Immunobiology 224 (2019) 307–315 Contents lists available at ScienceDirect Immunobiology journal homepage: www.elsevier.com/locate/imbio Expression of complement C3, C5, C3aR and C5aR1 genes in resting and activated CD4+ T cells T ⁎ Cecilie Bo Hansena, Anton Willera, Rafael Bayarri-Olmosa, Claudia Kemperb, Peter Garreda, a Laboratory of Molecular Medicine, Department of Clinical Immunology Section 7631, Faculty of Health and Medical Sciences, University Hospital of Copenhagen, Denmark b National Heart, Lung and Blood Institute, National Institute of Health, Bethesda, MD, 20814, USA ARTICLE INFO ABSTRACT Keywords: Complement activation is traditionally thought to occur in the extracellular space. However, it has been sug- T-cells gested that complement proteins are activated and function at additional locations. T cells contain intracellular Complement expression stores of C3 and C5 that can be cleaved into C3a and C5a and bind to intracellular receptors, which have been Intracellular complement shown to be of vital importance for the differentiation and function of these cells. However, whether the origin of C3 the complement proteins located within T cells is derived from endogenous produced complement or from an C5 uptake dependent mechanism is unknown. The presence of intracellular C3 in T cells from normal donors was investigated by fluorescence microscopy and flow cytometry. Moreover, mRNA expression levels of several genes encoding for complement proteins with primary focus on C3, C3aR, C5 and C5aR1 during resting state and upon activation of CD4+ T cells were investigated by a quantitative PCR technique. Furthermore, the gene expression level was evaluated at different time points. We confirmed the presence of intracellular C3 protein in normal T-cells. However, we could not see any increase in mRNA levels using any activation strategy tested. On the contrary, we observed a slight increase in C3 and C5aR1 mRNA only in the non-activated T-cells compared to the activated T cells, and a decrease in the activated T-cells at different incubation time points. Our results show that there is a baseline intracellular expression of the complement C3, C5, C3aR and C5aR1 genes in normal CD4+ T cells, but that expression is not increased during T-cell activation, but rather down regulated. Thus, the pool of intracellular complement in CD4+ T cells may either be due to accumulated complement due low-grade expression or arise from the circulation from an uptake dependent mechanism, but these possibilities are not mutually exclusive. 1. Introduction which leads to preferential opsonization (Gadjeva et al., 1998; Law et al., 1984). Deposition of C5b onto a target initiates the formation of The complement system is an important effector mechanism in both the C5b-9 terminal complex, which exists as the membrane attack the innate and adaptive immune defence. It is composed of three complex (MAC) targeting cells for lysis, and an inactive soluble version pathways – the classical, alternative and lectin pathway, which all work named sC5b-9 (Walport, 2001a; Morgan and Gasque, 1997; Klos et al., by initiating a proteolytic cascade that detects and eliminates invading 2009; Müller-Eberhard, 1985; Sahu et al., 1994; Ricklin et al., 2010). pathogens and altered, apoptotic and dangerous host cells (Walport, The complement system does not only participate in the elimination 2001a; Le and Kemper, 2009; Walport, 2001b). The three activation of microbes and altered cells, but has a wide variety of functions and pathways lead to the generation of the C3 and C5 convertase enzyme contributes in diverse processes such as synaptic pruning in the central complexes, which cleave C3 into the anaphylatoxin C3a and the op- nervous system (Schafer et al., 2012), bone formation (Matsuoka et al., sonin C3b, and C5 into the anaphylatoxin C5a and into C5b, respec- 2014), angiogenesis (Pio et al., 2013), supports the pluripotency of tively. C3a and C5a are potent inflammatory mediators targeting a human stem cells (hPSCs) (Hawksworth et al., 2014), tissue regenera- broad spectrum of immune and non-immune cells. C3b predominantly tion (Mastellos et al., 2001) and lipid metabolism (Ricklin et al., 2010); form ester bonds with hydroxyl groups on carbohydrates or proteins, (Cui et al., 2007). ⁎ Corresponding author at: Department of Clinical Immunology Section 7631, Rigshospitalet, Ole Maaloesvej 26, 2200, Copenhagen N, Denmark. E-mail address: [email protected] (P. Garred). https://doi.org/10.1016/j.imbio.2018.12.004 Received 16 November 2018; Received in revised form 21 December 2018; Accepted 21 December 2018 Available online 27 December 2018 0171-2985/ © 2018 Elsevier GmbH. All rights reserved. C.B. Hansen et al. Immunobiology 224 (2019) 307–315 Complement activation is traditionally thought to occur exclusively Michelle Elvington et al. (2017), the authors observed by Western blot in the extracellular space, however in recent years it has been suggested (WB) a clear distinction in the presence of C3 and C3 cleavage products that complement components play additional ‘non-canonical’ roles in between peripheral blood cells (PBC) and their respective cell lines (i.e. the immune system. Almost all immune cells - including T- and B-cells CD4+ T cells versus Jurkat cells). A major difference between PBC and have receptors that can bind complement fragments and through these cultured cells is that the latter had not been exposed to a source of complement receptors activate specific cellular processes (Kolev et al., human C3 for many generations. They demonstrated that many types of 2014; Köhl, 2006; Kolev et al., 2013; Liszewski et al., 2013). Ad- human cells and cell lines including CD4+ T are able to internalize the ditionally, complement molecules can be activated intracellularly and it hydrolytic product of C3 (C3(H2O)) from the extracellular milieu, in a has been shown that particular complement molecules are of vital im- manner that is rapid, saturable and sensitive to competition, indicating portance for the differentiation and function of specific lymphocyte a specific mechanism of loading. However the precise mechanism of populations such as T cells (Kolev et al., 2013; Liszewski et al., 2013; this internalization is currently unknown. Suresh et al., 2003). It has been shown that resting human CD4+ T cells Moreover, it is not clear whether complement genes are induced by contain intracellular stores of C3 and the protease cathepsin L (CTSL) in cellular activation and whether complement gene expression might be endosomal and lysosomal compartments, and that CTSL is able to related to cell survival. In this study we have investigated and com- cleave C3 into C3a and C3b in a convertase-independent manner (Köhl, pared gene expression in selected complement genes in a time depen- 2006). Intracellular C3a can thus bind to the lysosomal-localized re- dent fashion in resting and activated CD4+ T cells. ceptor, C3aR, and mediate homeostatic T cell survival through basal activation of mammalian target of rapamycin (mTOR) (Liszewski et al., 2. Material and methods 2013). This autocrine interaction between the complement system and the T cell will consequently determine T cell function and activity 2.1. Blood sampling and CD4+ T cell isolation (Köhl, 2006). The source of the intracellular stores of C3 and C5 has not been Blood was drawn from healthy donors in EDTA tubes (VACUETTE® rigorously investigated. It has been speculated that T cells, upon acti- K3EDTA, Greiner Bio One International GmbH). All samples were vation, may be able to produce complement components endogenously handled within one hour of blood sampling. CD4+ T lymphocytes were due to the fact that intracellular C3 and C3a protein can be observed in isolated from whole blood with The EasySep™ Human Whole Blood patients that lack C3 circulating in serum (Liszewski et al., 2013). CD4 Positive Selection and RoboSep™ (STEMCELL technologies) ac- Hence, such existing data suggest a potential disconnection between cording to the manufacturer’s instructions. All donors gave informed plasma (liver-derived) C3 and (immune cell-derived) intracellular C3 consent. The study was approved by the regional Health Ethics expression. Additionally, the initial study describing this observation Committee in the Capital Region of Denmark (reference no. H2-2011- concludes that CD46 is crucial in certain T cell activation regimes, 133). particularly those connected with Th1 induction (Köhl, 2006). CD46, or membrane cofactor protein (MCP), is a cell surface receptor, which is 2.2. Antibodies expressed by a variety of human nucleated cell types, including monocytes, B and T cells. CD46 functions as a cofactor for the de- Cell-stimulating antibodies were bought from Miltenyi Biotec (T gradation of complement fragments C3b and C4b deposited on cells and Cell Activation/Expansion Kit); anti-CD2, anti-CD3, anti-CD28 and anti- thereby protects human cells from lysis by autologous complement CD46 (REA312). Anti-CD4 (560158), anti-CD8 (641058), anti-CD14 (Seya et al., 1986). In addition to this complement regulatory function, (345786) were obtained from BD Biosciences and anti-CD19 (302233) CD46 is a receptor for a number of pathogenic bacteria and viruses, from Biolegend. Intracellular C3 was detected using chicken anti-C3 including measles virus, human herpesvirus 6, adenovirus of different (GW20073 F) from Sigma Aldrich and secondary antibody goat anti- serotypes, Streptococcus pyogenes and pathogenic Neisseria (Källström chicken IgG (A-11039)