Expression and function of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) in an in vitro model of the human blood–brain barrier

Abstract

The interaction of B7 molecules with their ligand provides important accessory signals for optimal T cell activation and proliferation. In this study the in vitro expression of B7-1 and B7-2 by human brain microvessel endothelial cells (HBMEC) was investigated by semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR) and immunocytochemistry. In addition, the contribution of B7 molecules to T cell proliferation on cerebral endothelial cells was studied by coincubating purified CD4+ T cells with resting or cytokine activated HBMEC. Untreated cultures constitutively expressed B7-2 RNA and surface protein, but lacked B7-1 expression. Treatment with TNF-α and IFN-γ upregulated B7-2 and induced de novo expression of B7-1. Monoclonal blocking antibodies to B7-1 or B7-2 and human CTLA-4Ig chimeric protein significantly reduced the ability of HBMEC to support α-CD3-induced proliferation of CD4+ T lymphocytes. Expression of B7 glycoproteins and the ability to provide secondary signals for T cell proliferation suggest a potential role of the human cerebral endothelium in T cell activation during the early stages of central nervous system inflammation.

Introduction

The central nervous system (CNS) exists in a state of relative immunologic isolation due to the presence of the blood–brain barrier (BBB) which normally restricts the entry of leukocytes into the brain. In the course of CNS inflammation the BBB is breached and circulating leukocytes migrate across the endothelium to accumulate at sites of antigenic challenge (Hauser et al., 1986, Cross et al., 1990, Hickey et al., 1997). Recent studies have convincingly demonstrated that interactions between adhesion molecules expressed by cytokine activated cerebral endothelial cells (EC) and their ligands on lymphocytes are key mediators of the adhesion and migration of T lymphocytes across the BBB (Yednock et al., 1992, Archellos et al., 1993, Baron et al., 1993, Wong et al., 1999). Furthermore, peripherally activated T cells readily cross the BBB, irrespective of the activation state of the endothelium, but leave the CNS if they do not encounter specific antigen (Hickey et al., 1991, Ludowyk et al., 1992). One of the many unresolved issues in the pathogenesis of autoimmune CNS inflammation is whether antigen presentation and T cell activation take place at the BBB. The present dogma dictates that T cell activation and proliferation requires two signals: a primary signal provided by recognition of antigen–MHC complex by the T cell receptor (TCR)/CD3 complex, and a secondary costimulatory signal resulting from the interaction of accessory molecules on antigen presenting cells (APC) with their ligands on T cells (Altman et al., 1990, Steinman and Young, 1991). Failure to engage this costimulatory interaction may result in antigen-specific anergy and lack of cytokine production (Harding et al., 1992). Several costimulatory receptors have been implicated including intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, lymphocyte function associated antigen (LFA)-3, and B7 molecules (Damle et al., 1992, Wingren et al., 1995).

B7-1 (CD80) and B7-2 (CD86) are transmembrane glycoproteins sharing 25% amino acid homology and belonging to the immunoglobulin superfamily (IgSF). B7 molecules bind to two homodimers also members of the IgSF: CD28 expressed on 90% of CD4+ and 50% of CD8+ T lymphocytes, and cytotoxic T lymphocyte-associated antigen (CTLA)-4 found on activated T cells (June et al., 1994, Linsley et al., 1994). B7–CD28 interactions have been shown to provide positive signals for T cell activation and proliferation resulting in: (1) elevated cytokine production, especially IL-2, due to increased gene transcription and mRNA stabilization (Lindsten et al., 1989, Fraser et al., 1991, Ragheb et al., 1999); (2) prevention of anergy or tolerance induction (Gimmi et al., 1993, Yi-qui et al., 1997); (3) induction of T cell survival promoting genes (Boise et al., 1995, Sperling et al., 1996); and (4) facilitation of entry into the cell cycle (Boonen et al., 1999). In contrast, engagement of the second receptor, CTLA-4, by B7 molecules seems to result in negative regulatory signals that diminish production of IL-2, and prevent T cells from entering the cell cycle (Gribben et al., 1995, Krummel and Allison, 1996, Blair et al., 1998, Brunner et al., 1999). B7-1 and B7-2, originally detected on B cells (Freedman et al., 1987, Azuma et al., 1993a), are also expressed by ‘classical’ professional APC (dendritic cells, macrophages/monocytes) (Young et al., 1992, Creery et al., 1996, Fleischer et al., 1996) and non-professional APC (T lymphocytes, epithelial cells) (Azuma et al., 1993b, Sansom and Hall, 1993, Ye et al., 1997, Jeannin et al., 1999). The expression of B7 molecules by EC has been investigated in vitro on human umbilical vein EC (HUVEC), human dermal microvascular EC (HDMEC), porcine aortic EC (PAEC) and murine sinusoidal EC (SEC) (Seino et al., 1995, Batten et al., 1996, Foreman et al., 1996, Lohse et al., 1996). In vivo, B7 expression has been demonstrated on SEC in fulminant hepatic failure (Leifeld et al., 1999). Within the CNS, B7 molecules have been found on cultured human microglia, but not astrocytes (Satoh et al., 1995, de Simone et al., 1995, Dangond et al., 1997). In situ, B7 immunoreactivity has been demonstrated on perivascular lymphocytes, infiltrating macrophages, and microglia in multiple sclerosis (MS) lesions and cerebral infarcts (Williams et al., 1994, de Simone et al., 1995, Windhagen et al., 1995). The importance of B7-mediated costimulation in the development of autoimmune CNS inflammation is indicated by studies showing amelioration of EAE after blocking CD28 or anti B7-1 treatment (Miller et al., 1995, Perrin et al., 1999).

Since EC are the first CNS cells to contact circulating T lymphocytes, interactions between these two cell types are considered important for T cell recruitment across the BBB. Previous studies from this laboratory have shown that human cerebral EC can be induced to express or upregulate ICAM-1, VCAM-1, E-selectin, class II MHC and LFA-3, and provide secondary signals for T cell proliferation through the LFA-3/CD2 pathway (Wong and Dorovini-Zis, 1992, Wong and Dorovini-Zis, 1995, Wong and Dorovini-Zis, 1996, Huynh and Dorovini-Zis, 1993, Omari and Dorovini-Zis, 1999). In order to further characterize the interactions between cerebral endothelium and lymphocytes in CNS inflammation, we investigated the expression and function of B7-1 and B7-2 in primary cultures of HBMEC before and after cytokine stimulation. Human brain microvessel EC in culture retain important morphological and functional characteristics of the cerebral endothelium in vivo including presence of tight junctions of high electrical resistance and paucity of cytoplasmic vesicles (Dorovini-Zis et al., 1991). Our data indicate that B7-1 and B7-2 expression by human cerebral EC provides costimulatory signals for T cell proliferation at the BBB.