Advances in Bioscience and Biotechnology, 2013, 4, 19-29 ABB http://dx.doi.org/10.4236/abb.2013.48A3003 Published Online August 2013 (http://www.scirp.org/journal/abb/) Macrophage PD-L1 strikes back: PD-1/PD-L1 interaction drives macrophages toward regulatory subsets Yun-Jung Lee, Young-Hye Moon, Kyeong Eun Hyung, Jong-Sun Yoo, Mi Ji Lee, Ik Hee Lee, Byung Sung Go, Kwang Woo Hwang* Laboratory of Host Defense Modulation, College of Pharmacy, Chung-Ang University, Seoul, Korea Email: *[email protected] Received 21 May 2013; revised 25 June 2013; accepted 19 July 2013 Copyright © 2013 Yun-Jung Lee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Keywords: Regulatory Macrophage; PD-1; PD-L1 Activated macrophages have been simply defined as cells that secrete inflammatory mediators and kill 1. INTRODUCTION intracellular pathogens until few years ago. Recent Macrophages are innate immune cells, and are the first studies have proposed a new classification system to line of defense against invading pathogens [1], and they separate activated macrophages based on their func- are also antigen presenting cells (APCs), participating in tional phenotypes: host defense, wound healing, and adaptive immunity. Macrophages have versatile abilities, immune regulation. Regulatory macrophages can including phagocytosis, antigen presentation, antimicro- arise following innate or adaptive immune responses bial toxicity, and tissue remodeling as well as the secre- and hinder macrophage-mediated host defense and tion of a wide range of growth factors, cytokines, com- inflammatory functions by inhibiting the production plement components, prostaglandins, and enzymes [2]. of pro-inflammatory mediators. In this study, we in- Monocytes developed from hematopoietic cells start to vestigated whether PD-1 and PD-L1 interaction be- circulate blood and enter tissues [3] and depend on the tween macrophages and T cells alters macrophage tissue environment they are capable of realizing different activities. Our data provide evidence for PD-1/PD-L1 physiological functions [4]. The signature role of macro- engagement inducing a regulatory profile in macro- phages in host defense is the production of inflammatory phages. Regulatory macrophages derived from PD- cytokines IL-1β, IL-6, TNF-α and nitric oxide (NO). In L1 signaling lost their host defense activity, which addition to this, macrophages can be activated alterna- consists of the production of pro-inflammatory cyto- tively and exhibit wound repair and immune regulation kine IL-6 and the exhibition of increased IL-10, functions [5]. SPHK1 and LIGHT gene levels in early phases of A recent system of classification was proposed by LPS stimulation. This differentiation seems to occur Mosser et al. in 2008 to separate activated macrophages through excessive activation of TLR4 downstream based on their functional phenotypes: host defense, MAPK signaling pathways. Regulatory macrophages wound healing, and immune regulation [6]. In a similar induced from PD-1/PD-L1 interaction decrease in- review, they also emphasize the importance of under- flammatory mediators and produce anti-inflamma- standing that macrophages exhibit not only the three cur- tory cytokines, so this macrophage subset has been rently recognized states of activation, but a broad spec- under considerable attention as a possible immune trum of activation states [7]. Since macrophages have regulation mechanism. Understanding and modulat- high plasticity [8], they are not restricted to a single ac- ing regulatory macrophages may lead to new appro- tivation state; there may be distinct macrophage states ches to treat or prevent auto-immune diseases such as which exhibit characteristics of two or more activation type I diabetes, rheumatic syndrome and hypersensi- states, termed “hybrid” macrophages [7]. tivity-related diseases, which are concerned with the Activation of antigen-specific T cells is dependent on overproduction of inflammatory cytokines in macro- T cell receptor (TCR) and major-histocompatibility-com- ages. plex (MHC) interactions, while crucial additional costi- *Corresponding author. mulatory signals are also necessary. In the absence of OPEN ACCESS 20 Y.-J. Lee et al. / Advances in Bioscience and Biotechnology 4 (2013) 19-29 costimulatory signals, lymphocytes fail to respond effec- It has been established that costimulatory molecules tively and are rendered anergy [9]. These signals are de- such as B7-1/B7-2 on APCs interacting with CD28/ livered to T cells by costimulatory cell surface mole- CTLA-4 on T cells regulate the T cell activation of tol- cules expressed on APCs. The best characterized positive erance through a unidirectional signal pathway, APCs to costimulatory receptor on T cells is CD28, which binds T cells [21-23]. However, recent studies have shown that B7-1/B7-2 and is mostly expressed on APCs and acti- these signals are not limited to T cell regulation, but also vated T cells. This concurrent engagement during TCR modulate APCs through costimulatory ligands B7-1/B7-2. ligation is necessary for T cell activation and B7-1/B7-2 Bidirectional signaling along the B7-CTLA-4 coreceptor and cytotoxic T lymphocyte attenuator-4 (CTLA-4) in- pathway enables reciprocal conditioning of T cells and teraction is needed for tolerance. Alongside this, Pro- dendritic cells. T cells can instruct dendritic cells to ma- grammed death 1 (PD-1) receptor on T cells and its nifest tolerogenic properties after CTLA-4 engagement ligands PD-L1 and PD-L2 on APCs interactions also to B7. In this manner, IDO expression is up-regulated play an important role in T cell activity regulation [10]. after engagement by CTLA-4 [8]. A separate study re- Programmed death 1 (PD-1) is an immunoreceptor of ported that B7-CD28 engagement induces IL-6, IFN-γ the CD28/CTLA-4 family whose expression is induced production through the p38 MAPK signal pathway in in activated T and B cells and in macrophages [11]. PD-1 DCs [24]. is a 50 - 55 kDa type I transmembrane glycoprotein com- Preceding research performed using PD-1 Tg mice posed of an IgV-type extracellular domain sharing 21% - showed that inhibitory effects of PD-1 prevent the induc- 33% sequence identity with CTLA-4, CD28 and ICOS tion of type 1 diabetes and these results were driven by [12,13]. The broader expression of PD-1 contrasts with changes in T cell function. Contrary to expectations, restricted expression of other CD28 family members to T while the number of T cells remained unchanged, the cells, suggesting that PD-1 regulates a wider spectrum of number of macrophages sharply decreased [25]. This al- immune response compared with other CD28 family tered macrophage population is induced by decreased members. PD-L1/PD-L2 are ligands known as bind to IFN-γ secretion from PD-1 Tg T cell or, surprisingly, it PD-1 [14], and share 20% amino acid identities with may be due to changes in the macrophage itself; thus the B7-1/B7-2 that are ligands for CD28 and CTLA-4 [15- possibility of overexpressed PD-1 on T cells acting on 17]. PD-L1 is constitutively expressed on T cells, B cells, macrophages came into the spotlight. To investigate macrophages and dendritic cells but PD-L2 expression is whether the interaction between PD-1 and its ligands regulated more tightly; and it is observed on activated alters macrophage activities, macrophages were stimu- macrophages and DCs [18]. lated with PD-1 human Ig or anti PD-L1 antibody. We There are several genetically modified mouse models found these engagements increase SPHK1, LIGHT, IL- that alter PD-1 expression. PD-1 deficient (PD-1-/-) mice 10 and decrease IL-6 in early phase stimulation. These were generated by deletion of the transmembrane and results suggest that signal transduction through PD-L1 cytoplasmic domain exons, and some studies show the drives macrophages toward regulatory subsets. inhibitory role of PD-1 in vivo using this mouse model [19]. PD-1-/- mice exhibit an increase in double positive 2. MATERIALS AND METHODS T cells, but a reduced number of single-positive CD8 T cells, suggesting a role for this pathway in setting thresh- 2.1. Cell Culture olds for thymocyte differentations [20]. PD-1-/- mice RAW 264.7 macrophage cell line was obtained from the develop various autoimmune diseases depending on their American Type Culture Collection (ATCC) and main- genetic background. Using NOD-PD-1-/- mice as an tained in DMEM medium (Cellgro) supplemented with efficient animal model of type 1 diabetes, diabetes-sus- 10% heat-inactivated FBS (Fetal bovine serum, Cellgro), ceptible loci were screened by genetic linkage analysis. 2 mM L-glutamine, 100 U/ml penicillin and streptomy- On the other hand, PD-1 transgenic mice, which were cin (Cellgro) at 37˚C in a 5% CO humidified incubator. engineered in our laboratory, constitutively express T 2 cell-restricted PD-1 under the control of the Lck proxi- 2.2. Mice mal promoter and CD2 locus control. PD-1 Tg mice did not develop gross abnormalities of thymic development Wild type male C57BL/6 mice, aged 6 to 12 weeks, were and displayed a normal number of thymocyte subsets and purchased from Orient Bio as controls, and PD-1 trans- peripheral T cells. In vitro, PD-1 Tg T cells had reduced genic mice, which overexpress PD-1, particularly on T function upon TCR stimulation and cross-linking of cells using the lck promoter, were generated by professor PD-1 resulted in diminished phosphorylation of protein Kwang Woo Hwang. kinase C-θ and Akt, as well as increased activation of the Animals were maintained under the controlled condi- phosphate and tensin homology [20]. tions of temperature (21˚C ± 3˚C), relative humidity Copyright © 2013 SciRes.