CSF-1−Dependent Red Pulp Macrophages Regulate CD4 T Cell Responses Daisuke Kurotaki, Shigeyuki Kon, Kyeonghwa Bae, Koyu Ito, Yutaka Matsui, Yosuke Nakayama, Masashi Kanayama, This information is current as Chiemi Kimura, Yoshinori Narita, Takashi Nishimura, of September 29, 2021. Kazuya Iwabuchi, Matthias Mack, Nico van Rooijen, Shimon Sakaguchi, Toshimitsu Uede and Junko Morimoto J Immunol published online 14 January 2011 http://www.jimmunol.org/content/early/2011/01/14/jimmun Downloaded from ol.1001345 Supplementary http://www.jimmunol.org/content/suppl/2011/01/14/jimmunol.100134 Material 5.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published January 14, 2011, doi:10.4049/jimmunol.1001345 The Journal of Immunology CSF-1–Dependent Red Pulp Macrophages Regulate CD4 T Cell Responses Daisuke Kurotaki,*,† Shigeyuki Kon,† Kyeonghwa Bae,† Koyu Ito,† Yutaka Matsui,* Yosuke Nakayama,† Masashi Kanayama,† Chiemi Kimura,† Yoshinori Narita,‡ Takashi Nishimura,‡ Kazuya Iwabuchi,x Matthias Mack,{ Nico van Rooijen,‖ Shimon Sakaguchi,# Toshimitsu Uede,*,† and Junko Morimoto† The balance between immune activation and suppression must be regulated to maintain immune homeostasis. Tissue macro- phages (MFs) constitute the major cellular subsets of APCs within the body; however, how and what types of resident MFs are involved in the regulation of immune homeostasis in the peripheral lymphoid tissues are poorly understood. Splenic red pulp MF (RPMs) remove self-Ags, such as blood-borne particulates and aged erythrocytes, from the blood. Although many scattered T cells Downloaded from exist in the red pulp of the spleen, little attention has been given to how RPMs prevent harmful T cell immune responses against self-Ags. In this study, we found that murine splenic F4/80hiMac-1low MFs residing in the red pulp showed different expression patterns of surface markers compared with F4/80+Mac-1hi monocytes/MFs. Studies with purified cell populations demonstrated that F4/80hiMac-1low MFs regulated CD4+ T cell responses by producing soluble suppressive factors, including TGF-b and IL-10. Moreover, F4/80hiMac-1low MFs induced the differentiation of naive CD4+ T cells into functional Foxp3+ regulatory T cells. hi low F Additionally, we found that the differentiation of F4/80 Mac-1 M s was critically regulated by CSF-1, and in vitro-generated http://www.jimmunol.org/ bone marrow-derived MFs induced by CSF-1 suppressed CD4+ T cell responses and induced the generation of Foxp3+ regulatory T cells in vivo. These results suggested that splenic CSF-1–dependent F4/80hiMac-1low MFs are a subpopulation of RPMs and regulate peripheral immune homeostasis. The Journal of Immunology, 2011, 186: 000–000. acrophages (MFs) play a critical role in innate and Once the circulating monocytes (Mo) enter various tissue com- acquired immunity and can contribute proinflamma- partments, they exhibit a high degree of heterogeneity and acquire M tory or anti-inflammatory responses (1). Recently, some specific functions (4, 5). Spleen is known to contain various resident MFs populations possessing regulatory function were MF subsets, such as white pulp tingible-body MFs, marginal discovered in the airway interstitium and intestinal lamina propria zone-residing MFs, and red pulp MFs (RPMs) (4–6). RPMs by guest on September 29, 2021 (2, 3), indicating that these suppressive MFs play important roles remove self-Ags, such as blood-borne particulate matter and aged in the maintenance of immune homeostasis. However, in the erythrocytes, from the blood (6). Although T cell responses usu- steady state, the role of resident splenic MFs in T cell immune re- ally occur in the T cell zone of the white pulp, it is well known sponses is poorly understood. that numerous small patches within red pulp parenchyma (making up a total volume comparable to that in the white pulp in humans) contain mainly T cells, B cells, and RPMs (6–8), indicating the possibility of interaction between RPMs and T cells in the red *Division of Matrix Medicine, Institute for Genetic Medicine, Hokkaido University, pulp. Therefore, T cell responses occurring in the red pulp should † Sapporo 060-0815, Japan; Division of Molecular Immunology, Institute for Genetic be strictly regulated, because the majority of Ags are derived from Medicine, Hokkaido University, Sapporo 060-0815, Japan; ‡Division of Immunoreg- ulation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, self. x Japan; Division of Immunobiology, Institute for Genetic Medicine, Hokkaido Uni- CSF-1, also known as M-CSF, is constitutively produced by versity, Sapporo 060-0815, Japan; {Department of Internal Medicine II, University Hospital Regensburg, 93042 Regensburg, Germany; ‖Department of Molecular Cell several types of cells, including fibroblasts, endothelial cells, stro- Biology, Vrije Universiteit Medical Center, 1081 BT Amsterdam, The Netherlands; mal cells, and MFs (9, 10). The major function of CSF-1 is to elicit # and Department of Experimental Pathology, Institute for Frontier Medical Sciences, the differentiation and development of various tissue-resident Kyoto University, Kyoto 606-8507, Japan MFs (9, 10). CSF-1–deficient mice, which are also known as Received for publication April 26, 2010. Accepted for publication December 14, 2010. op/op mutant mice, have a residual MF population (CSF-1– independent MFs). Previous studies showed that op/op mice were This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.U.). capable of inducing normal humoral and cellular immunity, Address correspondence and reprints requests to Dr. Toshimitsu Uede and Dr. Junko but they contained lower levels of TNF-a and G-CSF following Morimoto, Division of Molecular Immunology, Institute for Genetic Medicine, Hok- immunization with sheep RBCs, suggesting that the CSF-1– kaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail ad- independent MF population is primarily responsible for the dresses: [email protected] and [email protected] classical APC population, whereas the CSF-1–dependent MF The online version of this article contains supplemental material. population has the potential to regulate immune response (11, 12). Abbreviations used in this article: clod-lip, clodronate liposomes; FCM, flow cytom- etry; GMDC, bone marrow-derived dendritic cells induced by GM-CSF; IHC, im- Consistent with this notion, a recent report showed that in a model munohistochemistry; MF, macrophage; M-MF, bone marrow-derived macrophages of graft-versus-host disease, mice in which CSF-1–dependent induced by CSF-1; Mo, monocyte; MOG, myelin oligodendrocyte glycoprotein; MFs were depleted using an Ab against CSF-1R had a decreased RPM, red pulp macrophage; SSC, side scatter; Treg, regulatory T cell. number of Foxp3+ regulatory T cells (Tregs), which resulted in Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 accelerated pathology and exaggerated donor T cell activation www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001345 2 REGULATORY RED PULP MFs (13). Although RPMs in op/op mice were reduced to ∼50% of those negative selection system, and the cells were irradiated by x-ray (3000 in the normal littermates, these mice still had F4/80+ RPMs, sug- rad). gesting that RPMs are composed of CSF-1–dependent and -inde- Cell culture pendent populations (14–16). However, the phenotypical and func- 3 6 tional differences between CSF-1–dependent and -independent Isolated MFs were adjusted to 1.5 or 2.0 10 cells/ml in TIL media (IBL), supplemented with 10% FCS and cultured in 96-well flat-bottom RPMs remain largely unknown. dishes at 37˚C at 5% CO for 48 or 72 h. The cultured supernatants were hi low 2 Previous studies showed that splenic F4/80 Mac-1 MFs exist used for ELISA (48 h) and T cell-stimulation experiments (72 h). For TLR in the red pulp of the spleen (3, 5, 17–20). In this study, we first stimulation of MFs, purified F4/80hiMac-1low MFs and F4/80+Mac-1hi hi low 3 6 isolated splenic F4/80 Mac-1 MFs and analyzed how T cell Mo/MFs were adjusted to 1.0 10 cells/ml in TIL media, supplemented m responses are regulated by F4/80hiMac-1low MFs in vivo and with 10% FCS and LPS (1 g/ml; IBL) or CpG-B dinucleotides hi low (ODN1668, 3 mg/ml; system science) and cultured for 48 h. The culture in vitro. F4/80 Mac-1 MFs showed a different expression of supernatant was harvested and used for ELISA. To generate M-MFsor + hi surface molecules compared with splenic F4/80 Mac-1 Mo/MFs, bone marrow-derived dendritic cells (GMDCs), bone marrow cells were which are capable of inducing strong T cell immune responses. isolated from the femurs and tibias of mice and cultured in RPMI 1640 Purified F4/80hiMac-1low MFs produced suppressive cytokines, media, supplemented with 10% FCS, 100 U/ml penicillin, and 100 mg/ml + streptomycin in the presence of 20 ng/ml CSF-1 or GM-CSF (WAKO). such as TGF-b and IL-10, and suppressed CD4 T cell prolifera- After 3 d, the adherent cells were collected and cultured for 2 more days in hi low tion in vitro.