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The Journal of Immunology, 2007, 178: 1357-1362.
Copyright © 2007 by The American Association of Immunologists, Inc.

IL-12 Rapidly Alters the Functional Profile of Tumor-Associated and Tumor-Infiltrating Macrophages In Vitro and In Vivo1

Stephanie K. Watkins*, Nejat K. Egilmez*,{dagger}, Jill Suttles*,{dagger} and Robert D. Stout2,*,{dagger}

* Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY 40292; and {dagger} James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40292


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tumor-associated macrophages (TAMs) play a major role in promoting tumor growth and metastasis and in suppressing the antitumor immune response. Despite the immunosuppressive environment created by the tumor and enforced by tumor-associated macrophages, treatment of tumor-bearing mice with IL-12 induces tumor regression associated with appearance of activated NK cells and activated tumor-specific CTLs. We therefore tested the hypothesis that IL-12 treatment could alter the function of these tumor-associated suppressor macrophages. Analysis of tumor-infiltrating macrophages and distal TAMs revealed that IL-12, both in vivo and in vitro, induced a rapid (<90 min) reduction of tumor supportive macrophage activities (IL-10, MCP-1, migration inhibitory factor, and TGFbeta production) and a concomitant increase in proinflammatory and proimmunogenic activities (TNF-{alpha}, IL-15, and IL-18 production). Similar shifts in functional phenotype were induced by IL-12 in tumor-infiltrating macrophages isolated from the primary tumor mass and in TAMs isolated from lung containing metastases, spleen, and peritoneal cavity. Therefore, although TAMs display a strongly polarized immunosuppressive functional profile, they retain the ability to change their functional profile to proinflammatory activities given the appropriate stimulus. The ability of IL-12 to initiate this functional conversion may contribute to early amplification of the subsequent destructive antitumor immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages are capable of displaying diverse activities, many of which appear to be opposing in nature, such as inflammatory vs anti-inflammatory or destructive vs tissue restorative activities (1, 2, 3). Although the ligation of a variety of receptors, such as CD40 or TLR, will activate expression of macrophage functional activities, the pattern of functional activities being expressed is regulated by biological response modifiers, such as cytokines (1, 2, 3, 4). Thus, LPS activation of macrophages in the presence of IFN-{gamma} will elicit a pattern of functional activities quite distinct from that elicited in the presence of IL-4, TGFbeta, or other response modifiers (2, 4). We have hypothesized that macrophages retain a high degree of functional plasticity throughout these responses in that they can change their functional profile repeatedly through multiple cycles as the response modifiers in their environment sequentially change (2, 4). There are, however, pathological states in which macrophages display what appears to be a stable functional state (5, 6, 7).

An important example of stable regulation of macrophage function is the symbiotic relationship between macrophages and malignant tumors (8). Tumors produce an array of factors and cytokines which modify macrophage behavior (2, 8, 9, 10). Both tumor-infiltrating macrophages (TIM)3 and tumor-associated macrophages (TAM) have been described as suppressor macrophages that display anti-inflammatory and immunosuppressive activities (9, 11, 12, 13). TIM and TAM also display activities, which appear to be required for tumor growth and metastasis, including production of MCP-1, migration inhibitory factor (MIF), and TGFbeta (8, 14). Thus, if TIM and TAM retained functional plasticity, they would potentially be useful targets for tumor therapy regimens because redirecting their functional activities could reduce support for tumor metastasis and reduce suppressive influence of the tumor and TAM on the adaptive immune system.

IL-12 has been shown (15, 16) to stimulate antitumor responses in several models of solid tissue tumors. IL-12 is produced by macrophages and dendritic cells and promotes their inflammatory and proimmunogenic activities (17, 18). IL-12 activates IFN-{gamma} production by T and NK cells, enhances cytotoxicity by NK and NKT cells, and promotes the generation of type 1 CD4+ Th cells and cytotoxic CD8+ T cells (15, 19). Injection of IL-12 encapsulated in polymeric microspheres directly into subcutaneous tumors results in a vigorous NK and cytotoxic T cell response against the tumor and its metastases (16, 20, 21). Evidence suggests that quiescent NK and tumor-infiltrating lymphocytes are activated either directly or indirectly by the IL-12 and that these cells mediate the subsequent immune response (16, 20, 21). Although macrophages have been reported to function as antitumor effectors in the active antitumor immune response 7–10 days after IL-12 treatment (21, 22), no study to date has examined whether the functional profile of TIM and TAM is rapidly altered by IL-12 treatment. Given the documented systemic immunosuppressive influence of TAM, we considered it unlikely that a potent adaptive immune response could be initiated without first or concomitantly reducing the immunosuppressive influence of the tumor environment. Therefore, we investigated whether treatment with IL-12 could convert the suppressor macrophages of tumor-bearing mice into either neutral or inflammatory, proimmunogenic macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6J female mice were obtained from The Jackson Laboratory. All animal protocols received prior approval from the Institutional Animal Care and Use Committee and all experiments were performed in accordance with relevant guidelines and regulations.

Tumor models

Lewis lung carcinoma (3LLC) was obtained from Dr. G. Ross (Brown Cancer Center, Louisville, KY). Tumors were maintained by in vivo passage with limited intermittent culture in vitro. Tumor cells were injected s.c. into the left side of C57BL/6J mice. Tumors were measured at 3-day intervals with calipers at two bisecting diameters and an approximate volume was calculated by the formula (0.4) x (large diameter) x (small diameter)2. Tumors were excised at 0.5–1.0 cm3, diced, and passed through a sieve (Bellco Glass). The cells were washed with Dulbecco’s PBS plus 2% FBS and cultured for two to five 3-day cycles in RPMI 1640 with 5% FBS, HEPES, and gentamicin before repassage in vivo. Tumors with necrosis or with volumes 2-fold higher or lower than the mean of the group of mice were not used as a source of tissue for that experiment.

Macrophages

Macrophages were purified from single-cell suspensions of lung, spleen, peritoneal lavage, and tumor mass of tumor-bearing animals by magnetic bead separation using first a negative selection with anti-CD19 and anti-CD5 and then a positive selection with anti-CD11b (Mac-1; Miltenyi Biotec). The lungs were excised after perfusion with PBS-EDTA and removal of the mediastinal lymph nodes. Dispersed tumor cells were centrifuged over a discontinuous 30%/40%/70% Percoll gradient. Macrophages were purified by magnetic bead separation from the cells banding at the 40%/70% interface. Purity of >95% CD11b+ was confirmed by flow cytometry for peritoneal and splenic macrophages and >80% for lung and tumor macrophages; >90% of the CD11b+ cells coexpressed F4/80. Macrophages were cultured in RPMI 1640 supplemented with 5% FCS, HEPES, and gentamicin and were activated by addition of 100 ng/ml LPS (Escherichia coli 011:B4, chromatographically pure, low protein; Sigma-Aldrich) where indicated.

In vivo IL-12 treatment

IL-12-containing microspheres were prepared by phase inversion nanoencapsulation (20) and supplied by Dr. N. Egilmez (University of Louisville, Louisville, KY). Microspheres were injected at 200 µg IL-12 (1 mg microspheres)/100 µl in DMEM into the center of the tumor mass. Empty microspheres were used as a negative control.

Antibodies

Cytoplasmic IL-15 was assayed using the permeabilization and wash buffers obtained from BD Pharmingen according to their directions. Detection was achieved using biotinylated goat anti-mouse IgG IL-15 Ab (R&D Systems) and a PE-conjugated streptavidin. Macrophages were identified by surface labeling with allophycocyanin IgG2b anti-mouse CD11b Ab (BD Pharmingen). Appropriate isotype controls were used as well as an unconjugated rabbit anti-mouse IgG IL-15 Ab (Novus Biologicals) to block labeling of cytoplasmic IL-15.

Cytokine bead array (CBA) and IL-15 ELISA

Cytokines were assayed using a BD Biosciences cytokine bead array CBA kit and a BD FACSCalibur according to the manufacturers’ instructions. Sample supernatants were analyzed undiluted and also at a 1/50 dilution. The IL-15 ELISA was performed using rat anti-mouse IgG2a IL-15 Ab (R&D Systems) at 4 µg/ml as the capture Ab in combination with the biotinylated goat anti-mouse IgG IL-15 Ab (R&D Systems) at 400 ng/ml as the detection Ab.

SuperArray and quantitative real-time PCR analyses

For real-time PCR, cDNA was produced from mRNA from 106 cells from independent cell culture experiments (in treated and untreated tumor-bearing and normal mice) by using a µMACS One-Step cDNA kit (Miltenyi Biotec). The cDNA was used for quantitative real-time PCR amplification with SYBR Green using gene-specific primers for MIF (SuperArray), TGFbeta, IL-15, and IL-18 (Maxim). Duplicate cDNA template samples were amplified and analyzed in the MJ Opticon thermal cycler with conditions of 96°C for 15 min followed by 40 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s with a plate read for gene expression between each cycle. A standard curve of cycle thresholds using 10-fold serial dilutions of cDNA samples was established and used to calculate the relative abundance of the target gene of each cytokine and vehicle control samples. Values were normalized to the relative amounts of beta-actin mRNA (BD Biosciences) and GAPDH (Maxim), which were obtained from a similar standard curve. Differences between groups were analyzed using the REST program (Corbett Research) (23).

For GEArray, cells were purified and lysed in TRIzol for a chloroform extraction. Briefly, 200 µl of chloroform was added to cell lysates and centrifuged. The aqueous phase was collected and an equal amount of (25:24:1) phenol:chloroform:isoamyl alcohol was added and centrifuged. The aqueous phase was collected and washed with isopropanol, centrifuged, and washed with 75% ice-cold ethanol. After centrifugation, the pellet was air dried then resuspended in nuclease free water for further purification by the Qiagen MinElute RNeasy cleanup kit according to the manufacturer’s instructions and for quantification by spectrophotometry. The GEarray (SuperArray Q Series kit) was run according to the manufacturer’s instructions using 3 µg of the purified RNA and analyzed using software provided at SuperArray’s web site (http://www.superarray.com).

Statistical analysis

Data are expressed as mean ± SD of three individually assayed mice per group. Statistical comparisons were conducted via the Student’s t test and p values are indicated. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-12 treatment in vitro alters the functional profile of TIM and TAM

To obtain an overview of the differences in cytokine gene expression between normal and TAM, as well as the impact of IL-12 treatment, macrophages were isolated from the peritoneal cavity of normal and tumor-bearing mice and cultured overnight with or without IL-12. GEArray analysis indicated that a number of genes were down-regulated in TAM, most notably IFN-{gamma}, TNF-{alpha}, and several chemokine genes, and that a number of genes were up-regulated in TAM, most notably MIF, IL-10, and CCR2 (Fig. 1A). Overnight treatment with IL-12 appeared to enhance expression of the genes that were down-regulated in TAM and to reduce expression of most of the genes that were up-regulated in TAM (Fig. 1B). Two cytokines (TGFbeta and MIF) found to be significantly up-regulated by the presence of the tumor and two inflammatory cytokines (IL-15 and IL-18) found to be up-regulated by IL-12 treatment of tumor-bearing mice by the superarray (Fig. 1) and RNase Protection analysis (data not shown) were selected for quantitative analysis by real-time RT-PCR. TAM expressed highly elevated (>9-fold) expression of TGFbeta and MIF mRNA but not of IL-15 or IL-18 mRNA compared with normal peritoneal macrophages (Fig. 2). Overnight culture with IL-12 reduced the expression of TGFbeta and MIF genes to background levels and induced a 3-fold increase in expression of IL-18 mRNA and a 20-fold increase in expression of IL-15 mRNA (Fig. 2).


Figure 1
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  		FIGURE 1. Comparison of cytokine gene expression levels in peritoneal macrophages from normal vs tumor-bearing mice. Macrophages were purified from peritoneal lavage of tumor-bearing and normal mice and cultured overnight in the presence or absence of 5 ng/ml IL-12. RNA was purified and analyzed by a SuperArray inflammatory gene array kit. Relative expression of 20 of the genes displaying the greatest differences between normal macrophages (M{phi}s) and TAM or between untreated and IL-12-treated TAM are displayed.

 

Figure 2
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  		FIGURE 2. In vitro treatment of TAM with IL-12 alters profile of cytokine gene expression. Macrophages were purified from peritoneal lavage of tumor-bearing and normal mice and cultured overnight in the presence or absence of 5 ng/ml IL-12. RNA was extracted and analyzed for expression of the indicated cytokine genes by real-time PCR. Data are expressed as 1 pg mRNA/106 cells ± SD of three mice. *, p < 0.001 between normal macrophages and TAM or between untreated TAM and IL-12-treated TAM.

 
To determine the cytokine profile elicited by activation in vitro, the TIM, peritoneal, splenic, or lung macrophages were stimulated with 100 ng/ml LPS immediately ex vivo or after overnight culture with rIL-12. IL-12 treatment reduced or abrogated MCP-1 production and elevated TNF-{alpha} and IL-6 production by TIM, as well as by peritoneal, splenic, and lung TAM (Fig. 3). The IL-12 treatment reduced IL-10 production by splenic and lung TAM (Fig. 3, C and D) but elevated IL-10 production by peritoneal TAM (Fig. 3B). Therefore, for each tissue analyzed, IL-12 treatment altered the profile of their cytokine response to an activating stimulus. The difference in response to IL-12 in the different tissues is apparent and tissue environment likely contributes significantly, as suggested by the difference in response profile to LPS stimulation observed in peritoneal, spleen, and lung macrophages from normal mice (Fig. 3 and Refs. 1, 2, 3 and 25).


Figure 3
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  		FIGURE 3. In vitro culture with IL-12 changes the profile of cytokine production by TIM and TAM in response to LPS stimulation. Macrophages were purified from the primary tumor mass (A) or from peritoneal lavage (B), spleen (C), or lung (D) of tumor-bearing and normal mice. The cell cultures were stimulated with 100 ng/ml LPS immediately or after overnight culture with IL-12. The culture supernatants were collected and analyzed for cytokines by CBA. Data are displayed as mean ± SD of macrophages from three mice. *, p < 0.05; **, p < 0.01. Groups compared are TAM vs normal macrophages (M{phi}) and untreated TIM/TAM vs IL-12-treated TIM/TAM. n.d., No cytokine detected. The results are representative of four individual trials.

 
IL-12 treatment in vivo alters the functional profile of TIM and TAM

To determine whether IL-12 could alter the function of TAM in tumor-bearing mice, mice bearing 3LLC tumors were treated with IL-12-loaded microspheres. Peritoneal macrophages were harvested 90 min to 5 days later and assayed for cytokine production following LPS stimulation. IL-12 treatment induced a dramatic shift in cytokine production in the peritoneal TAM as early as 90 min after treatment (Fig. 4). The elevated production of MCP-1 and IL-10 elicited from peritoneal TAM from placebo-treated tumor-bearing mice was reduced 2- to 3-fold in TAM from IL-12-treated mice and the production of TNF-{alpha} was elevated. This shift in functional profile was sustained for 5 days after IL-12 treatment (Fig. 4).


Figure 4
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  		FIGURE 4. In vivo IL-12 treatment induces a rapid shift in the IL-10:TNF-{alpha} ratio that is sustained for 5 days. Peritoneal macrophages were purified 90 min to 120 h after injection of IL-12 microspheres s.c. or into the primary tumor mass. The cells were plated at 1 x 105/ml and stimulated with 100 ng/ml LPS and the supernatant was collected 17 h later for CBA analysis of cytokine protein secretion. Data are displayed as mean ± SD of three mice. *, p < 0.05; **, p < 0.01. Normal macrophages were compared with TAM or TAM from placebo-treated mice were compared with TAM from IL-12-treated mice. n.d., No cytokine detected. Results are representative of four independent trials.

 
Purified lung and splenic TAM (Fig. 5A) displayed the same shift in functional profile as peritoneal TAM after injection of the IL-12 microspheres. TAM from spleen and lung displayed elevated production of MCP-1 and IL-10 and decreased production of IL-6 relative to normal controls. Three hours after IL-12 treatment, the splenic and lung TAM displayed a 3-fold or greater reduction in MCP-1 and IL-10 production in response to LPS stimulation in vitro and a comparable increase in TNF-{alpha} and IL-6 production (Fig. 5A). Purified TIM from IL-12-treated mice displayed enhanced IL-6 production and dramatically decreased MCP-1 production as compared with placebo (Fig. 5B). Unlike TAM, TIM consistently failed to produce IL-10 in response to LPS stimulation in these studies (Figs. 3A and 5B).


Figure 5
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  		FIGURE 5. In vivo injection of IL-12 induces a rapid shift to proinflammatory functions systemically. A, Lung and splenic macrophages or TIM were purified 3 h after IL-12 injection (B). The cells were plated at 1 x 105/ml, stimulated with 100 ng/ml LPS, and the supernatant was collected 17 h later for CBA analysis of cytokine protein secretion. Data are displayed as mean ± SD of three mice. *, p < 0.05; **, p < 0.01. Normal macrophages were compared with TAM or TAM from placebo-treated mice were compared with TAM from IL-12-treated mice. n.d., No cytokine detected. Results are representative of four independent trials.

 
IL-12 induces release of IL-15 in tumor-bearing mice

Analysis of TAM from spleen, lung, and peritoneal cavity ex vivo for cytokine gene expression corroborated previous reports that genes for MIF and TGFbeta were significantly elevated (Fig. 6). Within 90 min after IL-12 treatment in vivo, the splenic, lung, and peritoneal TAM displayed almost a complete abrogation of expression of MIF and TGFbeta mRNA. This sharp down-regulation of tumor-supportive TGFbeta and MIF gene expression was accompanied by a >10-fold increase in expression of IL-15 and IL-18 mRNA relative to placebo (Fig. 6). Therefore, the same impact of IL-12 on expression of these four cytokine genes by TAM was observed both in vitro (Fig. 2) and in vivo (Fig. 6).


Figure 6
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  		FIGURE 6. IL-12 injection induces an increase in proinflammatory and a decrease in anti-inflammatory gene expression in TAM. Peritoneal, splenic, and lung macrophages were isolated and purified 90 min after IL-12 treatment. The cells were lysed immediately and mRNA was extracted for real-time RT-PCR. Data are expressed as nanograms of mRNA per 106 cells ± SD of thee mice. *, p < 0.05; **, p < 0.01. Normal macrophages were compared with TAM or TAM from placebo-treated mice were compared with TAM from IL-12-treated mice. Results are representative of two independent trials.

 
IL-15 administration to tumor-bearing mice has been reported (24) to rescue anergic/tolerant tumor-specific CTLs. The dramatic IL-12 mediated up-regulation of IL-15 gene expression (Fig. 6) was therefore of particular interest. To determine whether IL-15 protein was being released as a result of IL-12 treatment, a blood sample was obtained from mice 3 h after IL-12 treatment. Analysis revealed that IL-12 treatment induced rapid appearance of IL-15 in the serum of tumor-bearing mice, attaining >6-fold higher concentration than that observed in normal mice treated with IL-12 (Fig. 7A). The contribution of TAM to this IL-15 release was investigated by collecting peritoneal macrophages from the IL-12-treated mice and analyzing for the level of cytoplasmic IL-15. IL-12 treatment of normal mice induced an increase in cytoplasmic IL-15 in a small fraction of the macrophages, whereas IL-12 treatment of tumor-bearing mice resulted in nearly a complete depletion of cytoplasmic IL-15 (Fig. 7B).


Figure 7
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  		FIGURE 7. Macrophages release IL-15 in vivo in response to IL-12 treatment. A, A blood sample was collected from normal or tumor-bearing animals 3 h after IL-12 therapy. IL-15 was assayed by ELISA. Data are displayed as mean ± SD of three mice. p = 0.012 for the comparison of placebo vs IL-12-treated tumor-bearing mice. n.d., No cytokine detected. Results are representative of three independent trials. B, Peritoneal macrophages from normal (left panel) or tumor-bearing (right panel) animals were analyzed for cytoplasmic IL-15 3 h after IL-12 (black line) or placebo (dark gray line) microsphere injection. Specificity was controlled by prelabeling with cold anti-IL-15. Results are representative of two independent trials. C, The MFI of labeling of intracellular IL-15 was determined for each of the groups and the mean ± SD is displayed. The difference between IL-12-treated tumor-bearing mice and either untreated tumor-bearing mice or IL-12-treated normal mice was significant at p < 0.00001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Macrophages possess a large repertoire of diverse functions and are able to display a multitude of different functional patterns by differential and selective expression of those functions (2, 3, 4). The differential expression of these functions in response to environmental biological response modifiers likely contributes to the phenotypic and functional heterogeneity of macrophages within a tissue (e.g., Kupffer cells, pulmonary histiocytes, etc.) (25) or within an inflammatory site (26, 27). There are, however, chronic diseases in which macrophages display what appears to be a stable functional state (5, 6, 7, 13, 28). The development of solid tumors is usually associated with the appearance of anti-inflammatory and immunosuppressive macrophages (13). The TIM have been considered to be a unique macrophage subset whose suppressive function cannot be altered (29, 30). However, splenic TAM have been reported to rapidly lose their suppressive activity upon culture with GM-CSF and all-trans-retinoic acid (13). Given our previous demonstration (4) that macrophages which have been functionally polarized in vitro retained their functional plasticity and could rapidly convert to other functional phenotypes, we hypothesized that TIM and TAM could similarly be converted to nonsuppressive functional phenotypes by appropriate cytokine treatment. In this context, we noted that it seemed unlikely that IL-12 could elicit such a strong antitumor immune response, as has been reported (16, 20, 21), without countering the strong immunosuppressive influence of the TAM and the tumor environment.

TIM and TAM have been reported to produce a variety of cytokines in vivo which support tumor growth, including TGFbeta and MIF (8, 14). In the current study, this was extended to demonstrate that treatment of TAM with IL-12 in vitro or in vivo up-regulated IL-15 and IL-18 gene expression and down-regulated TGFbeta and MIF gene expression. Upon subsequent activation by LPS in vitro, the TAM treated with IL-12 either in vitro or in vivo responded with a more inflammatory profile as evidenced by the dramatic increase in the TNF-{alpha}:IL-10 ratio and release of IL-15. Thus, IL-12 was able to reprogram the functional profile of macrophages that had been chronically polarized in function by the tumor environment. The analysis of the macrophage functional phenotype reported herein establishes three significant conclusions. First, IL-12 treatment could overcome the influence of the tumor environment in vivo, shifting the functional response of TIM and TAM from a predominantly anti-inflammatory profile (elevated TGFbeta, IL-10, MIF, and low-level TNF-{alpha} expression) to a predominantly inflammatory profile (elevated TNF-{alpha}, IL-6, IL-15, IL-18 and diminished TGFbeta, MIF, and IL-10 expression). Second, this shift occurred very rapidly and was sustained for 5 or more days after a single injection of encapsulated IL-12. Third, the shift in macrophage functional response was systemic and was observed in tumor-infiltrating, peritoneal, splenic, and lung macrophages.

The persistence of the shift in functional phenotype of the macrophages may have several bases. The microencapsulation of IL-12 results in prolonged release of the IL-12 into systemic circulation (20). A more dramatic influence may be exerted by NK cells and an adaptive immune response. Treatment of tumor-bearing mice with IL-12 results in the appearance of activated, IFN-{gamma}-producing NK cells by 2–3 days after treatment and the appearance of activated CD8+ CTLs, also producing type 1 inflammatory cytokines, by 5–7 days after treatment (15, 16, 17, 18, 19, 20, 21). By 7 days posttreatment, macrophages have been reported to be actively participating in cytotoxic destruction of the tumor (21, 22). Our previous studies strongly suggest that as the type 1 adaptive immune response subsides, the resulting decline in inflammatory signaling will result in a conversion of the macrophage phenotype to that appropriate for a normal tissue environment (2, 4). The fate of the macrophages that accumulate during the response remains to be determined.

The rapid release of cytoplasmic IL-15 from macrophages and possibly other stromal cells upon IL-12 treatment may play a significant role in the efficacy of IL-12 therapy. IL-15 has been demonstrated to stimulate inflammatory activity in neutrophils and macrophages, to induce and support NK cell and CD8+ Tc activation, to restore cytotoxic activity to anergic tumor-specific CD8+ Tc, and to reverse tumor-induced inhibition of class I MHC processing by dendritic cells (24, 31, 32, 33). Neutralization of IL-15 has been reported to provide effective therapy for a variety of chronic autoimmune inflammatory diseases (34, 35). Thus, it is possible that TIM and/or TAM may play an important role in IL-12 initiation of antitumor cytotoxic responses, both by cessation of their anti-inflammatory suppressive activities (decreased TGFbeta and IL-10 production) and of their support for tumor growth and metastasis (decreased TGFbeta, MIF, and MCP-1 production) and by providing a rapid burst of proinflammatory activities (e.g., IL-15 release) which may contribute to the initiation or reactivation of innate and adaptive immune responses.

By demonstrating that functionally polarized TIM and TAM can be rapidly converted from a tumor-supportive and immunosuppressive functional phenotype to an inflammatory functional phenotype by IL-12 treatment in vivo as well as in vitro, this study offers supportive evidence for the macrophage functional plasticity hypothesis. This hypothesis is a simple restatement of the two signal paradigm of macrophage activation with the added dimension of time and the recognition that a multitude of biological modifiers exist which differentially regulate the macrophage response to activation (2, 4). An activation signal will induce macrophages to display a pattern of activities dictated by the type of biological response modifiers in the microenvironment. If the type of biological response modifiers in the microenvironment changes with time, the functional activities of the macrophages will change in response to those modifiers (4). If the type of biological response modifiers in the microenvironment remains constant, as seems to occur in chronic diseases such as solid tissue cancers or parasite infection (5, 13, 36), the functional phenotype of the macrophages will remain constant. This hypothesis sets the perspective that altering macrophage function via administration of biological response modifiers may have therapeutic benefit for chronic diseases involving pathological polarization of macrophage function.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by National Institutes of Health Grant CA100656 (to N.K.E.), the Commonwealth of Kentucky Lung Cancer Research Program (to R.D.S.), the American Lung Association of Kentucky (to S.K.W.), and the Commonwealth of Kentucky Research Challenge Trust Fund (to N.K.E., J.S., and R.D.S.). Back

2 Address correspondence and reprint requests to Dr. Robert D. Stout, Department of Microbiology and Immunology, University of Louisville, School of Medicine, 319 Abraham Flexner Way, Louisville, KY 40292. E-mail address: bobstout{at}louisville.edu Back

3 Abbreviations used in this paper: TIM, tumor-infiltrating macrophage; TAM, tumor-associated macrophage; MIF, migration inhibitory factor; CBA, cytokine bead array. Back

Received for publication August 12, 2006. Accepted for publication November 11, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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