Alternative activation of tumor-associated macrophages by IL-4: Priming for protumoral functions

Although macrophages were originally recognized as major immune effector cells, it is now appreciated that they also play many important roles in the maintenance of tissue homeostasis, and are involved in a variety of pathological conditions including cancer. Several studies have demonstrated the contributions of tumor-associated macrophages (TAMs) to tumor initiation, progression and metastasis. However, the detailed mechanisms underlying how TAMs differ molecularly from their normal counterparts and how the conversion to TAMs occurs have only just begun to be understood. TAMs have been proposed to exhibit phenotypes of ‘alternatively activated’ acrophages, though there has been limited evidence directly linking the phenotypes of TAMs to the alternative activation of macrophages. This review will focus on IL-4, the prototypic cytokine that induces the alternative activation of macrophages, and review current knowledge regarding the contributions of IL-4 to the phenotypes of TAMs and its effects on tumorigenesis.Key words: interleukin-4, tumor-associated macrophage, tumor microenvironment, cytokines, cathepsin proteases     

Aerobic glycolysis is a metabolic requirement to maintain the M2-like polarization of tumor-associated macrophages

Macrophages (MO) are versatile cells, assuming distinct functional phenotypes depending on the activating stimulus and the microenvironment. The differential activation of macrophages is supported by profound intracellular metabolic changes, being well accepted that the M1/M(LPS+IFN-γ) phenotype rely on aerobic glycolysis, while M2/M(IL-4) macrophages depend on oxidative metabolism. On the other hand, although tumor-associated macrophages (TAMs) are characterized by their high expression of M2/M(IL-4) markers, is currently unclear whether TAMs present the same oxidative metabolic profile of M2/M(IL-4) cells. Herein, we demonstrate for the first time that despite their high expression of M2/M(IL-4) markers, TAMs show high glycolytic activity, with high lactate secretion similar to the M1/M(LPS+ IFN-γ) phenotype. This activity seems to be essential for the M2 profile of TAMs, since the inhibition of glycolysis, but not the impairment of the oxidative phosphorylation or pentose phosphate pathway, diminished the expression of M2/M(IL-4) markers. These novel data indicate that TAMs, although are usually phenotyped as M2/M(IL-4)-like macrophages, they are metabolically distinct from these cells, being rather similar to M1/M(LPS+IFN-γ) macrophages, depending on the glycolytic metabolism to support their profile and functions.     

Emerging role of tumor-associated macrophages as therapeutic targets in patients with metastatic renal cell carcinoma

Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruited into the renal cell carcinoma (RCC) microenvironment. In response to inflammatory stimuli, macrophages undergo M1 (classical) or M2 (alternative) activation. M1 cells produce high levels of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12, IL-23 and IL-6, while M2 cells produce anti-inflammatory cytokines, such as IL-10, thus contributing to RCC-related immune dysfunction. The presence of extensive TAM infiltration in RCC microenvironment contributes to cancer progression and metastasis by stimulating angiogenesis, tumor growth, and cellular migration and invasion. Moreover, TAMs are involved in epithelial–mesenchymal transition of RCC cancer cells and in the development of tumor resistance to targeted agents. Interestingly, macrophage autophagy seems to play an important role in RCC. Based on this scenario, TAMs represent a promising and effective target for cancer therapy in RCC. Several strategies have been proposed to suppress TAM recruitment, to deplete their number, to switch M2 TAMs into antitumor M1 phenotype and to inhibit TAM-associated molecules. In this review, we summarize current data on the essential role of TAMs in RCC angiogenesis, invasion, impaired anti-tumor immune response and development of drug resistance, thus describing the emerging TAM-centered therapies for RCC patients.     

CDDO-Me Redirects Activation of Breast Tumor Associated Macrophages

Tumor-associated macrophages can account for up to 50% of the tumor mass in breast cancer patients and high TAM density is associated with poor clinical prognosis. Because TAMs enhance tumor growth, development, and metastatic potential, redirection of TAM activation may have significant therapeutic benefit. Our studies in primary human macrophages and murine breast TAMs suggest that the synthetic oleanane triterpenoid CDDO-methyl ester (CDDO-Me) reprograms the activation profile of TAMs from tumor-promoting to tumor-inhibiting. We show that CDDO-Me treatment inhibits expression of IL-10 and VEGF in stimulated human M2 macrophages and TAMs but increases expression of TNF-α and IL-6. Surface expression of CD206 and CD163, which are characteristic of M2 activation, is significantly attenuated by CDDO-Me. In contrast, CDDO-Me up-regulates surface expression of HLA-DR and CD80, which are markers of M1 activation, and importantly potentiates macrophage activation of autologous T cells but inhibits endothelial cell vascularization. These results show for the first time that CDDO-Me redirects activation of M2 macrophages and TAMs from immune-suppressive to immune-stimulatory, and implicate a role for CDDO-Me as an immunotherapeutic in the treatment of breast and potentially other types of cancer.     

Activation state of stromal inflammatory cells in murine metastatic pancreatic adenocarcinoma

The histologic presence of macrophages (tumor-associated macrophages, TAMs) and neutrophils (tumor-associated neutrophils, TANs) has been linked to poor clinical outcomes for solid tumors. The exact mechanism for this association with worsened prognosis is unclear. It has been theorized that TAMs are immunomodulated to an alternatively activated state and promote tumor progression. Similarly, TANs have been shown to promote angiogenesis and tumor detachment. TAMs and TANs were characterized for activation state and production of prometastatic mediators in an immunocompetent murine model of pancreatic adenocarcinoma. Specimens from liver metastases were evaluated by immunofluorescence and immunoblotting. TAMS have upregulated expression of CD206 and CD163 markers of alternative activation, (4.14 ± 0.55-fold and 7.36 ± 1.13-fold over control, respectively, P < 0.001) but do not have increased expression of classically activated macrophage markers CCR2 and CCR5. TAMs also express oncostatin M (OSM). We found that TANs, not TAMs, predominantly produce matrix metalloproteinase-9 (MMP-9) in this metastatic tumor microenvironment, while MMP-2 production is pan-tumoral. Moreover, increased expression of VEGF colocalized with TAMs as opposed to TANs. TAMs and TANs may act as distinct effector cells, with TAMs phenotypically exhibiting alternative activation and releasing OSM and VEGF. TANs are localized at the invasive front of the metastasis, where they colocalize with MMP-9. Improved understanding of these interactions may lead to targeted therapies for pancreas adenocarcinoma.     

Regulation of tumor-associated macrophage (TAM) differentiation by NDRG2 expression in breast cancer cells

Macrophages are a major cellular component of innate immunity and are mainly known to have phagocytic activity. In the tumor microenvironment (TME), they can be differentiated into tumor-associated macrophages (TAMs). As the most abundant immune cells in the TME, TAMs promote tumor progression by enhancing angiogenesis, suppressing T cells and increasing immunosuppressive cytokine production. N-myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor gene, whose expression is down-regulated in various cancers. However, the effect of NDRG2 on the differentiation of macrophages into TAMs in breast cancer remains elusive. In this study, we investigated the effect of NDRG2 expression in breast cancer cells on the differentiation of macrophages into TAMs. Compared to tumor cell-conditioned medium (TCCM) from 4T1-mock cells, TCCM from NDRG2-overexpressing 4T1 mouse breast cancer cells did not significantly change the morphology of RAW 264.7 cells. However, TCCM from 4T1-NDRG2 cells reduced the mRNA levels of TAM-related genes, including MR1, IL-10, ARG1 and iNOS, in RAW 264.7 cells. In addition, TCCM from 4T1-NDRG2 cells reduced the expression of TAM-related surface markers, such as CD206, in peritoneal macrophages (PEM). The mRNA expression of TAM-related genes, including IL-10, YM1, FIZZ1, MR1, ARG1 and iNOS, was also downregulated by TCCM from 4T1-NDRG2 cells. Remarkably, TCCM from 4T1-NDRG2 cells reduced the expression of PD-L1 and Fra-1 as well as the production of GM-CSF, IL-10 and ROS, leading to the attenuation of T cell-inhibitory activity of PEM. These data showed that compared with TCCM from 4T1-mock cells, TCCM from 4T1-NDRG2 cells suppressed the TAM differentiation and activation. Collectively, these results suggest that NDRG2 expression in breast cancer may reduce the differentiation of macrophages into TAMs in the TME.     

Tumor associated macrophages and neutrophils in tumor progression

Tumor‐associated macrophages (TAMs) are a key component of the tumor microenvironment and orchestrate various aspects of cancer. Diversity and plasticity are hallmarks of cells of the monocyte–macrophage lineage. In response to distinct signals macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a spectrum of activation states. Metabolic adaptation is a key component of macrophage plasticity and polarization, instrumental to their function in homeostasis, immunity and inflammation. Generally, TAMs acquire an M2‐like phenotype that plays important roles in many aspects of tumor growth and progression. There is now evidence that also neutrophils can be driven towards distinct phenotypes in response to microenvironmental signals. The identification of mechanisms and molecules associated with macrophage and neutrophil plasticity and polarized activation provides a basis for new diagnostic and therapeutic strategies. J. Cell. Physiol. 228: 1404–1412, 2013. © 2012 Wiley Periodicals, Inc.     

Decoy receptor 3 enhances tumor progression via induction of tumor-associated macrophages

Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues. Decoy receptor 3 (DcR3), a member of the TNFR superfamily, is overexpressed in tumor cells and is capable of modulating host immunity as either a neutralizing decoy receptor or an effector molecule. Upregulation of DcR3 has been observed to correlate with a poor prognosis in various cancers. However, the mechanisms underlying the DcR3-mediated tumor-promoting effect remain unclear. We previously demonstrated that DcR3 modulates macrophage activation toward an M2-like phenotype in vitro and that DcR3 downregulates MHC class II expression in TAMs via epigenetic control. To investigate whether DcR3 promotes tumor growth, CT26-DcR3 stable transfectants were established. Compared with the vector control clone, DcR3-transfectants grew faster and resulted in TAM infiltration. We further generated CD68 promoter-driven DcR3 transgenic (Tg) mice to investigate tumor growth in vivo. Compared with wild-type mice, macrophages isolated from DcR3-Tg mice displayed higher levels of IL-10, IL-1ra, Ym1, and arginase activity, whereas the expression of IL-12, TNF-α, IL-6, NO, and MHC class II was downregulated. Significantly enhanced tumor growth and spreading were observed in DcR3-Tg mice, and the enhanced tumor growth was abolished by arginase inhibitor N-ω-hydroxy-l-norarginine and histone deacetylase inhibitor sodium valproate. These results indicated that induction of TAMs is an important mechanism for DcR3-mediated tumor progression. Our findings also suggest that targeting DcR3 might help in the development of novel treatment strategies for tumors with high DcR3 expression.     

In Vitro Generation of Monocyte-Derived Macrophages under Serum-Free Conditions Improves Their Tumor Promoting Functions

The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs), reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM) in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy.     

Hydrazinocurcumin Encapsuled Nanoparticles “Re-Educate” Tumor-Associated Macrophages and Exhibit Anti-Tumor Effects on Breast Cancer Following STAT3 Suppression

Tumor-associated macrophages (TAMs) are essential cellular components within tumor microenvironment (TME). TAMs are educated by TME to transform to M2 polarized population, showing a M2-like phenotype, IL-10^high, IL-12^low, TGF-β^high. STAT3 signaling triggers crosstalk between tumor cells and TAMs, and is crucial for the regulation of malignant progression. In our study, legumain-targeting liposomal nanoparticles (NPs) encapsulating HC were employed to suppress STAT3 activity and “re-educate” TAMs, and to investigate the effects of suppression of tumor progression in vivo. The results showed that TAMs treated by HC encapsuled NPs could switch to M1-like phenotype, IL-10^low, IL-12^high, TGF-β^low, and the “re-educated” macrophages (M1-like macrophages) considerably demonstrated opposite effect of M2-like macrophages, especially the induction of 4T1 cells migration and invasion in vitro, and suppression of tumor growth, angiogenesis and metastasis in vivo. These data indicated that inhibition of STAT3 activity of TAMs by HC-NPs was able to reverse their phenotype and could regulate their crosstalk between tumor cells and TAMs in order to suppress tumor progression.     

Regulation of alternative macrophage activation by galectin-3

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-gamma/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-beta-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.     

IL-8 secreted by tumor associated macrophages contribute to lapatinib resistance in HER2-positive locally advanced breast cancer via activation of Src/STAT3/ERK1/2-mediated EGFR signaling

Locally advanced breast cancer (LABC) is an aggressive disease characterized by late clinical presentation, large tumor size, treatment resistance and low survival rate. Expression of EGFR/HER2 and activation of intracellular tyrosine kinase domains in LABC are associated with poor prognosis. Thus, target therapies such as the anti-receptor tyrosine kinases lapatinib drug have been more developed in the past decade. The response to lapatinib involves the inhibition of RTKs and subsequently signaling molecules such as Src/STAT3/Erk1/2 known also to be activated by the cytokines in the tumor microenvironment (TME). The aim of the present study is to identify the major cytokine that might contribute to lapatinib resistance in EGFR+/HER2+ LABC patients. Indeed, tumor associated macrophages (TAMs) are the main source of cytokines in the TME. Herein, we isolated TAMs from LABC during modified radical mastectomy (MRM). Cytokine profile of TAMs revealed that IL-8 is the most prominent highly secreted cytokine by TAMs of LABC patients. Using in-vitro cell culture model we showed that recombinant IL-8 (50 and 100 ng/mL) at different time intervals interfere with lapatinib action via activation of Src/EGFR and signaling molecules known to be inhibited during treatment. We proposed that to improve LABC patients' response to lapatinib treatment it is preferred to use combined therapy that neutralize or block the action of IL-8.     

Proangiogenic TIE2+/CD31+ macrophages are the predominant population of tumor-associated macrophages infiltrating metastatic lymph nodes

Tumor-associated macrophages (TAMs) accumulate in various cancers and promote tumor angiogenesis and metastasis, and thus may be ideal targets for the clinical diagnosis of tumor metastasis with high specificity. However, there are few specific markers to distinguish between TAMs and normal or inflammatory macrophages. Here, we show that TAMs localize in green fluorescent protein-labeled tumors of metastatic lymph nodes (MLNs) from B16F1 melanoma cells but not in necrotic tumor regions, suggesting that TAMs may promote the growth of tumor cells and the progression of tumor metastasis. Furthermore, we isolated pure populations of TAMs from MLNs and characterized their gene expression signatures compared to peritoneal macrophages (PMs), and found that TAMs significantly overexpress immunosuppressive cytokines such as IL-4, IL-10, and TGF-β as well as proangiogenic factors such as VEGF, TIE2, and CD31. Notably, immunological analysis revealed that TIE2⁺/CD31⁺ macrophages constitute the predominant population of TAMs that infiltrate MLNs, distinct from tissue or inflammatory macrophages. Importantly, these TIE2⁺/CD31⁺ macrophages also heavily infiltrated MLNs from human breast cancer biopsies but not reactive hyperplastic LNs. Thus, TIE2⁺/CD31⁺ macrophages may be a unique histopathological biomarker for detecting metastasis in clinical diagnosis, and a novel and promising target for TAM-specific cancer therapy.     

Role of the ubiquitin–proteasome system on macrophages in the tumor microenvironment

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment, and their different polarization states play multiple roles in tumors by secreting cytokines, chemokines, and so on, which are closely related to tumor development. In addition, the enrichment of TAMs is often associated with poor prognosis of tumors. Thus, targeting TAMs is a potential tumor treatment strategy, in which therapeutic approaches such as reducing TAMs numbers, remodeling TAMs phenotypes, and altering their functions are being extensively investigated. Meanwhile, the ubiquitin–proteasome system (UPS), an important mechanism of protein hydrolysis in eukaryotic cells, participates in cellular processes by regulating the activity and stability of key proteins. Interestingly, UPS plays a dual role in the process of tumor development, and its role in TAMs deserve to be investigated in depth. This review builds on this foundation to further explore the multiple roles of UPS on TAMs and identifies a promising approach to treat tumors by targeting TAMs with UPS.     

Sphingosine 1-phosphate induced anti-atherogenic and atheroprotective M2 macrophage polarization through IL-4

Sphingosine 1-phosphate (S1P) has been implicated in anti-atherogenic properties of high-density lipoproteins. However, the roles and signaling of S1P in macrophages, the main contributor to atherosclerosis, have not been well studied. Furthermore, pro-inflammatory M1 and anti-inflammatory M2 macrophage phenotypes may influence the development of atherosclerosis. Therefore, we investigated the effects of S1P on macrophage phenotypes, especially on M2 polarization and its signaling in relation to the anti-atherogenic properties of S1P. It was found that S1P induced anti-inflammatory M2 polarization via IL-4 secretion and its signaling, and induced IL-4Rα and IL-2Rγ. In addition, down-stream signalings, such as, stat-6 phosphorylation, SOCS1 induction, and SOCS3 suppression were also observed in macrophages in response to S1P. Furthermore, S1P-induced ERK activation, and the inhibitions of p38 MAPK and JNK were found to be key signals for IL-4 induction. Moreover, the anti-atherogenic effect of S1P in HDL was confirmed by the observation that oxidized LDL-induced lipid accumulation was attenuated in S1P-treated M2 macrophages. Furthermore, the atheroprotective effect of S1P was demonstrated by its anti-apoptotic effect on S1P-treated macrophages. The present study shows that S1P-induced M2 polarization of macrophages could be mediated via IL-4 signaling, and suggests that M2 polarization by S1P is responsible for the anti-atherogenic and atheroprotective properties of high-density lipoproteins in vivo.     

Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences

Recent studies have described the development of distinct functional subsets of macrophages in association with cancer, autoimmune disease, and chronic infections. Based on the ability of Th1 vs Th2 cytokines to promote opposing activities in macrophages, it has been proposed that macrophages develop into either type 1 inflammatory or type 2 anti-inflammatory subsets. As an alternative to the concept of subset development, we propose that macrophages, in response to changes in their tissue environment, can reversibly and progressively change the pattern of functions that they express. As demonstrated herein, macrophages can reversibly shift their functional phenotype through a multitude of patterns in response to changes in cytokine environment. Macrophages display distinct functional patterns after treatment with IFN-gamma, IL-12, IL-4, or IL-10 and additional functional patterns are displayed depending on whether the cytokine is present alone or with other cytokines and whether the cytokines are added before or concomitantly with the activating stimulus (LPS). Sequential treatment of macrophages with multiple cytokines results in a progression through multiple functional phenotypes. This ability to adapt to changing cytokine environments has significant in vivo relevance, as evidenced by the demonstration that macrophage functional phenotypes established in vivo in aged or tumor-bearing mice can be altered by changing their microenvironment. A concept of functional adaptivity is proposed that has important implications for therapeutic targeting of macrophages in chronic diseases that result in the dominance of particular functional phenotypes of macrophages that play a significant role in disease pathology.     

IL-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo

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.     

Correlation between PD-1/PD-L1 expression and polarization in tumor-associated macrophages: A key player in tumor immunotherapy

Tumor immunotherapy, such as PD-1/PD-L1 blockade, has shown promising clinical efficacy in patients with various types of tumors. However, the response to PD-1/PD-L1 blockade in a majority of malignancies is limited, indicating an urgent need for a deeper understanding of the mechanisms of PD-1/PD-L1 axis-mediated tumor tolerance. As the most abundant immune cells in the tumor stroma, macrophages display multiple phenotypes and functions in response to the stimuli of the tumor microenvironment. PD-1/PD-L1 has been demonstrated to be highly expressed in tumor-associated macrophages (TAMs), and TAM polarization has been shown to be important during tumor progression. In this review, we outline the relationship between TAM PD-1/PD-L1 expression and polarizations, summarize the involvement of M2 TAMs in PD-1/PD-L1-mediated T-cell exhaustion, and discuss improved approaches for overcoming PD-1/PD-L1 blockade resistance by inducing M2/M1 switching of TAMs.     

IL-10 induces gene expression in macrophages: partial overlap with IL-5 but not with IL-4 induced genes

The hypothesis that IL-10, in addition to down-regulating pro-inflammatory activities of macrophages, induces an alternative state of macrophage reactivity was tested. We therefore conducted a systematic search for genes induced by IL-10 using the method of suppression subtractive hybridisation. Of an initial 1,300 candidate clones obtained, several screening rounds led to the identification of 51 clones which were reproducibly at least twofold up-regulated in mouse J774 macrophages in response to treatment with IL-10. Of these, 41 genes were homologous to known genes involved in cell metabolism or immunoregulation, five contained novel sequences and another five were homologous to ESTs without known function. One major finding was that about 25% of the IL-10 genes were also found expressed in response to IFNgamma, and several of these also reappeared in IL-4 or IL-5 induced mRNA species. Hence, Th1 and Th2 type cytokines may elicit a common basal activation response in macrophages. The second major finding was that 57% of IL-10 induced genes reappeared in IL-5 induced mRNA but no more than 18% were also found in IL-4 induced mRNA of J774 cells. We conclude that the gene expression response to IL-10 in macrophages is partially different from the response to IL-5 and is substantially different from the response to IL-4, which suggests an unexpected diversity of biological phenotypes induced by different Th2 type cytokines.