Molecular Immunology 46 (2009) 2938–2946 Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm A model system to study Connexin 43 in the immune system Thien D. Nguyen, Steven M. Taffet ∗ Department of Microbiology, SUNY Upstate Medical University, 750 E Adams St., Syracuse, NY 13210, United States article info Article history: Received 14 May 2009 Received in revised form 11 June 2009 Accepted 18 June 2009 Available online 15 July 2009 Keywords: Connexin 43 Immunity Cell proliferation Cell trafficking Inflammation abstract Connexin 43 (Cx43) is the predominant gap junction protein expressed in immune cells. Previous manuscripts have stated that gap junctions may play a role in antigen cross-presentation, dendritic cell maturation, T cell development, and regulatory T cell function. Many of these previous studies were per- formed in vitro. In vivo studies were not directly possible in adult mice because Cx43-/- mice die shortly after birth due to a cardiac malformation. To overcome these drawbacks, we have developed a mouse model that deletes Cx43 in the immune system while maintaining normal cardiac function. In our model, irradiated CD45.1+ wild-type mice were reconstituted with Cx43WT, Cx43±, or Cx43-/- hematopoietic fetal liver cells that were derived from CD45.2+ mice. The presence of CD45.2 allowed us to identify and track the donor cells following reconstitution. We determined that Cx43± and Cx43-/- hematopoietic cells were able to reconstitute irradiated mice as well as Cx43WT cells. Reconstitution was nearly 100% in the thymus and over 90% in the spleen. There appeared to be no difference in thymocyte development or in the ability of lymphocytes to transmigrate to peripheral lymphoid organs. However in response to inflammation, Cx43± radiation chimeras had increased peritoneal infiltration compared to Cx43WT and Cx43-/- groups. IgG responses were normal in all groups but the Cx43-/- reconstituted mice had an elevated IgM response. Our data suggests that Cx43 may not be involved in the normal development of the immune system but may regulate certain effector functions in vivo. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Connexins are a family of membrane proteins that are the build- ing blocks of intercellular channels called gap junctions (Yeager and Harris, 2007). Gap junction channels allow for the diffusion of small molecules between neighboring cells. These small molecules are generally restricted to less than 1 kDa in size, which can include molecules such as ATP, charged ions, cAMP, and peptides. Signals that gap junctions may convey include metabolic, activation, and apoptotic information between cells. There are at least 20 different connexins that are expressed in mice (Willecke et al., 2002) with Connexin 43 (Cx43) being the predominant species expressed in most cells including cells of the immune system (Neijssen et al., 2007; Wong et al., 2004). Recently, much attention has been focused on various roles gap junctions may play in the immune system (Alves et al., 1998; Chanson et al., 2005; Neijssen et al., 2007; Oviedo-Orta et al., 2001; Oviedo-Orta and Howard Evans, 2004; Wong et al., 2004). These roles may include antigen cross-presentation (Mendoza- Abbreviations: Cx43, Connexin 43; AGA, 18-alpha-glycyrrhetinic acid; CBX, car- benoxylone; KLH, keyhole limpet hemacyanin. ∗ Corresponding author. Tel.: +1 315 464 5419; fax: +1 315 464 7668. E-mail address: taffets@upstate.edu (S.M. Taffet). Naranjo et al., 2007; Neijssen et al., 2005), dendritic cell activation (Matsue et al., 2006), immunoglobulin isotype switching (Oviedo- Orta et al., 2001), and regulatory T cell function (Bopp et al., 2007). In addition, gap junctions may have important roles in immune system-dependent functions such as inflammatory transmigration (Eugenin et al., 2003; Oviedo-Orta et al., 2002; Zahler et al., 2003), hematopoiesis (Cancelas et al., 2000; Montecino-Rodriguez and Dorshkind, 2001; Montecino-Rodriguez et al., 2000), wound heal- ing (Chanson et al., 2005; Coutinho et al., 2003; Hodgins, 2004; Wang et al., 2007a), and progression of atherosclerosis (Kwak et al., 2002; Kwak et al., 2003; Wong et al., 2003). Some connexins may also form dynamic pores in the cell membrane called hemichan- nels that allow the release of small molecules into the extracellular environment (De Vuyst et al., 2007; Eltzschig et al., 2006; Evans et al., 2006; Wong et al., 2006). For example, Cx43 and Cx37 may form hemichannels that have been shown to release ATP from neu- trophils and monocytes (Eltzschig et al., 2006; Evans et al., 2006; Wong et al., 2006). Although gap junctions were first described in the immune system in the 1970s, we are only now beginning to unravel their true role. To date, many of the previous studies describing the role of gap junctions in immune cells use in vitro or ex vivo methods. The primary reason for this is that Cx43 knock out mice die at birth due to a cardiac defect. It is unknown if these changes in func- tion produced in vitro will translate into in vivo differences. Many 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.06.022