VIP and PACAP inhibit Fas ligand-mediated bystander lysis by CD4⁺ T cells

Abstract

FasL/Fas-mediated lysis represents the major cytotoxic mechanism for CD4⁺ effectors, with important consequences for immune cell homeostasis. Upon stimulation by specific antigen-presenting cells (APCs), CD4⁺ effectors can lyse the cognate APCs (direct targets) and neighboring innocent bystanders. Previously we showed that the neuropeptides VIP and PACAP prevent FasL expression and activation-induced cell death in T cells. In this study we investigated the effects of VIP and PACAP on FasL expression and subsequent direct and bystander lysis by CD4⁺ effectors generated in vivo. VIP/PACAP inhibit FasL expression in allogeneic effectors, and reduce Fas-mediated cytotoxicity against specific allotargets and syngeneic bystanders. VIP/PACAP also inhibit FasL expression in antigen-specific CD4⁺ effectors, and reduce their cytotoxic activity against both the stimulatory APC, and syngeneic or allogeneic bystanders. Since bystander lysis is involved in the pathogenesis of several autoimmune and inflammatory diseases, the identification of regulatory factors that limit this process is highly significant.

Introduction

Two independent lytic pathways mediate T-cell cytotoxicity (Berke, 1995, Nagata and Golstein, 1995): a calcium-dependent degranulation pathway, in which exocytosis of perforin in combination with granzymes results in lysis of target cells (Shiver et al., 1992), and a calcium-independent pathway, which involves interactions between Fas ligand (FasL) expressed on cytotoxic T cells (CTLs) and the Fas protein present on target cells (Rouvier et al., 1993, Kagi et al., 1994, Walsh et al., 1994, Takahashi et al., 1994). Fas-mediated lysis is controlled mainly through the regulation of FasL expression. FasL expression and subsequent Fas-mediated cytotoxicity are induced following treatment of T cells with a variety of stimuli, including appropriate Ag–MHC complexes, TCR cross-linking with anti-CD3 Ab, and pharmacological agents that mimic TCR activation, such as phorbol esters plus calcium ionophores (Rouvier et al., 1993, Ju et al., 1994, Stalder et al., 1994). The perforin/granzyme pathway plays a major role in the elimination of virally-infected cells or allogeneic grafts, whereas Fas-mediated cytotoxicity appears to be the major player in immune homeostasis and tolerance induction (Vignaux and Golstein, 1994, Bellgrau et al., 1995).

CD8⁺ T cells using the perforin/granzyme pathway are the classical effectors in response to viral antigens. However, Th1 CD4⁺ T cells display cytolytic activity through the FasL/Fas pathway, lysing activated APCs (direct targets), and neighboring bystanders (Fas-expressing non-APCs) (Erb et al., 1990, Lancki et al., 1991, Ju, 1991, Lowin et al., 1994, Kojima et al., 1994, Ju et al., 1994, Stalder et al., 1994). The APC activates the CD4⁺ T cell in a MHC II-restricted manner, leading to the up-regulation of FasL, which then enables the CD4⁺ T cell to lyse Fas-bearing targets found in the vicinity, in a MHC-nonrestricted and Ag-nonspecific manner. In vitro, bystander lysis by CD4⁺ T cells requires the simultaneous presence of the CD4⁺ cytotoxic T cells, the cognate APC, and the bystander (Wang et al., 1996, Smyth, 1997).

Although Fas-mediated cytotoxicity plays an important role in maintaining T cell homeostasis, the Fas-mediated lysis of bystander targets has been implicated in the pathogenesis of several autoimmune and inflammatory diseases, especially in tissues with limited or restricted MHC II expression (reviewed in De Maria and Testi, 1998). The mechanisms limiting the killing of innocent bystanders are not understood, and the identification of factors that regulate this process represents the focus of considerable research.

Vasoactive intestinal peptide (VIP), and the structurally related peptide, the pituitary adenylate cyclase-activating polypeptide (PACAP), are two neuropeptides released in the immune microenvironment (Leceta et al., 1996, Bellinger et al., 1996), affecting both natural and acquired immunity, predominantly as anti-inflammatory agents (Ganea, 1996, Delgado et al., 1999a, Pozo et al., 2000). Recently we reported that VIP and PACAP inhibit activation-induced FasL expression and subsequent cell death in T cells and T cell hybridomas (Delgado and Ganea, 2000a), as well as the FasL-mediated cytotoxic activity of alloreactive CD8⁺ CTLs (Delgado and Ganea, 2000b). In this study, we investigate the effects of VIP and PACAP on FasL/Fas-mediated cytotoxicity of in vivo primed effector CD4⁺ T cells against direct and bystander targets. Our studies indicate that VIP/PACAP inhibit both the direct and bystander lysis of syngeneic targets by CD4⁺ T cells, through the down-regulation of FasL expression on the cytotoxic effectors.