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(Journal of Leukocyte Biology. 2002;71:271-278.)
© 2002 by Society for Leukocyte Biology

Macrophages from IL-12p40-deficient mice have a bias toward the M2 activation profile

Karina R. B. Bastos*, José M. Alvarez*, Cláudio R. F. Marinho*, Luiz V. Rizzo*,{dagger} and Maria Regina D’Império Lima*

* Department of Immunology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil; and
{dagger} Fundação E. J. Zerbini, São Paulo, Brazil

Correspondence: Dr. Maria Regina D’Império Lima, Departamento de Imunologia, ICB, Av. Prof. Lineu Prestes, 1730, Universidade de São Paulo, São Paulo, SP, Brazil, CEP-05508-900. E-mail: relima{at}usp.br


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have provided evidence that macrophages from Th1-prone mouse strains respond with an M1 profile, and macrophages from Th2-prone mouse strains respond with an M2 profile, characterized by the dominant production of NO or TGF-ß1, respectively. We have shown that peritoneal macrophages from IL-12p40 gene knockout mice have a bias toward the M2 profile, spontaneously secreting large amounts of TGF-ß1 and responding to rIFN-{gamma} with weak NO production. Moreover, IL-12p40KO macrophages are more permissive to Trypanosoma cruzi replication than their wild-type littermate cells. Prolonged incubation with rIL-12 fails to reverse the M2 polarization of IL-12p40KO macrophages. However, TGF-ß1 is directly implicated in sustaining the M2 profile because its inhibition increases NO release from IL-12p40KO macrophages. IFN-{gamma} deficiency is apparently not the reason for TGF-ß1 up-regulation, because rIFN-{gamma}KO macrophages produce normal amounts of this cytokine. These findings raise the possibility that IL-12 has a central role in driving macrophage polarization, regulating their intrinsic ability to respond against intracellular parasites.

Key Words: macrophage polarization • TGF-ß1 • nitric oxide • IL-12p40KO • Trypanosoma cruzi


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-12 [1 ] is a heterodimeric glycoprotein cytokine composed of p35 and p40 subunits encoded by genes located in different chromosomes. Coexpression of both genes is required to generate the bioactive molecule (p70) [2 ]. This molecule is produced by dendritic cells [3 ] and peripheral blood monocytes [4 ], among other cell types. Recently, a novel cytokine that shares the p40 subunit with IL-12 has been described. This molecule, termed IL-23, is also released by dendritic cells, binds to the IL-12 receptor (IL-12R) ß1, and exhibits some of the biological activities of IL-12 [5 , 6 ]. The role of IL-12 in the development of cellular and humoral responses in vivo has been demonstrated after administration of recombinant IL-12 (rIL-12) or IL-12 cDNA and in studies on mice genetically deficient in their p35 or p40 subunit. Treatment with rIL-12 or IL-12 cDNA induces and sustains in vivo-generated memory/effector T helper cell type 1 (Th1) cells [7 , 8 ], up-regulates the synthesis of antigen-specific complement-fixing antibodies [9 ], and protects against tumors and infectious diseases [10 11 12 ]. Conversely, the IL-12 gene knock out (KO) mice have inadequate Th1 responses [13 ] and increased susceptibility to infectious diseases in which protection is primarily mediated by interferon-{gamma} (IFN-{gamma}), such as leishmaniasis [14 ] and tuberculosis [15 ]. The ability of IL-12 to direct the differentiation pattern of T cells suggests that this cytokine bridges innate and adaptive immunity, influencing the development of immune responses and, therefore, the degree of susceptibility to infection [16 ].

It is generally accepted that the central role of IL-12 in host defense against many intracellular pathogens arises from its capacity to stimulate IFN-{gamma} secretion by natural killer (NK) and Th1 cells, which in turn activate phagocytes to control parasite growth [17 ]. Nonetheless, in recent years, macrophages have also been recognized to respond to IL-12, raising the possibility that this cytokine can induce macrophage activation through an autocrine pathway [18 19 20 21 22 23 24 25 26 ]. Although minor contamination with T or NK cells has not always been excluded completely, these studies demonstrate unequivocally that macrophages not only express IL-12 ß1 and ß2 receptors but also respond to IL-12 by producing IFN-{gamma}, tumor necrosis factor {alpha} (TNF-{alpha}), and nitric oxide (NO). Besides having these effects on macrophages, IL-12 has been shown to down-regulate the expression of transforming growth factor (TGF)-ß1 mRNA in monocytes and bone marrow-adherent cells [27 ]. Therefore, it is possible that IL-12 influences the macrophage-activation profile directly, driving them to react against foreign stimuli with a prominent IFN-{gamma}, TNF-{alpha}, and NO response or a dominant TGF-ß1 response.

Recent studies comparing NO and TGF-ß1 secretion among different mouse strains have provided evidence that macrophages can be divided into two types, M1 and M2, based on their ability to produce different types of response [28 ]. M1 macrophages from Th1-prone mouse strains (C57BL/6 and B10D2) are more easily activated to produce NO with rIFN-{gamma} or lipopolysaccharides (LPS). In contrast, M2 macrophages from Th2-prone mouse strains (BALB/c and DBA/2) secrete TGF-ß1 preferentially. Moreover, macrophages from the "Th1" mouse strains are more resistant to intracellular parasites than those from "Th2" mouse strains [29 30 31 ]. The mechanism underlying M1/M2 polarization is not known, but T and B lymphocytes do not seem to participate in this process because C57BL/6 and BALB/c severe combined immunodeficiency (SCID) macrophages also exhibit a distinct activation profile [28 ]. Unpublished data from our laboratory have shown that macrophages from IL-12p40-deficient mice secrete little NO in response to malaria infection. Surprisingly, this deficient NO production could not be attributed to the inability of IL-12p40KO-infected mice to set up a Th1 response, because their spleen cells produce considerable amounts of IFN-{gamma}. These data raised the possibility that the absence of IL-12p40 may have favored macrophage polarization toward the M2 profile, a possibility supported by the findings described above concerning the direct effects of IL-12 on macrophages. Most important, the hypothetical role of IL-12 in M1/M2 polarization could explain the polarized macrophage profile observed in Th1/Th2-prone mouse strains, because it has been shown that C57BL/6 macrophages produce more IL-12 than BALB/c macrophages [32 ].

To test this hypothesis, we evaluated the capacity of peritoneal macrophages from IL-12p40KO and C57BL/6 mice to produce NO and TGF-ß1 and to kill intracellular parasites before and after rIFN-{gamma} and/or LPS activation. These molecules were investigated, because their relative production distinguishes M1 from M2. To assess the involvement of IL-12p40 in the macrophage ability to kill intracellular parasites, we evaluated the growth of Trypanosoma cruzi, the etiological agent of Chagas’ disease. The T. cruzi model was chosen based on previous studies showing that IL-12 mediates resistance against these parasites [33 , 34 ]. Moreover, killing of T. cruzi amastigotes is dependent on the ability of macrophages to release NO, which is stimulated by IFN-{gamma} and inhibited by TGF-ß [35 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and parasites
Six- to 8-week-old IL-12p40-/- (IL-12p40KO) [15 ], IFN-{gamma}-/- (IFN-{gamma}KO; Jackson Laboratory, Bar Harbor, ME), C57BL/6 (wild-type), 129/Sv, and BALB/c female mice were bred at the Biotério de Camundongos Isogênicos, ICB/USP (São Paulo, Brazil) under standard pathogen-free conditions. T. cruzi trypomastigotes of the Sylvio-X10/4 strain were purified from a monkey epithelial cell line (LLC-MK2).

Peritoneal cell suspensions
Four to six mice were injected intraperitoneally (i.p.) with 5 ml 3% starch (Sigma Chemical Co., St. Louis, MO). Five days later, cells were obtained by peritoneal lavage with chilled RPMI 1640 (Sigma Chemical Co.).

Adherent-peritoneal cell cultures
Peritoneal cells (106) were incubated for 4 h in RPMI 1640 supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 2-mercaptoethanol (50 µM), L-glutamine (2 mM), sodium pyruvate (1 mM), and 3% heat-inactivated fetal calf serum (FCS). All supplements were purchased from Life Technologies (Rockville, MD). Nonadherent cells were removed by three vigorous washes with medium.

Phenotypic analysis of total and adherent-peritoneal cells
Adherent-peritoneal cells (106) were stained with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or Cy-chrome-labeled monoclonal antibodies (mAb) to CD4 (H129.19), CD8 (53-6.7), CD45R (B220, RA3-6B2), CD11b (Mac-1, M1/70), I-Ab (AF6-120.1), CD16/CD32 (Fc{gamma}R II/III, 2.4G2), CD80 (B7.1, 1G10), CD86 (B7.2, GL-1), CD119 (IFN-{gamma}R {alpha} chain, GR20), and/or Pan NK cells (DX5) from PharMingen (San Diego, CA). Cells were analyzed by flow cytometry using a Facscalibur (Becton Dickinson, Mountain View, CA).

Detection of NO and TGF-ß1 in culture supernatants
Adherent-peritoneal cells were cultured with 10–1000 pg/ml rIFN-{gamma} (PharMingen) and/or 10 µg/ml LPS (Sigma Chemical Co.), and the supernatants were harvested 48 h later. Culture supernatants were assayed for NO by the Griess reaction. Briefly, 50 µl supernatant was incubated with 50 µl Griess reagent for 5 min at room temperature, and NO2 concentration was determined by measuring the optical density at 550 nm in reference to a standard NaNO2 solution. Latent plus bioactive TGF-ß1 was quantified by the TGF-ß1 EmaxTM ImunoAssay system (Promega, Madison, WI). All supernatant samples were activated by acid treatment before TGF-ß1 determination. TGF-ß1 measured in culture medium supplemented with 3% FCS ranged from 450 to 550 pg/ml. The amount of TGF-ß1 produced by adherent cells was determined by subtracting the TGF-ß1 value of the culture medium from that obtained in the supernatant sample.

Killing of intracellular T. cruzi parasites
Peritoneal cells (106) were added to tissue culture chambers (Lab-Tek Chamber Slide, Nunc, Rochester, NY) and incubated for 4 h in supplemented RPMI medium. Adherent cells were further cultured for 48 h with rIFN-{gamma} (100–1000 pg/ml) or medium, infected with T. cruzi at 1:1 ratio for 120 min, and washed six times to remove extracellular parasites. Cultures were then kept for an additional 48 h with rIFN-{gamma} or medium. After this period, supernatants were examined for extracellular parasites, and adherent cells were stained with Giemsa to count intracellular amastigotes.

rIL-12 and anti-TGF-ß1 treatment of adherent-peritoneal cells
Adherent-peritoneal cells (106) were cultured with 2500 pg/ml rIL-12p70 (Bacullo rIL-12 from DNAX). Neutralizing anti-TGF-ß1 and control chicken immunoglobulin (Ig; R & D Systems, Minneapolis, MN) were used at 50 µg/ml. Cells were stimulated with rIFN-{gamma} (1000 pg/ml). Some of the cultures were infected with T. cruzi as described above. Supernatants were harvested 48 h later to determine NO levels.

Statistical analysis
Statistical analysis was performed by unpaired analysis of variance (ANOVA). Differences between two groups were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adherent-peritoneal cells have a macrophage phenotype
Initially, peritoneal-adherent cells from IL-12-deficient and control mice were characterized by flow cytometry. For IL-12p40KO and C57BL/6 mice, this population was found to be homogeneously positive for CD11b (>99%) and negative for T (CD4 and CD8), B (CD45R), and NK (DX5) cell markers ( Fig. 1 A) Class II major histocompatibility complex (MHC; I-Ab), Fc{gamma}R II/III (CD16/CD32), B7.1 (CD80), and B7.2 (CD86) were also expressed uniformly on adherent cells from both groups of mice (Fig. 1B) . Moreover, a similar number of CD11b+CD4-CD8-CD45R- cells were found in these cultures (unpublished results). Microscopy analysis and staining for nonspecific esterase (>98% positive) were consistent with the flow cytometry analysis. These results indicate that the great majority of adherent cells from the peritoneum of IL-12p40KO and C57BL/6 mice has a macrophage phenotype, with undetectable numbers of T and B lymphocytes and NK cells.



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  		Figure 1. Phenotypic analysis of adherent-peritoneal cells (106) from IL-12p40KO and C57BL/6 mice. (A) Adherent cells were harvested and stained for macrophage (CD11b), B (CD45R), T (CD4 and CD8), and NK (Pan NK) cell markers. (B) Expression of Class II MHC (I-Ab), IFN-{gamma}R {alpha} chain, B7.1 (CD80) and B7.2 (CD86), and Fc{gamma}R II/III (CD16/CD32) was determined in total adherent cells from IL-12p40KO (–) and C57BL/6 (—) mice.

 
rIFN-{gamma}-stimulated IL-12p40KO peritoneal macrophages produce little NO compared with C57BL/6 macrophages
To evaluate the possibility that IL-12p40 deficiency influences the macrophage activation profile, we verified the ability of IL-12p40KO and C57BL/6 macrophages to secrete NO, a major IFN-{gamma}-induced effector molecule. Adherent-peritoneal cells from IL-12p40KO and C57BL/6 mice were activated in vitro with different amounts of rIFN-{gamma}, LPS, or both stimuli, and NO levels were determined in the culture supernatants. Peritoneal macrophages from BALB/c mice were included as counter-control because they have an M2 profile [28 ]. Macrophages from 129/Sv mice were also analyzed because 129/Sv genes could eventually contribute to the IL-12p40KO background. Compared with C57BL/6 and 129/Sv cells, IL-12p40KO macrophages secreted little NO in response to exogenous IFN-{gamma} (Fig. 2 A ). The amount of NO released by IL-12p40KO macrophages was similar to that produced by BALB/c macrophages stimulated with the same dose of rIFN-{gamma}. The activation of macrophages with LPS plus rIFN-{gamma} had a synergistic effect on NO production, independent of the macrophage origin (Fig. 2B) . Under these conditions, the amount of NO secreted by IL-12p40KO macrophages was significantly lower than that produced by C57BL/6 cells. Our findings indicate that macrophages from IL-12p40-deficient mice have a lower ability to secrete NO in response to rIFN-{gamma}, even when concomitantly activated by LPS.



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  		Figure 2. NO production by adherent-peritoneal cells (106) from IL-12p40KO, C57BL/6, 129/Sv, and BALB/c mice. (A) Spontaneous and rIFN-{gamma} (250, 500, and 1000 pg/ml)-induced NO production. (B) NO release after stimulation with LPS (10 µg/ml) and/or rIFN-{gamma} (100, and 1000 pg/ml). NO levels were determined in the supernatants of cells cultured for 48 h. Experiments were repeated three times with the same pattern of results. Data represent the means ± SD of the different experiments. *, P < 0.05.

 
IL-12p40KO peritoneal macrophages spontaneously secrete high amounts of TGF-ß1
TGF-ß is a powerful inhibitor of NO secretion by macrophages [36 37 38 ], and its increased production is associated with the M2 functional profile [28 ]. To determine whether TGF-ß1 could antagonize NO production by IL-12p40KO macrophages, we measured the total (bioative plus latent) levels of this cytokine in adherent-peritoneal cell supernatants. IL-12p40KO macrophages spontaneously produced significantly higher amounts of TGF-ß1 compared with C57BL/6 and 129/Sv cells, which reached levels similar to those of BALB/c macrophages (Fig. 3 ). After rIFN-{gamma} stimulation, TGF-ß1 levels remained unchanged, and differences among groups persisted. Regarding TGF-ß1 and NO production, the activation pattern of IL-12p40KO macrophages was similar to the M2 profile of BALB/c macrophages.



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  		Figure 3. Spontaneous and rIFN-{gamma}-induced TGF-ß1 production by peritoneal-adherent cells (106) from IL-12p40KO, C57BL/6, 129/Sv, and BALB/c mice. TGF-ß1 levels were determined in the supernatants of cells cultured for 48 h with or without rIFN-{gamma} (500 pg/ml). Experiments were repeated three times with the same pattern of results. Data represent the means ± SD of a representative experiment. Standard deviations were calculated from triplicate samples. *, P < 0.05.

 
IL-12p40KO peritoneal macrophages are more permissive to T. cruzi replication than C57BL/6 macrophages
NO and TGF-ß play a positive and negative role in the ability of macrophages to kill T. cruzi amastigotes, respectively [35 ]. To test whether the activation profile of IL-12p40KO macrophages results in increased susceptibility to T. cruzi infection, adherent-peritoneal cells stimulated or not with rIFN-{gamma} were infected with trypomastigotes. Parasite replication was monitored by counting intracellular amastigotes and released trypomastigotes. Nonstimulated macrophages from IL-12p40KO mice were permissive to T. cruzi replication (Fig. 4 B ) compared with C57BL/6 macrophages (Fig. 4A) , harboring 2.5 times more amastigotes per cell (Fig. 4C) . When stimulated with rIFN-{gamma}, macrophages of both strains increased their killing capacity (Fig. 4C) . However, higher numbers of live amastigotes were still observed inside IL-12p40KO cells. Moreover, 48 h post-infection, trypomastigotes were found in culture supernatants from IL-12p40KO macrophages stimulated with 100 pg/ml rIFN-{gamma} at a concentration of 1.6 x 105/ ml, whereas no parasites were detected in the corresponding C57BL/6 supernatants (unpublished results). In these experiments, we also evaluated NO production by T. cruzi-infected macrophages (unpublished results). T. cruzi alone did not induce NO production but increased IFN-{gamma}-induced NO production, and persisted of the differences was between IL-12p40KO and C57BL/6 macrophages.



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  		Figure 4. T. cruzi replication in adherent-peritoneal cells (106) from IL-12p40KO and C57BL/6 mice. (A and B) Microphotographs (60x magnification) from nonstimulated C57BL/6- and IL-12p40KO-infected cells, respectively. (C) Amastigote/macrophage ratios in nonstimulated and rIFN-{gamma}-stimulated macrophages. Cells were cultured for 48 h with rIFN-{gamma} (100, 500, and 1000 pg/ml) or medium, infected with T. cruzi at a 1:1 ratio for 120 min, and washed six times to remove extracellular parasites. Experiments were repeated three times with the same pattern of results. Data represent the means ± SD of a representative experiment. Standard deviations were calculated from triplicate samples. *, P < 0.05.

 
The M2 bias of IL-12p40KO peritoneal macrophages cannot be reversed by the addition of rIL-12
Experiments were designed to determine whether a prolonged incubation with rIL-12 could restore the ability of IL-12p40KO peritoneal macrophages to secrete NO. Adherent cells were pretreated with rIL-12 for 1, 3, or 5 days and then incubated for an additional 48 h with rIFN-{gamma} or rIFN-{gamma} plus rIL-12. Cells kept in culture without rIL-12 were used as control (day 0). All cells remained in culture for a total period of 7 days. The dose of rIL-12 used in these experiments (2500 pg/ml rIL-12p70) stimulated a huge production of IFN-{gamma} by spleen cells (unpublished results). We observed that activation of IL-12p40KO macrophages in the presence rIL-12 did not improve their ability to secrete NO (Fig. 5 C , day 0). Moreover, pretreatment with rIL-12 for 1, 3, or even 5 days was unable to rescue IL-12p40KO macrophages for the NO response induced by rIFN-{gamma} (Fig. 5B) or rIFN-{gamma} plus rIL-12 (Fig. 5C) . Unexpectedly, the prolonged rIL-12 treatment resulted in a marked inhibition of rIFN-{gamma}-induced NO response by C57BL/6 macrophages. Another experiment using a rIL-12 dose of 250 pg/ml showed similar results (unpublished results). We also treated T. cruzi-infected IL-12p40KO and C57BL/6 macrophages with rIL-12 (unpublished results). Confirming the previous results with noninfected cells, this treatment failed to improve NO production by IL-12p40KO-infected macrophages.



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  		Figure 5. Effect of rIL-12 treatment on the rIFN-{gamma}-induced NO response of IL-12p40KO and C57BL/6 adherent-peritoneal cells (106). Cells were pretreated with rIL-12 (2500 pg/ml rIL-12p70) for 1, 3, or 5 days and then incubated for an additional 48 h with (A) medium, (B) rIFN-{gamma} (500 pg/ml), or (C) rIFN-{gamma} (500 pg/ml) plus rIL-12 (2500 pg/ml). Cells kept in culture without rIL-12 were used as control (day 0). All cells remained in culture for a total period of 7 days. Experiments were repeated three times with the same pattern of results. Data represent the means ± SD of a representative experiment. Standard deviations were calculated from triplicate samples. *, P < 0.05.

 
TGF-ß1 is responsible for the inhibition of the IFN-{gamma}-induced NO response of IL-12p40KO macrophages
TGF-ß1 and NO were produced inversely by IL-12p40KO and C57BL/6 macrophages, suggesting that these mediators could be regulated reciprocally. Because TGF-ß1 has been shown to suppress IFN-{gamma}-induced NO production by M2 macrophages from Th2 mouse strains [28 ], we evaluated whether this cytokine is implicated directly in the maintenance of the M2 profile of IL-12p40KO peritoneal macrophages. The results showed that in the presence of anti-TGF-ß1 antibodies, the amount of NO released by IFN-{gamma}-activated IL-12p40KO macrophages was significantly greater than that secreted by control Ig-treated cells, whereas only a modest increment occurred in corresponding C57BL/6 cultures (Fig. 6 ). These data indicate that TGF-ß1 suppresses the IFN-{gamma}-induced NO response of IL-12p40KO macrophages.



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  		Figure 6. Effect of anti-TGF-ß1 treatment on the rIFN-{gamma}-induced NO response of adherent-peritoneal cells (106) from IL-12p40KO and C57BL/6 mice. Anti-TGF-ß1 and control chicken Ig were added at 50 µg/ml to macrophages stimulated or not with rIFN-{gamma} (500 pg/ml) for 48 h. Experiments were repeated three times with the same pattern of results. Data represent the means ± SD of a representative experiment. Standard deviations were calculated from triplicate samples. *, P < 0.05.

 
Up-regulation of macrophage TGF-ß1 does not result from a deficient IFN-{gamma} production
The majority of the demonstrated effects of IL-12 on macrophages is IFN-{gamma}-dependent [18 19 20 21 22 23 24 25 26 ]. To evaluate whether IFN-{gamma} is involved in the up-regulation of TGF-ß1, we measured its production by peritoneal macrophages from IFN-{gamma}KO, IL-12p40KO, and C57BL/6 mice stimulated or not with rIFN-{gamma}. As shown in Figure 7 , TGF-ß1 production was not augmented in IFN-{gamma}KO macrophages compared with C57BL/6 macrophages, suggesting that IFN-{gamma} deficiency is not responsible for the increased TGF-ß1 production observed in IL-12p40KO macrophages. These findings are consistent with the fact that IFN-{gamma} could not be detected in the supernatants of nonstimulated macrophages and that in those from rIFN-{gamma}-stimulated cells, the levels were below the rIFN-{gamma} amount added to the cultures (unpublished results).



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  		Figure 7. Spontaneous and rIFN-{gamma}-induced TGF-ß1 production by peritoneal-adherent cells (106) from IFN-{gamma}KO, IL-12p40KO, and C57BL/6 mice. TGF-ß1 levels were determined in the supernatants of cells cultured for 48 h with or without rIFN-{gamma} (500 pg/ml). Experiments were repeated three times with the same pattern of results. Data represent the means ± SD of a representative experiment. Standard deviations were calculated from triplicate samples. *, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that IL-12p40KO peritoneal macrophages have a bias in their activation profile, characterized by high, spontaneous TGF-ß1 secretion, a low IFN-{gamma}-induced NO response, and an intrinsic deficiency in inhibiting intracellular T. cruzi growth. Therefore, mice lacking IL-12p40 have a defective IFN-{gamma} production [13 ] and an inadequate macrophage IFN-{gamma} response. Regarding the secretion of TGF-ß1 and NO, the activation pattern assumed by IL-12p40KO macrophages corresponds to the M2 functional profile developed by macrophages from Th2 mouse strains [28 ]. M2 polarization of IL-12p40KO macrophages cannot be reversed by exogenous administration of rIL-12. Conversely, TGF-ß1 up-regulation appears to be implicated in sustaining their M2 profile. This effect does not seem to result from deficient IFN-{gamma} production but is likely to be a direct consequence of the absence of IL-12p40. These observations raise the possibility that IL-12 can play a dual role in the host defense against intracellular pathogens; i.e., in addition to its involvement in the induction of IFN-{gamma} secretion by NK and Th1 cells [1 , 3 , 10 , 11 ], IL-12 could influence M1/M2 polarization prior to the onset of immune responses, rendering macrophages prone to react with an effector (NO-dominant or M1) or a tolerant (TGF-ß1-dominant or M2) profile. In contrast to the current view, macrophages would not only be influenced by the milieu created during immune responses but also by the cytokines they produce constitutively. In this regard, IL-12 would gain a new function in the immune system by orchestrating the continuous macrophage crosstalk.

The mechanisms involved in IL-12p40-mediated polarization of macrophages or their precursors toward a NO- or TGF-ß1-dominant profile are speculative but exciting. Because IL-12 and IL-23 share IL-12p40, both cytokines are putative candidates to induce this process. The possibility that constitutive levels of IL-12 directly drive M1/M2 polarization is supported by recent data showing that macrophages not only express IL-12R ß1 and ß2 [22 , 26 ] but also respond to this cytokine through an autocrine pathway [18 19 20 21 22 23 24 25 26 ]. Because IL-23 binds to IL-12Rß1 [5 ], this cytokine could also be involved in macrophage differentiation. Based on our findings, IL-12 and/or IL-23 seem to mediate M1/M2 polarization by directly regulating the production of TGF-ß1, which in turn exerts a negative control over the macrophage NO response. Although IL-23 biological activity is still poorly understood, we discussed our data, taking into account current knowledge concerning the direct effects of IL-12 on macrophages.

Supporting the role of IL-12 in down-regulating TGF-ß1 secretion, it has recently been shown that IL-12 suppresses TGF-ß1 mRNA expression in K562, monocytes, and bone marrow cells [27 ]. Therefore, the absence of a negative control exerted by IL-12 on TGF-ß1 synthesis could be a reason why IL-12p40KO peritoneal macrophages constitutively produce increased amounts of TGF-ß1. The lack of IL-12-mediated control of TGF-ß1 production could determine M2 polarization in the early stages of macrophage differentiation, when these cells are more open to regulation [27 ]. The fact that we failed to rescue IL-12p40KO peritoneal macrophages in their ability to release NO by supplying the cultures with rIL-12, even during a prolonged period of time, could be explained by the inhibitory effect exerted by TGF-ß1 that protects more differentiated cells from the regulatory effects of IL-12 [27 ]. In this case, modulation of the TGF-ß1 promoter activity by IL-12 does not occur in the presence of rTGF-ß1, suggesting that in situations where TGF-ß1 is in excess, the effect of TGF-ß1 on autoregulation [39 ] predominates over the effects of other cytokines such as IL-12. Studies with rat bone marrow-derived cells confirm and extend this idea by showing that the functional response of macrophages to cytokines is determined by the first cytokine to which they are exposed [40 ]. These studies showed that pretreatment of macrophages with TGF-ß completely inhibited NO generation induced by IFN-{gamma} plus TNF-{alpha}, but administration of this cytokine a few hours after IFN-{gamma} priming had no effect. The mechanism by which TGF-ß interferes with IL-12-induced activation has been analyzed in T lymphocytes and includes inhibition of the Jak-STAT pathway [41 ] and down-regulation of the IL-12 receptor [42 ]. Based on the above considerations, we postulate that basal levels of IL-12, produced by macrophages themselves or other cell types, may act during the early phase of macrophage differentiation and polarize these cells toward the M1 activation profile. This constitutive IL-12 may play a negative control over the TGF-ß1 promoter, a phenomenon demonstrated for bone marrow-derived cells treated with IL-12 [27 ]. In M2 macrophages, IL-12 may have no effect on rescuing their ability to secrete NO, probably because of the excess TGF-ß1 produced by these cells. Experiments with bone marrow-derived cells are in progress to identify the developmental stages that are targets for this IL-12 regulatory effect and the activation pathway involved in macrophage polarization.

Although IFN-{gamma} was not detected in our experiments, production of small amounts of this cytokine during macrophage differentiation may have influenced their activation profile. However, our data showing that IL-12p40KO macrophages, but not IFN-{gamma}KO macrophages, produce increased amounts of TGF-ß1 confirm the major role of IL-12/IL-23 in this process, ruling out the indirect participation of IFN-{gamma}. Mills and coworkers [28 ] addressed the possibility that M1/M2 polarization is a consequence of the cytokine milieu in which macrophages develop. They analyzed the influence of lymphocytes on M1/M2 polarization by comparing macrophages from SCID mice with a C57BL/6 or BALB/c background. Their results showed that differences among macrophages persist in the absence of lymphocytes, supporting the idea that cytokines produced by Th1 or Th2 cells are not responsible for the distinct profile developed by these macrophages. The possibility that IL-12p40KO macrophage phenotype is governed by a gene originated from the 129/Sv background was also ruled out by the fact that 129/Sv macrophages have an M1 profile. This is in consonance with data showing that 129/Sv mice are able to mount an effective Th1 response against Leishmania major [14 ].

That treatment with anti-TGF-ß1 is able to partially restore the NO response of IL-12p40KO macrophages indicates that continuous production of this cytokine is necessary to maintain NO suppression. These findings support the hypothesis that TGF-ß1 plays an important role in down-regulating the NO released by M2 cells [28 ]. The inefficient production of NO by IL-12p40KO macrophages could be responsible for their inability to kill intracellular T. cruzi parasites. This idea is based on a previous observation showing that TGF-ß inhibits NO-mediated, T. cruzi killing by IFN-{gamma}-activated macrophages [35 ]. Therefore, our data open the possibility that endogenous IL-12/IL-23 levels regulate TGF-ß1 production and consequently the macrophage susceptibility to intracellular parasites. Theoretically, TGF-ß1 could limit NO release in IL-12p40KO macrophages by several mechanisms. TGF-ß1 has been shown to down-regulate the IFN-{gamma} receptor [43 ], suppress the synthesis of cytokines involved in NO production such as TNF-{alpha} [38 ], and directly inhibit iNOS expression [37 ] or up-regulate arginase activity [44 ]. We have not observed down-regulation of IFN-{gamma}R {alpha} chain expression in IL-12p40KO macrophages. The other possibilities are currently under investigation in our laboratory.

Regardless of the mechanism underlying the M1/M2 bias, the idea that constitutive levels of cytokines produced by macrophages influence their own activation profile may represent a missing element in understanding parasite-host relationships and the development of immune diseases. Macrophage polarization may determine the general pattern of innate immune response against foreign stimuli and consequently the feature of adaptive immunity. Therefore, the notion that the M1/M2 balance is governed by constitutive levels of macrophage cytokines could be a reason why individuals maintaining poor contact with microorganisms have increased chances to suffer from allergic diseases. Further studies on IL-12p40-mediated macrophage polarization may clarify its mechanisms, improving our understanding of immune responses and our ability to modify the outcome of infectious and immune diseases.


   ACKNOWLEDGEMENTS
 
This study was supported by grants from FAPESP and CNPq (Brazil). We thank Ulisses Rodrigues da Silva for technical assistance, Dr. Ises A. Abrahamsohn for providing rIL-12, and Dr. Gustavo P. Amarante-Mendes and Dr. Momtchilo Russo for helpful discussion and revision of the manuscript.

Received August 2, 2001; revised October 7, 2001; accepted October 11, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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