G Model MIMM-4749; No. of Pages 9 ARTICLE IN PRESS Molecular Immunology xxx (2015) xxx–xxx Contents lists available at ScienceDirect Molecular Immunology j ournal homepage: www.elsevier.com/locate/molimm CD100 and plexins B2 and B1 mediate monocyte-endothelial cell adhesion and might take part in atherogenesis a,b a a a,b,c Maria Carolina A. Luque , Paulo S. Gutierrez , Victor Debbas , Jorge Kalil , d,∗ Beatriz S. Stolf a Heart Institute of São Paulo (InCor), HC-FMUSP, São Paulo, SP, Brazil b Clinical Immunology and Allergy, Department of Clinical Medicine, University of São Paulo Medical School—HC-FMUSP, São Paulo, SP, Brazil c Institute for Investigation in Immunology – INCT – National Institute of Science and Technology, São Paulo, SP, Brazil d Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil a r t i c l e i n f o a b s t r a c t Article history: Leukocyte migration is essential for the function of the immune system. Their recruitment from the Received 28 April 2015 vessels to the tissues involves sequential molecular interactions between leukocytes and endothelial Received in revised form 23 June 2015 cells (ECs). Many adhesion molecules involved in this process have already been described. However, Accepted 22 July 2015 additional molecules may be important in this interaction, and here we explore the potential role for Available online xxx CD100 and plexins in monocyte-EC binding. CD100 was shown to be involved in platelet-endothelial cell interaction, an important step in athero- Keywords: genesis and thrombus formation. In a recent work we have described CD100 expression in monocytes Monocyte adhesion SEMA4D and in macrophages and foam cells of human atherosclerotic plaques. In the present work, we have iden- Plexins tified plexin B2 as a putative CD100 receptor in these cells. We have detected CD100 expression in the Atherosclerosis endothelium as well as in in vitro cultured endothelial cells. Blocking of CD100, plexin B1 and/or B2 in adhesion experiments have shown that both CD100 and plexins act as adhesion molecules involved in monocyte-endothelial cell binding. This effect may be mediated by CD100 expressed in both cell types, probably coupled to the receptors endothelial plexin B1 and monocytic plexin B2. These results can bring new insights about a possible biological activity of CD100 in monocyte adhesion and atherosclerosis, as well as a future candidate for targeting therapeutics. © 2015 Published by Elsevier Ltd. 1. Introduction inflammatory diseases such as atherosclerosis. In fact, atheroscle- rosis is characterized by monocyte and macrophage accumulation Immune system functions depend largely on leukocyte in the vascular intima of the vessels (van Gils et al., 2009). migration from the vessels to the tissues, which involves sequen- Endothelial cell (EC) activation occurs in the presence of proathero- tial molecular interactions between leukocytes and endothelial genic insults such as oxLDL, proinflammatory cytokines, oxidative cells (ECs) (Imhof and Aurrand-Lions, 2004). While transient stress, hypertension, hyperglycemia, aging, and shear stress (Sun leukocyte–endothelial cell interactions are mediated by the et al., 2013) and result in the increase of the mentioned adhesion endothelial E- and P-selectins and leukocyte L-selectin and PSGL- molecules on the surface of the arteries (Hansson and Hermansson, 1, firm adhesion relies on the binding of activated integrins to 2011). Concurrently, activated monocytes and T lymphocytes endothelial Intercellular Adhesion Molecule 1 (ICAM-1) and Vas- express cell surface molecules that interact with the endothelia and cular Cell Adhesion Molecule (VCAM-1) (Heemskerk et al., 2014). mediate their transmigration into the arterial wall (Mehta et al., Because of their ability to differentiate into phagocytes and 1998). antigen-presenting cells, monocytes have important roles in CD100/SEMA4D belongs to the class 4 of the semaphorin pro- tein family, and has important roles in the nervous and immune systems. It is expressed by the majority of hematopoietic cells (B, T, NK and myeloid cells), and its levels generally increase after cell Abbreviations: CD100, cluster of differentiation 100; CD72, cluster of differenti- activation (Delaire et al., 1998). In T lymphocytes it is expressed ation 72; SEMA4D, semaphorin-4D. ∗ as membrane and soluble forms with distinct functional properties Corresponding author. Tel.: +55 1130917271; fax: +55 1130917417. E-mail address: [email protected] (B.S. Stolf). (Delaire et al., 1998). T cells exhibit the highest levels of CD100 (Hu http://dx.doi.org/10.1016/j.molimm.2015.07.028 0161-5890/© 2015 Published by Elsevier Ltd. Please cite this article in press as: Luque, M.C.A., et al., CD100 and plexins B2 and B1 mediate monocyte-endothelial cell adhesion and might take part in atherogenesis. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.07.028 G Model MIMM-4749; No. of Pages 9 ARTICLE IN PRESS 2 M.C.A. Luque et al. / Molecular Immunology xxx (2015) xxx–xxx et al., 2008; Ji et al., 2009), followed by platelets (Zhu et al., 2007, School of Medicine (controls with no vascular disease). Researchers 2009) and monocytes (Bougeret et al., 1992; Luque et al., 2013). involved in the project were present during heart removal from Human CD100 was shown to influence migration and cytokine the donor and received a fresh fragment of approximately 5 cm production in monocytes (Chabbert-de Ponnat et al., 2005), and is of the carotid that would not be used for transplantation. The involved in T cell activation and B cell survival. It has also been artery fragments were fixed for 24 or 48 h in buffered solution shown to participate in cell-to-cell communication and adhesion with 10% formalin for posterior inclusion in paraffin and prepa- in different contexts (Mizrahi et al., 2007; Witherden et al., 2012; ration of tissue sections (∼7 ␮m). Donor’s family had previously Zhu et al., 2007). Distinct receptors have been identified for CD100, signed an authorization form specific for this project donating the depending on the cell type and organism. In humans, plexin B1 is artery fragment to the “Laboratory of Immunology, Heart Institute”. the CD100 high affinity receptor in a broad range of non-immune Collection of all human samples was approved by the Heart Insti- cells (Tamagnone et al., 1999). In the immune system plexin B1 is tute Scientific Committee and CAPPesq (Hospital das Clinicas Ethics expressed only in follicular dendritic cells, bone marrow stromal Committee). cells and (at lower levels) in activated T cells, but not in mono- cytes, macrophages, other dendritic cells and quiescent T and B 2.3. Cell culture lymphocytes (Ishida et al., 2003; Granziero et al., 2003). In fact, no high affinity receptor for CD100 was precisely identified in PBMCs were obtained from healthy subjects by density gradient human monocytes. Plexin B2 has been described in mouse ger- centrifugation with Ficoll (Invitrogen, Carlsbad, CA) and mono- minal center B cells (Yu et al., 2008) and in mouse macrophages, cytes were purified by positive selection using anti-CD14 magnetic conventional and plasmacytoid dendritic cells, and its cDNA was beads, as recommended by the manufacturer (Miltenyi Biotec, present in human myeloid cells (Roney et al., 2011). Germany). Purity of monocytes was above 98% as verified by FACS In atherosclerosis, CD100 was shown to be involved in platelet- analysis. Human T lymphocytes were purified by negative selection, endothelial cell interaction, an important step in atherogenesis and using Pan T Isolation kit (Miltenyi Biotec, Germany). THP-1 mono- thrombus formation (Zhu et al., 2007). In the mice model the lack of cytes (ATCC, Manassas, VA) and PBMC monocytes, macrophages CD100 reduces platelet hyperactivity, therefore conferring protec- and foam cells were cultured in RPMI-1640 (Gibco, NY) containing tion against atherosclerosis (Zhu et al., 2007). Sema4D−/−ApoE−/− 10% calf serum (Hy Clone, Utah), 10 mM HEPES, 1 mM glutamine, mice present decreased lipid staining, macrophage infiltration and 200 U/ml penicillin, 2 mg/ml streptomycin, 1 mM sodium pyru- intimal neovascularization in the aortic plaques (Yukawa et al., vate. T lymphocytes were cultured in DMEM (Gibco, NY) with 2010). A recent work from our group has shown CD100 expres- 10% calf serum (Hy Clone, Utah), 10 mM HEPES, 1 mM glutamine, sion in human atheromas, more specifically in plaque macrophages 200 U/ml penicillin, 2 mg/ml streptomycin, 1 mM sodium pyru- and foam cells (Luque et al., 2013). In vitro cultured monocytes (Hu vate and IL-2, IL-7 e IL-15 (R&D, Minneapolis, MN) 40 U/ml each. et al., 2008) and differentiated macrophages and foam cells were HUVECs (human umbilical vein endothelial cells) (ATCC, Manas- shown to express CD100, mainly under pro-inflammatory stimuli sas, VA) were cultured in 199 medium (Earle’s) supplemented with (Luque et al., 2013). In addition, we have shown that CD100 reduces 0.6 mg/ml penicillin, 60 mg/ml streptomycin, 2 mM glutamine, oxLDL incorporation by macrophages by decreasing CD36 expres- 4 20 mM HEPES and 10% FCS on coverslips in 24-well plates (5 × 10 sion, pointing to a specific antiatherogenic effect of the molecule cells/well in 500 ␮l) for immunocytochemistry experiments or in 6 (Luque et al., 2013). 5 well plates (4 × 10 cells/well in 2 ml of medium) for qRT-PCR and Since adherence of monocytes to endothelium is among the ear- Western blot experiments. For activation, HUVECs were cultivated liest events in atherogenesis (Ross, 1993) and since CD100 acts in 6-well plates until confluence and then incubated with 1 ng/ml as an adhesion molecule in platelet-EC interaction (Zhu et al., ◦ LPS (Sigma) and 10 ng/ml TNF-␣ (Peprotech) for 6 h at 37 C and 5% 2007), we analyzed whether CD100 could also participate in CO2.