sign in sign up
<<
Home , Dendritic cell, Macrophage

ANTICANCER RESEARCH 32: 2257-2262 (2012)

Short Review Reconsideration of and Classification

TAKESHI KADOWAKI1, MISATO SHIMADA1, HIROYUKI INAGAWA1,2,3, CHIE KOHCHI1,2, MITSUOMI HIRASHIMA1 and GEN-ICHIRO SOMA1,3,4

1Faculty of Medicine, Kagawa University, Kagawa, Japan; 2Macrophi Inc., Hayashi-cho, Takamatsu-shi, Kagawa-ken, Japan; 3Institute for Drug Delivery Systems, Tokyo University of Science, Chiba, Japan; 4Institute for Health and Sciences, Tokushima Bunri University, Nishihama, Tokushima, Japan

Abstract. It is well known that the activation of innate are dynamic and heterogeneous cells that immune cells, especially -presenting cells such as exhibit different roles through their production and macrophages and dendritic cells, can ameliorate or presentation of microbial components. Currently, exacerbate various diseases, including cancer. Currently, the and macrophages are divided into several groups: myeloid- macrophages and dendritic cells are categorized into several derived suppressor cells (MDSCs), M1 macrophages groups by their cell surface and intracellular molecules. (classically activated), M2 macrophages (alternatively However, the detailed classification of the differences activated) and (Mreg) (1, 2). They between macrophages and dendritic cells has still not been have also been classified on the basis of cell surface markers, established. Here, we summarized and reviewed the previous cytokine production, expression of transcription factors and studies on the classification of macrophages and dendritic their effects on other cells (1-3). cells. In addition, the previous classification of monocytes, It is well known that the differentiation of tumour- macrophages and dendritic cells is discussed based on our associated macrophages (TAMs) and MDSCs are associated findings of macrophage activation, which has both with derived from tumour cells such as macrophage conventional and plasmacytoid dendritic cell phenotype. colony stimulating factor (M-CSF), macrophage- colony stimulating factor (GM-CSF) and vascular endothelial Categorization of and (VEGF). These proteins can induce the Macrophage Subsets expansion and accumulation of TAMs and MDSCs in tumour lesions and may also accelerate tumour growth by Macrophages play crucial roles as sentinels in first-line alerts immunosuppressive activities. Some groups have suggested against and as mediators of host defence. In that these characteristics of TAMs should categorize them as addition, they assist in maintaining through M2 macrophages or Mregs (2, 4-6). MDSCs, which express immune regulation, by eliminating foreign substances and the myeloid lineage markers CD11b and Granulocyte- unwanted apoptotic cells. Although macrophages are found differentiation antigen-1 (Gr-1) on their surfaces and include in virtually all tissues, these cells display remarkable granulocyte and monocyte lineage cells, are a heterogeneous plasticity that allow them to efficiently respond to various population of cells that expand during cancer, environmental signals and change their phenotype. and infection, and that have a remarkable ability to suppress T-cell responses (7-9). Unfortunately, the differences between TAMs and MDSCs are not clear because the detailed definition of what Correspondence to: Gen-Ichiro Soma, Institute for Health Sciences, comprises a TAM is still obscure. Tokushima Bunri University, Nishihama, Yamashiro-cho, Usually, macrophages that are differentiated in vitro due Tokushimashi, 770-8514, Japan. Tel: +81 886028103, Fax: +81 886028103, e-mail: [email protected] to the actions of M-CSF or GM-CSF are designated as either M2 macrophages or M1 macrophages, respectively (10, 11). Key Words: Macrophage, dendritic cell, marker, classification, pDC, In fact, we confirmed that the basal expression levels of review. (IL)-10 and arginase I mRNA in macrophages

0250-7005/2012 $2.00+.40 2257 ANTICANCER RESEARCH 32: 2257-2262 (2012)

Figure 1. Basal , inducible synthase (iNOS) and arginase (arg I) mRNA expression in macrophages differentiated with macrophage-colony stimulating factor (M-CSF, I) and granulocyte macrophage-colony stimulating factor (GM-CSF, I). marrow derived mononuclear cells were cultured in the presence of M-CSF and GM-CSF for seven days according to previous reports (11, 13) with slight modifications. Total RNA of the cells was extracted and reverse transcripted using a commercial kit. The obtained cDNA was used as template for real-time quantitative PCR. The expression levels of each RNA were normalized to that of β2 microglobulin. Results are means±SEM in tripricate experiments. For statistical comparison, Student’s t-test was performed. *p<0.05, NS: not significant, IL: interleukin, TNF: tumour factor.

that were differentiated with M-CSF were higher than in Categorization of Dendritic Cell Subsets macrophages that were differentiated with GM-CSF in vitro (Figure 1). By comparison, the basal expression level of Dendritic cells (DCs) are well known antigen presenting inflammatory cytokines mRNA such as IL-1β and IL-12, and cells that express MHC class II and CD86. Currently, DCs inducible nitric oxide synthase (iNOS), in macrophages are divided into two main groups: myeloid DCs (mDCs, also differentiated with GM-CSF were higher than those in known as DC-1 or classical DCs cDCs) and plasmacytoid macrophages differentiated with M-CSF. Thus, these results DCs (DC-2, pDCs). mDCs express the cell surface markers suggest that it is reasonable to apply the categorizations of CD11c and do not express B220. In addition, mDCs are also M1 and M2 macrophages in this case. divided into several groups: lymphoid organ resident DCs, When focusing on their cell surface molecule expression, interstitial DCs and monocyte-derived DCs. macrophages that were differentiated with either GM-CSF or Immature mDCs are usually differentiated with GM-CSF M-CSF expressed CD11b and F4/80, which are used as and IL-4 in vitro (14), and these immature DCs are known macrophage markers except for intestinal macrophages (12) to mature after stimulation with agents such as tumor (Figure 2A). In addition, major histocompatibility complex necrosis factor-α (TNF-α), LPS, and other immune class II (MHC class II, I-A/I-E) expression on macrophages stimulators (15, 16). The confirmation of DCs is usually differentiated with GM-CSF was higher than on based on the type of inflammatory cytokines production, macrophages differentiated with M-CSF (Figure 2B). Based such as IL-12, and the expression of cell surface markers, on previous studies, those macrophages differentiated with such as CD80, CD86 and MHC class II molecules. As GM-CSF and M-CSF undoubtedly could be defined as mentioned above, macrophages can also be differentiated macrophages, whereas the macrophages differentiated with with GM-CSF (i.e. categorized as M1 macrophages). In M-CSF expressed Ly-6C, which belongs to Gr-1. In addition, addition, in our experiments, macrophages differentiated Ly-6C expression was up-regulated by with GM-CSF expressed CD11b, F4/80, CD11c and MHC (LPS) and co-culture with tumour cells (13). If M2 class II molecules on their surfaces (Figure 2A and B and macrophages belong to a category that is different from Figure 3). Therefore, it is possible that macrophages MDSCs, then M2 macrophages, such as TAMs, can possibly differentiated with GM-CSF and immature DCs (or DCs) change their phenotype to MDSCs under certain have partly similar characteristics. However, differences physiological conditions. This suggests that the differences between macrophages differentiated with GM-CSF and DCs between TAMs and MDSCs cannot be defined based on their have not been reported previously. This has led us to surface marker expressions. hypothesize that immature DCs (or DCs) are one type of

2258 Kadowaki et al: Classification of Macrophage and Dendritic Cell (Review)

were also differentiated with a high concentration of GM- CSF (17). Therefore, it is possible that macrophages differentiated with GM-CSF or immature DCs are easily changed to an MDSC phenotype. It is well known that the surface marker profile that defines pDCs is B220+ CD11c+ or intermediate, Ly-6C+ and plasmacytoid dendritic cell antigen 1+ (PDCA-1+). Immature pDCs also express toll like receptor (TLR)7 or TLR9 and produce type I (IFNs), such as IFN-α and IFN- β, via myeloid differentiation factor 88 (MyD88) and regulatory factor (IRF) 7 signalling cascade (18, 19). The functional roles of type I IFNs are the activation of CD8+ T- and natural killer (NK) cells and the induction of Th1 response (20-22). In contrast, mature pDCs reduce production of type I IFNs, which also leads to immune tolerogenic activity, such as inducing the production of IL- 10 by CD8+ T-cells and Tregs, or inducing Th2 responses (23). Therefore, pDCs play crucial roles linking the innate and adaptive arms of the . Recently, many investigators have reported the frequency of pDCs in various diseases. Although pDCs classically belong to the lymphoid lineage, they are currently defined as a subset of DCs derived from both myeloid and lymphoid precursors (24). We also confirmed the frequency of pDCs in tumour-bearing mice. CD11c+ PDCA-1+ cells infiltrated into Figure 2. analyses of macrophages differentiated with the and were also detected in tissue macrophage-colony stimulating factor (M-CSF) and granulocyte after tumour inoculation (13, 25). However, these cells in the macrophage-colony stimulating factor (GM-CSF). cells peritoneal cavity also expressed F4/80 and Ly-6C on their were differentiated into macrophages in the presence of M-CSF (20 surfaces, suggesting the possibility that macrophages had ng/ml) or GM-CSF (20 ng/ml) for seven days. After seven days, non- changed and had begun to display a pDC phenotype in this adherent cells were vigorously removed by washing and adherent cells were used. Adherent cells were collected and washed with phosphate physiological state. As we expected, a previous study had buffered saline (PBS) containing 2% fetal bovine serum (FBS), and reported that the pDC markers of macrophages differentiated incubated with anti-CD16/32 monoclonal to reduce with M-CSF were up-regulated by LPS stimulation, and co- nonspecific binding. Cells were then stained with the appropriate culture with tumour cells (13). Based on these recent studies fluorochrome-labeled monoclonal and analyzed by flow and our findings, there is a possibility that the pDCs cytometry using a Becton Dickinson FACSCalibur. A: The macrophages were stained with anti-F4/80 (clone BM8) and previously categorized in vivo and in vitro were contaminated anti-CD11b (clone M1/70) monoclonal antibody. B: The macrophages with macrophages, and this may have confused evaluation of were stained with anti-F4/80 monoclonal antibody and anti-I-E/I-E pDC functions in various physiological states. monoclonal antibody (clone M5/114.15.2). Acquired data were analyzed with FlowJo software (Tree Star, Ashland, OR). Studies of Macrophage and DC Classification in Re-considerations of Immune Conditions macrophage that arises from macrophage activation. In Immune system inflammation is thought to be triggered by addition, macrophages differentiated with GM-CSF appear effector cells, such as macrophages, , mast cells to be separated into CD11b+ CD11cintermediate and CD11b+ and other cell types involved in innate (NK, NKT CD11chigh cells (Figure 3). In the case of macrophage and γδT cells). It is well known that the activation of these differentiated with M-CSF, CD11b+ CD11cintermediate and individual cell types leads to individual inflammation. For CD11b+ CD11chigh cells were differrent in cytokine gene example, activated macrophages produce IL-12, which is a expression, cell surface marker and respond for various known , leading to Th1 polarization. stimuli (13), suggesting that the macrophage differentiated In addition, activation of mast cells leads to Th2 polarization, with M-CSF and GM-CSF were functionally separated into and activation of neutrophils leads to Th17 polarization. If two major subsets at least. It has been reported that MDSCs these categorizations were matched completely to our

2259 ANTICANCER RESEARCH 32: 2257-2262 (2012)

Figure 3. Flow cytometry analysis of macrophages differentiated with macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage- colony stimulating factor (GM-CSF). The detailed methods are given in legend of Figure 2. The macrophages were stained with anti-CD11b monoclonal antibody and CD11c (clone N418) monoclonal antibody.

understanding of inflammatory conditions, then in the future immunity, they can promote tumour progression by inducing it will be reported that many types of inflammation are the formation of abnormal vessels (26). Based on these adaptable to a number of effector cells. findings, it can be said that macrophages in the tumour With regard to macrophage plasticity, heterogeneity and environment not only have suppressive effects, but are also activation states, there are innumerable types of immune known to induce hyper-immune responses. Therefore, it is cells in acquired immunity based on previous definitions. obviously difficult to functionally define whether there are Although we can easily analyse the cell types and functions hypo-immune or hyper-immune conditions because the of molecules including transcription factors using flow immune responses differ for each cell type. Thus, it is cytometry and knock-out mice, the results often appear to be considered that the hypo-immune condition is not sufficient interpreted under uncertain physiological conditions (i.e. in to elicit appropriate inflammation systemically. vitro experiments or non-disease conditions). This problem Based on our definition, , viral infections and cancer possibly makes it quite confusing and difficult to understand can be categorized as hypo-immune conditions, whereas the fundamental significance of their activation. LPS-mediated inflammation (acute injury), , Nowadays, we believe that the current definition of hypo- autoimmune diseases, helminth infections and metabolic immune diseases is not sufficient to elicit appropriate syndromes as hyper-immune conditions. Generally, sepsis inflammation systemically in host immunity. For example, appears to be categorized as a hyper-immune disease. cancer is well known as a hypo-immune disease. Many However, we believe that the article by Ogiku et al. should investigators have reported that CD4+ CD25+ FOXP3+ cells be carefully re-evaluated. The mortality of IL-17-deficient (so-called regulatory T- cells; Tregs) are induced in various mice in their study was higher than that of wild-type mice tumour-bearing mice models, leading to bidirectional after cecal ligation puncture – a model used for sepsis (27). suppressive effects against cytotoxic T-cells, NK cells, In conclusion, it is essential to define the categorization macrophages, DCs and other cell types. Focusing on of disease based on systemic action and carefully re-evaluate monocytes and macrophages involved in metastasis, MDSCs the importance of functional analyses of innate immune and TAMs are known to accumulate in tumour lesions. cells, particularly macrophages, in their regulation of Although MDSCs and TAMs also suppress antitumour homeostatic inflammation and diseases.

2260 Kadowaki et al: Classification of Macrophage and Dendritic Cell (Review)

References 16 Dai SY, Nakagawa R, Itoh A, Murakami H, Kashio Y, Abe H, Katoh S, Kontani K, Kihara M, Zhang SL, Hata T, Nakamura T, 1 Benoit M, Desnues B and Mege JL: in Yamauchi A and Hirashima M: -9 induces maturation of bacterial infections. J Immunol 181: 3733-3739, 2008. human monocyte-derived dendritic cells. J Immunol 175: 2974- 2 Mosser DM and Edwards JP: Exploring the full spectrum of 2981, 2005. macrophage activation. Nat Rev Immunol 8: 958-969, 2008. 17 Ribechini E, Greifenberg V, Sandwick S and Lutz MB: Subsets, 3 Manrique SZ, Correa MA, Hoelzinger DB, Dominguez AL, Mirza expansion and activation of myeloid-derived suppressor cells. N, Lin HH, Stein-Streilein J, Gordon S and Lustgarten J: Foxp3- Med Microbiol Immunol 199: 273-281, 2010. positive macrophages display immunosuppressive properties and 18 Cao W and Liu YJ: Opn: key regulator of pDC interferon promote tumor growth. J Exp Med 208: 1485-1499, 2011. production. Nat Immunol 7: 441-443, 2006. 4 Solinas G, Germano G, Mantovani A and Allavena P: Tumor- 19 Kawai T, Sato S, Ishii KJ, Coban C, Hemmi H, Yamamoto M, associated macrophages (TAM) as major players of the cancer- Terai K, Matsuda M, Inoue J, Uematsu S, Takeuchi O and Akira related inflammation. J Leuk Biol 86: 1065-1073, 2009. S: Interferon-alpha induction through Toll-like receptors involves 5 Mantovani A, Sozzani S, Locati M, Allavena P and Sica A: a direct interaction of IRF7 with MyD88 and TRAF6. Nat Macrophage polarization: tumor-associated macrophages as a Immunol 5: 1061-1068, 2004. paradigm for polarized M2 mononuclear . Trends 20 Kolumam GA, Thomas S, Thompson LJ, Sprent J and Murali- Immunol 23: 549-555, 2002. Krishna K: Type I interferons act directly on CD8 T cells to 6 Kurahara H, Shinchi H, Mataki Y, Maemura K, Noma H, Kubo allow clonal expansion and memory formation in response to F, Sakoda M, Ueno S, Natsugoe S and Takao S: Significance of viral infection. J Exp Med 202: 637-650, 2005. M2-polarized tumor-associated macrophage in . 21 Wenzel J, Worenkamper E, Freutel S, Henze S, Haller O, Bieber J Surgi Res 167: e211-219, 2011. T and Tuting T: Enhanced type I interferon signalling promotes 7 Nagaraj S, Schrum AG, Cho HI, Celis E and Gabrilovich DI: Th1-biased inflammation in cutaneous erythematosus. J Mechanism of T-cell tolerance induced by myeloid-derived Pathol 205: 435-442, 2005. suppressor cells. J Immunol 184: 3106-3116, 2010. 22 Zhu J, Huang X and Yang Y: A critical role for type I IFN- 8 Kusmartsev S, Nagaraj S and Gabrilovich DI: Tumor-associated dependent NK cell activation in innate immune elimination of CD8+ T-cell tolerance induced by bone marrow-derived adenoviral vectors in vivo. Mol Ther 16: 1300-1307, 2008. immature myeloid cells. J Immunol 175: 4583-4592, 2005. 23 Matta BM, Castellaneta A and Thomson AW: Tolerogenic 9 Gabrilovich DI, Velders MP, Sotomayor EM and Kast WM: plasmacytoid DC. Eur J Immunol 40: 2667-2676, 2010. Mechanism of immune dysfunction in cancer mediated by 24 Naik SH, Corcoran LM and Wu L: Development of murine immature Gr-1+ myeloid cells. J Immunol 166: 5398-5406, plasmacytoid dendritic cell subsets. Immunol Cell Biol 83: 563- 2001. 570, 2005. 10 Fleetwood AJ, Dinh H, Cook AD, Hertzog PJ and Hamilton JA: 25 Nobumoto A, Oomizu S, Arikawa T, Katoh S, Nagahara K, GM-CSF- and M-CSF-dependent macrophage phenotypes Miyake M, Nishi N, Takeshita K, Niki T, Yamauchi A and display differential dependence on type I interferon signaling. J Hirashima M: Galectin-9 expands unique macrophages Leuk Biol 86: 411-421, 2009. exhibiting plasmacytoid dendritic cell-like phenotypes that 11 Kuroda E, Ho V, Ruschmann J, Antignano F, Hamilton M, Rauh activate NK cells in tumor-bearing mice. Clin Immunol 130: MJ, Antov A, Flavell RA, Sly LM and Krystal G: SHIP 322-330, 2009. represses the generation of IL-3-induced M2 macrophages by 26 Sasajima J, Mizukami Y, Sugiyama Y, Nakamura K, Kawamoto inhibiting IL-4 production from . J Immunol 183: T, Koizumi K, Fujii R, Motomura W, Sato K, Suzuki Y, Tanno S, 3652-3660, 2009. Fujiya M, Sasaki K, Shimizu N, Karasaki H, Kono T, Kawabe 12 Gordon S, Hamann J, Lin HH and Stacey M: F4/80 and the J, Ii M, Yoshiara H, Kamiyama N, Ashida T, Bardeesy N, Chung related -GPCRs. Eur J Immunol 41: 2472-2476, 2011. DC and Kohgo Y: Transplanting normal vascular proangiogenic 13 Kadowaki T, Arikawa T, Shinonaga R, Oomizu S, Inagawa H, cells to tumor-bearing mice triggers vascular remodeling and Soma G, Niki T and Hirashima M: Galectin-9 signaling prolongs reduces in tumors. Cancer Res 70: 6283-6292, 2010. survival in murine lung-cancer by inducing macrophages to 27 Ogiku M, Kono H, Hara M, Tsuchiya M and Fujii H: differentiate into plasmacytoid dendritic cell-like macrophages. Interleukin-17A plays a pivotal role in polymicrobial sepsis Clin Immunol 142: 296-307, 2012. according to studies using IL-17A knockout mice. J Surgi Res 14 Bender A, Sapp M, Schuler G, Steinman RM and Bhardwaj N: 174: 281-291, 2010. Improved methods for the generation of dendritic cells from nonproliferating progenitors in human . J Immunol Meth 196: 121-135, 1996. 15 Landi A, Babiuk LA and van Drunen Littel-van den Hurk S: Dendritic cells matured by a E2-containing cocktail can produce high levels of IL-12p70 and are more Received April 4, 2012 mature and Th1-biased than dendritic cells treated with TNF-α Revised May 18, 2012 or LPS. Immunobiol 216: 649-662, 2011. Accepted May 18, 2012

2261