustrated by the fact that lymphocyte-deficient to stimulate a Th2 response instead of a Th1 response (22). In a
rodents or humans succumb rapidly to infection. In line with the related vein, it has been reported recently that dendritic cells have
increasing lymphocentricity of immune systems, a paradigm has the potential to shepherd T lymphocytes into Th1- or Th2-domi-
developed over the last decade or so in which T or other types of nant phenotypes (23–26). That macrophage and dendritic cells can
lymphocytes control the type of immune responses generated by both influence lymphocyte responses is in keeping with the knowl-
the profile of cytokines they secrete (10 –12). In particular, im- edge that macrophages are a precursor of dendritic cells (27).
mune responses highlighted by T lymphocyte production of IFN-␥, In the course of our ongoing investigations into factors that reg-
which causes macrophage activation, are called Th1. Th2 immune ulate macrophage arginine metabolism, we found evidence that
responses are associated with IL-4, IL-5, and IL-10, which, in Th1 and Th2 macrophages not only differ in their ability to be
contrast to IFN-␥, inhibit macrophage activation and instead stim- activated in the classical sense, but made qualitatively different
ulate Ab production. Leishmania major is the prototypical model responses to the same stimuli. For example, we confirmed that
of Th1 and Th2 responses. Resistant C57BL/6 T lymphocytes pro- macrophages from Th1-like strains are more easily activated to
duce IFN-␥ that activates macrophages to produce NO and kill the produce NO than macrophages from Th2-like strains. More im-
parasite, while susceptible BALB/c T lymphocytes instead pro- portantly, however, we also discovered that in response to certain
duce more IL-4 that suppresses macrophages (13, 14). In addition, stimuli (LPS), Th2 macrophages not only do not produce NO, but
IL-4 and other Th2 cytokines have been reported to increase mac- instead increase arginine metabolism to ornithine; Th1 macro-
rophage arginine metabolism via argininase, which produces or- phages do not. Because NO inhibits cell replication (7, 28, 29),
nithine and urea (15). T lymphocytes of other strains of mice have while ornithine (as a precursor of polyamines) can stimulate rep-
also been reported to have a tendency to produce Th1 (B10D2) or lication, these results suggested that macrophages from Th1 and
Th2 (DBA/2) cytokine profiles (16). Th2 mice can influence immune reactions in opposite ways. The
Whereas the aforementioned results clearly indicate that T lym- results to follow provide evidence to support this postulate and will
phocytes from different strains of mice have a tendency to produce show that macrophages from Th1 and Th2 strains differentially
influence whether Th1, Th2, or other immune response occurs. We
propose that these different macrophage responses be termed M-1
Department of Surgery, University of Minnesota, Minneapolis, MN 55455 and M-2.
Received for publication January 3, 2000. Accepted for publication March 30, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Animals
This study was supported by National Institutes of Health Grants R01DK51495
and 5P01GM50150. C57BL/6, B10D2, DBA/2, BALB/c, B6D2F1, CB6F1, C57BL/6J-
2
Address correspondence and reprint requests to Dr. Charles D. Mills, Department of Hfh11nu, BALB/cByJ-Hfh11nu, C57BL/6J-Prkdcscid/SzJ, and BALB/cByJ-
Surgery, University of Minnesota Hospital and Clinic, Box 120, 420 Delaware St. Prkdcscid/J female mice were purchased from The Jackson Laboratory (Bar
S.E., Minneapolis, MN 55455. E-mail address: mills002@tc.umn.edu Harbor, ME). Mice used for experiments were between 9 and 14 wk old.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

The Journal of Immunology 6167

They were routinely tested for common murine pathogens by the Univer-
sity of Minnesota Research Animal Resources (Minneapolis, MN). Mice
were euthanized with carbon dioxide.

Spleen cell culture

Spleens from five mice were pooled and cultured for 3 days in the presence
of 5 ␮g/ml Con A, as previously described (30). The medium employed is
described below. Supernatants were collected and assayed for IFN-␥
and IL-4.

Resident peritoneal macrophage culture

Resident peritoneal macrophages (PEC)3 were cultured as in preceding
studies (30, 31). Briefly, PEC from five or more mice were harvested by
three injections of 5 ml PBS containing antibiotics. PEC were resuspended
in RPMI 1640 supplemented with antibiotics, 1 ⫻ 10⫺2 M morpholinopro-
pane sulfonic acid (buffer), 5 ⫻ 10⫺5 M 2-ME, BSA (2.5 mg/ml), trans-
ferrin (10 ␮g/ml), and insulin (1 ␮U/ml). Tissue culture reagents were
purchased from Life Technologies (Gaithersburg, MD) or Sigma (St.
Louis, MO). All experiments were performed in this serum-free medium
(SFM), except for the spleen cell culture in Fig. 1. In this experiment, the
basal medium was supplemented with 10% FBS (HyClone, Logan, UT), as FIGURE 1. Spleen cytokine profiles of the Th1- and Th2-type mice
in preceding studies (30). All medium components contained less than used in this study. Spleen cells (3 ⫻ 105) were cultured in 5 ␮g/ml Con A
0.015 ng endotoxin/ml. Cells were plated at 3 ⫻ 106 or 1 ⫻ 106/ml in 0.1 for 3 days, and supernatants were collected for IFN-␥ or IL-4 measure-
ml in quadruplicate flat-bottom microtiter wells. Adherent and nonadherent ment. Other concentrations of Con A gave the same pattern.
cells were separated by repeated washing after a 2-h incubation at 37°C in
an atmosphere of 7% CO2. PEC were then cultured for 48 h before super-
natant collection to measure NO, cytokine production, or amino acid me- were added per well (⬃50 beads/cell). A test plate (37°C) and a control
tabolism. IFN-␥ was purchased from Life Technologies. LPS 055:B5 was plate (4°C) were incubated for 2 h. At that time, the wells were washed
purchased from Difco (Detroit, MI). Unless indicated, macrophages were three times each with 0.2 ml of PBS to remove beads not actively phago-
cultured with 500 pg/ml IFN-␥ (10 U/ml) and/or 1 ng/ml LPS. Any ex- cytosed. A final 0.1 ml of PBS was added and the plate was read at 480 nm
periment comparing strains of mice was head to head, in that it was con- on a Perkin-Elmer Luminescence Spectrometer (Perkin-Elmer, Bea-
ducted on the same day with the same medium, reagents, etc. consfield, Buckinghamshire, U.K.). Fluorescence in replicate control wells
Nitrite assay was subtracted from the test wells to calculate the phagocytic index.

NO production by PEC was measured in supernatants collected after 48 h Macrophage/lymphocyte coculture

of culture, as described previously (32). Briefly, 0.05 ml of Griess reagent
PEC from C57BL/6 or BALB/c SCID mice were collected, irradiated
(prepared with reagents from Sigma) was added to 0.05 ml of supernatant,
(1000 rad), and then cultured at 1 ⫻ 105/well in 0.2 ml. After 2 days, 0.1
and absorbance was read at 550 nm using an automated plate reader. Nitrite
ml was removed and 3 ⫻ 105 (C57BL/6 ⫻ BALB/c)F1 spleen cells de-
concentration was calculated from a NaNO2 standard curve.
pleted of macrophages by adherence were added in 0.05 ml to the macro-
IFN-␥, IL-4, and TGF-␤1 determination phage cultures. Con A (0.06 ␮g/ml) was added in 0.05 ml. After 2 days, 1
␮Ci [3H]thymidine (6.7 Ci/mmol) was added in 0.025 ml. Cells were har-
Cytokines in cell culture supernatants were measured using commercially vested 16 h later, and [3H]thymidine uptake was determined.
available kits from Genzyme (Cambridge, MA) (Figs. 1 and 3) or R&D
Systems (Minneapolis, MN) (Fig. 6). The two companies’ kits behaved Data presentation
similarly. We switched companies by necessity; R&D Systems purchased
Data reported are means ⫾ 1 SD from a representative experiment. The
Genzyme’s ELISA kit division. The sensitivity limits for the assays were
error bars represent the average ⫾ SD of quadruplicate microtiter wells. If
5 pg/ml (IFN-␥ and IL-4) and 7 pg/ml (TGF-␤1). Latent plus active TGF-␤
the SD was smaller than the width of the bar, it is not shown. All of the
was measured. Experimental values were determined from standard curves
experiments reported in this work were repeated two to three times with the
calculated in Excel. The SFM employed contained undetectable quantities
same pattern of results. Student’s unpaired t test was used to analyze sta-
of these cytokines.
tistical significance. Inverse correlation statistics (Pearson’s correlation co-
Amino acid analysis efficient (␳) and p value) were obtained from a simple regression analysis
(exponential model) using GB-Stat PPC 5.5.
Amino acids were measured as in previous publications (31). Briefly, sam-
ples for amino acid analysis were deproteinized with a solution containing Results and Discussion
sulfosalicylic (Seraprep; Pickering Labs, Mountain View, CA) and 250
␮M norleucine internal standard. Denatured protein is removed by centrif- Th1/Th2 profiles of selected inbred mice
ugation at 14,000 ⫻ g for 5 min. The pH of the supernatant was adjusted Before undertaking a detailed comparison of macrophages from
to 2.2 by the addition of a lithium-based buffer solution. Amino acids were
measured using a Dionex BioLC Amino Acid Analyzer System (Dionex,
selected Th1- and Th2-type mouse strains, it was considered im-
Sunnyvale, CA) with lithium eluents. Samples with amino acid concentra- portant to first characterize their lymphocyte cytokine propensities.
tions greater then 5 ␮M can be measured with this procedure. Variation It can be seen first in Fig. 1 that Con A stimulation of spleen cells
between replicate amino acid runs routinely averaged less than 5%. from C57BL/6 mice results in a Th1 response characterized by
Anti-TGF-␤1 Ab high IFN-␥/low IL-4 production, while BALB/c spleen cells ex-
hibit a Th2 response with low IFN-␥/high IL-4 production. B10D2
Polyclonal neutralizing Ab against TGF-␤1 (catalog AB-101-NA) and con- spleen cells also exhibit the Th1 profile of high IFN-␥/low IL-4
trol chicken Ig were purchased from R&D Systems and were used at a
concentration of 50 ␮g/ml. production. DBA/2 spleen cells exhibit something of an interme-
diate phenotype, producing a significant quantity of both IFN-␥
Macrophage phagocytosis and IL-4. B10D2 and DBA/2 were chosen for analysis because
After 48 h of culture, 0.05 ml of Fluoresbrite Plain YG 2 ␮m microspheres they share major histocompatibility type H-2d with BALB/c mice
(Polysciences, Warrington, PA) at a concentration of 3 ⫻ 108 beads/ml and they are two other strains that have been reported to exhibit
Th1 and Th2 T lymphocyte tendencies, respectively (16). Because
3
Abbreviations used in this paper: PEC, resident peritoneal macrophages; iNOS, the polyclonal activator Con A was used as the stimulant, the re-
inducible NO synthase; SFM, serum-free medium. sults also demonstrate that the tendencies of mice to produce
6168 M-1/M-2 MACROPHAGES AND Th1/Th2 RESPONSES

provision of polyamines promotes healing (38, 39). Also, ornithine

has been reported to be a proline precursor in healing wounds (40).
Thus, the pathway macrophages utilize to metabolize arginine
could alter the outcome of inflammation in opposite ways. There-
fore, it was also of interest to compare not only the iNOS, but also
the arginase pathway in Th1/Th2 macrophages. Results in Fig. 2
show that macrophages from Th1 and Th2 mice can activate op-
posing pathways of arginine metabolism in response to the same
stimuli. Specifically, the results in Fig. 2 demonstrate that in this
experiment, Th2 macrophages not only do not produce NO in re-
sponse to LPS, but instead increase their production of ornithine/
urea. Specifically, there is a 91% increase for DBA/2 and a 103%
increase for BALB/c in the extracellular ornithine concentration.
In contrast, Th1 macrophages, if anything, slightly decreased or-
nithine production in response to LPS. Thus, these results directly
demonstrate that under the same circumstances, Th1 and Th2 mac-
rophages can be activated to express qualitatively different meta-
bolic programs, which could affect inflammatory outcomes in op-
FIGURE 2. Th1 macrophages preferentially produce NO from arginine; posite ways. In vivo evidence of such differential changes in
Th2 macrophages produce ornithine. PEC from Th1 or Th2 strains of mice macrophage arginine metabolism includes the findings that mac-
were cultured for 48 h, and supernatants were measured for NO2⫺ and rophages up-regulate NO production during tumor rejection, and
amino acids. RPMI 1640 contains 1100 ␮M arginine. Stimuli, 500 pg/ml
up-regulate ornithine production during progressive tumor growth
IFN-␥ and/or 1 ng/ml LPS.
(41). From the results in this section, we propose that macrophages
predominately expressing the iNOS or arginase pathway be termed
M-1 or M-2, respectively.
IFN-␥ or IL-4 are Ag independent. These results provide a back- Another difference observed between the way that M-1 or M-2
ground for the macrophage studies by defining the T lymphocyte macrophages respond is that LPS strongly synergizes with IFN-␥
cytokine profiles of the strains of mice to be used. in DBA/2 and BALB/c macrophages, but much less so with
C57BL/6 or B10D2. How can these results be explained in terms
Differential arginine metabolism by Th1 and Th2 macrophages of the two-signal concept of macrophage activation (42, 43)? Part
Results such as those in Fig. 1 suggest that lymphocytes from of the explanation seems to be that some of the original experi-
certain inbred mice have a predilection to produce IFN-␥ and/or ments were performed with BALB/c mice, in which strong syn-
IL-4. However, if differences in cytokines produced by lympho- ergy between IFN-␥ and LPS is observed. Also, most investigators
cytes could wholly explain susceptibility to diseases like Leishma- use medium supplemented with 10% FBS, whereas the experi-
nia major, then macrophages from C57BL/6 and BALB/c should ments reported in this work and in certain other recent reports (44)
be similarly responsive to IFN-␥, the primary Th1 cytokine that have used SFM. SFM was selected because results to follow will
activates macrophages. Fig. 2 shows that this is not the case. It can show that 10% FBS contains enough TGF-␤ to inhibit macrophage
be seen, in agreement with some recent reports (17–21), that PEC activation. In turn, it may only be necessary to use both IFN-␥ and
from two Th1-like strains (C57BL/6 and B10D2) are more easily LPS to optimally stimulate macrophages if there is a significant
activated by IFN-␥ to produce NO than macrophages from the two quantity of TGF-␤ in the medium.
Th2-like strains (BALB/c, DBA/2). The concentration of citrul-
line, the other product of the iNOS pathway, parallels that of NO, Macrophage TGF-␤1 production is inversely proportional to
as would be expected. It can also be seen that C57BL/6 and B10D2 macrophage NO production in M-1 and M-2 strains of mice
macrophages respond more readily to LPS than DBA/2 or BALB/c Results in the preceding section indicate that macrophages from
macrophages, in agreement with a recent report (21). The simplest M-1 and M-2 strains have a propensity to activate the iNOS or
explanation for these data is that Th1 macrophages become acti- arginase pathway, respectively. To probe the molecular basis for
vated and Th2 do not. these differences, TGF-␤1 production by M-1 and M-2 macro-
However, in addition to their role in host defense, macrophages phages was measured. TGF-␤1 was selected for study because it is
are known to perform critical functions in Ag presentation (33, known to be a powerful inhibitor of macrophage NO production
34), tissue repair (35), angiogenesis (36), and other constructive (45, 46). In addition, TGF-␤1 has been reported to increase argi-
processes. One would predict that macrophages performing these nase activity (47). In vivo, the level of TGF-␤ correlates with
functions are not activated in the classical sense, which involves susceptibility to Leishmania (20, 48, 49). Also, inhibition of
destructive processes (3). Evidence to support the concept of mac- TGF-␤1 has been shown to heighten resistance to diseases, in
rophage activation for either defense or nondefense purposes in- which macrophages are the likely effector (50). On the other hand,
cludes observations from this laboratory (37) that during wound NO has been reported to inhibit macrophage TGF-␤ production
healing, macrophages produce little NO/citrulline, but instead me- (44). Thus, although the foregoing results indicate that a chicken or
tabolize arginine primarily to ornithine/urea via arginase. This pat- egg question remains regarding who regulates whom, important
tern of arginine metabolism makes sense for constructive pro- links exist between TGF-␤ and NO. In turn, it was considered
cesses such as wound healing because excess NO would inhibit important to determine whether there is a relationship between NO
cell replication/healing, while ornithine could promote cell repli- and TGF-␤ production in M-1 and M-2 macrophages.
cation/healing because it is a precursor for polyamines and colla- It can be seen first in Fig. 3 that C57BL/6 and B10D2 macro-
gen (7, 28, 29). That ornithine plays a role in healing is supported phage again responded more readily to IFN-␥ by production of
by findings that inhibition of ornithine decarboxylase, the rate- NO. It can be seen that BALB/c macrophages in this experiment
limiting enzyme in polyamine synthesis, inhibits healing, while produced NO in response to LPS. We do observe some day to day
The Journal of Immunology 6169

(200 – 400 pg/ml); no consistent difference between strains has

been observed. However, more importantly, it can be seen that the
quantity of NO produced by M-1 and M-2 macrophages stimulated
with different concentrations of IFN-␥ or LPS is inversely propor-
tional to the quantity of TGF-␤1 produced. That there is a causal
link between NO and TGF-␤1 production is suggested by the find-
ings that in all four strains of mice examined, and with essentially
all of the different stimuli, a highly significant inverse relationship
is observed (␳ ⫽ ⫺0.81; p ⬍ 0.0001). That macrophage TGF-␤ is
physiologically important is suggested by the fact that the quantity
of TGF-␤1 produced (400 pg/ml) is in the range that has been
shown to inhibit NO production (45). Although total (latent plus
active) TGF-␤1 was measured in this study, macrophages are
known to activate latent TGF-␤1, so the values are in the correct
range (51). Together, these results are consistent with the conclu-
sion that M-1- and M-2-dominant responses occur, in part, because
of autocrine macrophage regulation by TGF-␤1.

FIGURE 3. Inverse relationship between macrophage TGF-␤1 produc-

tion and macrophage NO production. PEC from M-1 or M-2 mouse strains Macrophage TGF-␤1 is an endogenous inhibitor of NO
were cultured for 48 h, and TGF-␤1 and NO2⫺ were measured in the production
supernatants.
The results in Fig. 3 suggested that TGF-␤1 produced by macro-
phages plays a role in down-regulating NO production, or vice
variation in responsiveness to LPS, but not IFN-␥. The variability versa. To measure TGF-␤1, it was necessary to use SFM because
occurs despite using medium components from the same lot and 10% FBS that is used by most investigators provides about 750
with very low endotoxin, and using mice of the same age, etc. On pg/ml TGF-␤1, and thus would obscure that produced by macro-
average, however, BALB/c and DBA macrophages both produce phages. Evidence that the quantity of TGF-␤1 contained in me-
significantly less NO than C57BL/6 or B10D2 in response to either dium supplemented with 10% FBS is sufficient to inhibit macro-
IFN-␥ or LPS in agreement with the results of others (17–21). phage NO production is shown in Fig. 4A. Macrophages cultured
Most importantly, BALB/c macrophages again responded in a in a medium containing 10% FBS produce less NO than macro-
qualitatively different manner to LPS than C57BL/6, as evidenced phages cultured in SFM only supplemented with BSA, transferrin,
by increased ornithine production. Specifically, medium ornithine and insulin. Also, addition of FBS to SFM inhibits NO production,
concentrations (␮M) in the experiment in Fig. 3 were: BALB/c, indicating that inhibitory components of FBS rather than stimula-
NONE ⫽ 564; 500 IFN-␥ ⫽ 659; LPS ⫽ 761; IFN-␥ ⫹ LPS ⫽ tory components in SFM are responsible. That the inhibitory com-
394. C57BL/6, NONE ⫽ 320; 500 IFN-␥ ⫽ 239; LPS ⫽ 236; ponent in FBS is TGF-␤1 is further suggested by the finding that
IFN-␥ ⫹ LPS ⫽ 165. the addition of 750 pg/ml TGF-␤1 to SFM (the same concentration
As for the production of TGF-␤ by M-1 and M-2 macrophages, provided by 10% FBS) inhibits NO production. Thus, the use of
it can be seen that both produce a significant quantity of TGF-␤1 SFM, in addition to allowing TGF-␤1 to be measured, also serves

FIGURE 4. Macrophage TGF-␤1 is an en-

dogenous inhibitor of NO production. A,
NO2⫺ production medium (M, RPMI 1640,
MOPS, 2-ME, antibiotics) with 10% FBS or in
SFM or in SFM supplemented with 10% FBS
(CM). B, NO2⫺ production stimulated by anti-
TGF-␤1 Ab. C, D, and E, PEC after 2 days of
culture in SFM (None), IFN-␥, or IFN-␥ plus
TGF-␤1. Resident C57BL/6 ⫻ DBA/2 F1
macrophages were used in these experiments.
6170 M-1/M-2 MACROPHAGES AND Th1/Th2 RESPONSES

to demonstrate that culturing macrophages in standard FBS-con-

taining medium may be misleading because the TGF-␤1 artifi-
cially inhibits macrophage activation.
To directly test the hypothesis that TGF-␤1 is an endogenous
autocrine inhibitor of macrophage NO production, anti-TGF-␤1
Ab (50 ␮g/ml) was added to macrophage cultures and NO pro-
duction was measured. It can be seen first in Fig. 4B that IFN-␥
stimulates significant NO production. However, more importantly,
addition of anti-TGF-␤1 Ab without IFN-␥ also increases NO pro-
duction. That endogenously produced TGF-␤1 inhibits macro-
phage activation is further evidenced by the additional finding that
addition of IFN-␥ and anti-TGF-␤1 results in the highest level of
NO production. Control Ab (nonimmune chicken Ig) does not
stimulate NO production. Also, anti-TGF-␤1 increases NO pro-
duction in C3H/He macrophages, indicating that endotoxin is not
responsible for the activity observed (not shown). Thus, the results
in this section indicate that macrophages produce a quantity of
TGF-␤1 that is sufficient to endogenously down-regulate their ac-
tivation state. FIGURE 5. Macrophages from NUDE or SCID mice on the C57BL/6
The impact of TGF-␤1 on macrophages is not subtle. Fig. 4, C, or BALB/c background exhibit exaggerated expression of the iNOS or
D, and E, shows what PEC look like after 2 days of culture in SFM arginase pathway, respectively. PEC were cultured with 500 pg/ml IFN-␥
alone (C), SFM plus IFN-␥ (D), or SFM plus IFN-␥ and TGF-␤1 and/or 1 ng/ml LPS for 48 h, and NO2⫺ or ornithine was measured.
(E). It can be seen that TGF-␤1 (1 ng/ml) induces a dramatic
change in macrophage morphology, causing them to take on a
fibroblast-like appearance. In addition to their reduced NO pro-
duction, these TGF-␤-induced long slender cells have markedly lymphocytes could have been present in the macrophage-enriched
reduced phagocytic capacity. Specifically, macrophages cultured cultures. To rule out these potentialities, we compared NUDE or
alone or with IFN-␥ had a phagocytic index of 3.5 ⫾ 0.4 and 5.5 ⫾ SCID macrophages with a C57BL/6 or BALB/c background. It can
0.5, respectively, while macrophages cultured in IFN-␥ plus be seen first in Fig. 5 that macrophages from C57BL/6 NUDE
TGF-␤ had an index of only 1.5 ⫾ 0.5 ( p ⫽ 0.0034 and p ⬍ mice spontaneously produce significant quantities of NO. Addition
0.0001, respectively). In this regard, it has been previously re- of IFN-␥, LPS, or both does not further increase NO production.
ported that TGF-␤ did not decrease phagocytic activity (52). The C57BL/6 SCID macrophages spontaneously produce even more
most likely explanation for this discrepancy is that we have ob- NO, and again, stimuli do not further elevate production. Thus,
served that 10% FBS (containing TGF-␤) as used in other studies absence of T/B lymphocytes strongly enhances the M-1 pheno-
itself causes macrophages to assume this fibroblast-like appear- type. Theoretically, NK cells could be involved here. However, if
ance. In turn, addition of exogenous TGF-␤ may not have further they are, the results still indicate that differences between M-1 and
decreased phagocytosis under these conditions. It is not clear at M-2 macrophages are not dependent on Th1/Th2 influence. It can
present what functions these fibroblast-like cells induced by also be seen in Fig. 5 that in contrast to the up-regulation of the
TGF-␤ do perform. One possibility being explored is that they iNOS pathway in C57BL/6 mice, the arginase pathway is prefer-
exhibit an enhanced Ag presentation capability because TGF-␤ has entially expressed in BALB/c NUDE and SCID macrophages. For
been shown to promote the formation of dendritic cells (53). example, BALB/c NUDE and SCID macrophages produce about
As for the molecular basis of how TGF-␤1 so dramatically alters twice the ornithine as age-matched C57BL/6 NUDE macrophages
macrophage morphology, there are suggestions that its effect may measured on the same day. BALB/c SCID macrophages did pro-
result from changes in arginine metabolism. For example, in a duce NO in response to IFN-␥, and produced about the same max-
previous publication, we observed that macrophages cultured in a imum quantity of NO as C57BL/6 SCID macrophages. Although
low arginine environment also exhibited this fibroblast-like mor- an explanation for these results requires more investigation, one
phology (54). Also, there have been repeated suggestions that mac- might speculate that an additional NO-inhibiting cytokine is lost in
rophages in culture eventually transform into fibroblasts (55, 56): SCID as compared with NUDE BALB/c mice. Together, the re-
circumstances again in which the arginine concentration in the sults in this section suggest that the qualitative differences in
medium would have been greatly reduced by the macrophages. C57BL/6 and BALB/c macrophage arginine metabolism are not
Relatedly, TGF-␤1 not only down-regulates the iNOS pathway, dependent on T or B lymphocytes. Indeed, M-1 and M-2 pheno-
but also up-regulates the arginase pathway (47, 57). Thus, perhaps types seem exaggerated in their absence, indicating that T and B
the stimulus for the change in macrophage morphology is low NO lymphocytes play a modulating role.
concentration, either because of a decrease by TGF-␤ or a low
extracellular arginine concentration. M-1 or M-2 macrophages differentially influence lymphocyte
responses
Macrophages from C57BL/6 and BALB/c NUDE or SCID mice Evidence in this communication indicates that macrophages from
display exaggerated M-1 or M-2 phenotypes Th1- and Th2-type mice can respond in qualitatively different
Results in the preceding sections indicated that macrophages from ways to stimuli, and therefore could differentially influence what
Th1 or Th2 mice can respond very differently when confronted type of lymphocyte responses occur. To test this hypothesis, we
with the same stimuli. Although these results suggest that the mac- again took advantage of SCID mice with an M-1 (C57BL/6) or
rophages themselves are different, other explanations were possi- M-2 (BALB/c) background. Macrophages (1 ⫻ 105) from
ble. For example, macrophages could have been bathed in a Th1 or C57BL/6 or BALB/c SCID mice were cultured alone or in the
Th2 atmosphere before culture. Also, a significant number of T presence of IFN-␥ or LPS or both for 2 days. The macrophages
The Journal of Immunology 6171

In addition to affecting lymphocyte proliferation to different de-

grees, C57BL/6 and BALB/c SCID macrophages also influence
whether Th1- or Th2-associated cytokines are produced by lym-
phocytes. Specifically, it can be seen in Fig. 6B that C57BL/6
SCID macrophages precultured with IFN-␥ and LPS cause a
marked increase in IFN-␥ production by CB6F1 lymphocytes.
BALB/c SCID macrophages caused no increase. The vast majority
of the IFN-␥ measured in the supernatants was produced by CB6F1
lymphocytes (T or NK) because only 500 pg/ml IFN-␥ was added
to precultured macrophages, and about 6000 pg/ml was produced
in the secondary cultures. As for a molecular explanation for the
IFN-␥-inducing ability of C57BL/6 SCID macrophages, it has
been reported that C57BL/6 macrophages produce more IL-12
than BALB/c macrophages (58). We have also observed that
C57BL/6 macrophages produce more IFN-␥-inducing IL-12 than
BALB/c macrophages (C. D. Mills, unpublished).
In contrast to IFN-␥, BALB/c SCID macrophages, whether cul-
tured alone or with IFN-␥ and LPS, caused CB6F1 lymphocytes to
increase production of TGF-␤1 more than C57BL/6 SCID macro-
phages (Fig. 6C). Although not strictly a Th2 cytokine, TGF-␤
does favor the production of Th2 responses by inhibiting Th1-
induced macrophage activation. The majority of the TGF-␤ pro-
duced in the cocultures seems to be from CB6F1 lymphocytes (the
amount produced in macrophage cultures without lymphocytes is
shown under the histobars).
The quantity of the Th2 cytokine IL-4 produced by CB6F1 lym-
phocytes, whether cocultured with C57BL/6 or BALB/c SCID
macrophages, was barely detectable (C57BL/6, 25–30 pg/ml;
BALB/c, 10 –20 pg/ml). This is much less IL-4 than is even pro-
FIGURE 6. M-1 and M-2 macrophages differentially affect lymphocyte duced by the low IL-4 parent, C57BL/6, as shown in Fig. 1. In this
responses. Adherent macrophages (1 ⫻ 105) in 0.2 ml from C57BL/6 or regard, offspring of M-1 and M-2 mice ((C57BL/6 ⫻ BALB/c)F1
BALB/c SCID mice were cultured for 2 days, 0.1 ml supernatant was or C57BL/6 ⫻ DBA/2)F1) have intermediate phenotypes (not
removed, and then C57BL/6 ⫻ BALB/c F1 spleen cells and Con A were
shown). Therefore, the reason for low IL-4 does not appear to be
added. After an additional 2 days, 0.1 ml of supernatant was removed for
the mouse strain employed, but rather that the serum-free culture
cytokine analyses and the cultures were pulsed with [3H]thymidine. A,
Proliferation; B, IFN-␥; C, TGF-␤1, numbers under histobars are TGF-␤1 conditions do not support IL-4 production like cultures containing
in macrophage cultures with no CB6F1 lymphocytes added; D, IL-4. 10% FBS, as in Fig. 1. In summary, the results in this section
demonstrate that M-1 or M-2 macrophages when confronted with
the same stimuli: 1) can affect subsequent lymphocyte proliferation
in different ways; 2) influence whether Th1- or Th2-dominant cy-
were irradiated (1000 rad) before culture to prevent any contribu- tokines are produced.
tion to proliferation. Macrophage-depleted spleen cells (3 ⫻ 105) The results in this communication provide evidence that mac-
from (C57BL/6 ⫻ BALB/c)F1 mice (CB6F1) were then added, and rophages play a more important role in orchestrating immune re-
the macrophage/lymphocyte cocultures were stimulated with Con sponses than is currently appreciated. If one views immune re-
A. F1 lymphocytes were employed so they would not respond to sponses in temporal terms, however, it is not really surprising that
parental Ags. After 2 days, supernatants were removed for cyto- macrophages play a pivotal role in determining immunological
kine analyses and the cultures were pulsed with [3H]thymidine to outcomes because they are typically the first cells to receive danger
measure cell proliferation. It can be seen first in Fig. 6A that Con signals (59). Also, T lymphocytes require signals normally pro-
A-induced proliferation of CB6F1 lymphocytes is inhibited to a vided by macrophages or dendritic cells to respond to Ags (27, 33,
greater degree by unstimulated C57BL/6 SCID than BALB/c 34, 60). In this regard, although the results in this communication
SCID macrophages ( p ⫽ 0.03). Specifically, there is a 59% de- are described in terms of M-1 or M-2 macrophage responses, den-
crease in proliferation upon coculture with unstimulated C57BL/6 dritic cells are also likely to be involved in these responses because
SCID macrophages, whereas proliferation with unstimulated macrophages are a precursor for dendritic cells (27). Evidence to
BALB/c SCID macrophages is the same as with CB6F1 lympho- support this postulate includes results discussed earlier that den-
cytes cultured without exogenous macrophages. These results are dritic cells can stimulate Th1 or Th2 cytokine profiles in vivo (26).
consistent with the findings in Fig. 5 showing that unstimulated Perhaps M-1- or M-2-dominant responses evolve into dendritic
C57BL/6 NUDE or SCID macrophages endogenously produce cell responses that stimulate Th1 or Th2 responses, respectively.
NO, and therefore would be expected to express more suppressor At the same time, because M-1 equates with the production of
macrophage activity (30) than BALB/c SCID macrophages. It can destructive molecules such as NO, overexpression of this pathway
also be seen in Fig. 6A that proliferation is strongly inhibited by could inhibit lymphocyte responses through a suppressor macro-
either C57BL/6 or BALB/c SCID macrophages if they have been phage effect (30, 61– 63), as suggested in Fig. 6.
pretreated with IFN-␥ and LPS. These findings are also in keeping Our proposed classification of macrophage propensities into
with results in Fig. 5, which showed that BALB/c macrophages M-1 and M-2, while useful for conceptualizing immune responses,
can produce as much NO as C57BL/6 macrophages when stimu- certainly could be an oversimplification. In particular, it cannot be
lated with both IFN-␥ and LPS. concluded from the present results that M-1 or M-2 are clonally
6172 M-1/M-2 MACROPHAGES AND Th1/Th2 RESPONSES

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