Differential regulation of soluble and membrane CD40L proteins in T cells

Abstract

CD40 ligand is an important immunoregulatory protein expressed by T cells. This protein exists as two isoforms, a membrane glycoprotein and a truncated soluble form. Here we demonstrate that membrane and soluble CD40L (sCD40L) are differentially regulated depending upon the activation stimulus. In T cell receptor activated cells, both membrane and sCD40L proteins are expressed and CD28 costimulation further increases their expression. The dissection of TCR generated signals into calcium and PKC-dependent pathways demonstrates that calcium is sufficient to induce membrane CD40L yet insufficient for sCD40L. In contrast, sCD40L is preferentially induced by PKC. Moreover, sCD40L production is blocked by Zn²⁺-dependent metalloproteinase inhibitors while membrane CD40L is concurrently increased. This profile suggests the potential involvement of the ADAM-10 protease which was subsequently shown to cleave membrane CD40L to generate sCD40L. Given the role of sCD40L in numerous disease pathologies and its ability to activate proximal and distal immune responses, the regulated cleavage of CD40L may likely contribute to disease mechanisms.

Introduction

Several members of the TNF superfamily critically influence the course of inflammation [1]. These structurally related proteins [2] control a variety of cellular functions ranging from the regulation of proliferation, differentiation and programmed cell death. In particular, the family member CD40 ligand (CD40L, CD154, gp39, or TRAP), plays an important role in the regulation of humoral and cell mediated immune responses by controlling the balance of proliferative, maturation, and apoptotic processes [3]. On normal T cells, the expression of CD40L is tightly regulated and only transiently expressed [4]. The interaction of this ligand with its receptor, CD40, constitutively expressed on B cells and antigen presenting cells (APCs) triggers a myriad of signaling events affecting multiple cell types. CD40 ligation on APCs induces a marked up-regulation of adhesion and costimulatory molecules which increases their APC activity and results in enhanced T cell costimulation [5], [6], [7]. CD40 ligation also promotes APC induction of cytokines, increases macrophage tumoricidal activity, and promotes dendritic cell growth and survival [7], [8], [9]. Actions of CD40 ligation to promote humoral immunity occur by increasing B cell growth and survival and by triggering antibody isotope class switching in vivo[10]. Thus, reciprocal interactions between T cells and APCs induce their mutual activation that is central to the initiation and regulation of immunity.

The expression of CD40L is recognized as a rate-limiting event controlling the immune response. Even modest changes in CD40L expression can have significant biological effects as a 1.1- to 2.0-fold increase in CD40L produces a 4- to 5-fold increase in B cell antibody production [11] and a similar increase in B cell proliferation and monocyte matrix metalloproteinase (MMP) production [11], [12], [13]. Conversely, the lack of a functional CD40L gene in individuals suffering from X-linked hyper-IgM syndrome results in immunodeficiency characterized by recurrent infections, generalized defects in cell-mediated immunity, abnormal lymph node architecture, and an inability to produce IgG, IgA, and IgE antibodies [10].

Many cytokine receptors and members of the TNF receptor superfamily are naturally expressed as both membrane-linked and soluble forms [14]. A number of soluble proteins corresponding to the extracellular portions of membrane-bound receptors have been identified in biological fluids including a recently described soluble form of the CD40L protein [15]. Soluble CD40L (sCD40L) is a truncated form (15-18 kDa) of the protein that is cleaved at methionine 113 and lacks the transmembrane domain of the full-length 28–33 kDa protein [16]. Despite this truncation, sCD40L retains considerable biological activity and the ability to bind to the CD40 receptor as a trimeric ligand [16], [17], [18]. Elevated serum levels of sCD40L have been associated with various diseases ranging from atherosclerosis, unstable angina, hypercholesterolemia, systemic lupus erythematosus, and inflammatory bowel disease [15], [19], [20], [21], [22]. Soluble CD40L is speculated to play a role in the pathogenesis of these diseases by acting on CD40 receptor bearing bystander cells in a cytokine-like fashion. In these circumstances, sCD40L may either regulate distinct signaling pathways or potentially complement the biological activity of membrane bound CD40L.

To date, very limited information regarding mechanisms that control the production of sCD40L have been described although it has been suggested that proteolytic cleavage of membrane CD40L on T cells may give rise to the 18 kDa protein [23], [24]. In support of this hypothesis, this mechanism has been shown to occur for platelet-derived sCD40L [25]. The proteolytic release of the extracellular domains of many cell-surface proteins, a process known as shedding, is becoming increasingly recognized as a common regulatory mechanism in mammalian cells. The modification of the cell surface in this manner can alter the cell’s responsiveness to its environment and release biologically active soluble regulatory factors. In many cases, protein shedding is enhanced by stimulation with the protein kinase C (PKC) agonist, phorbol-12-myristate-13-acetate (PMA). This mechanism is central for the release of a soluble form of TNF-α from its membrane-bound precursor. Although CD40L belongs to the TNF superfamily, no direct evaluation of this mechanism in the generation of sCD40L from T cells has been reported. Here, we demonstrate that various activation stimuli independently regulate the production of soluble and membrane CD40L expression, that phorbol ester stimulation enhances sCD40L production, and that this process is blocked by metalloproteinase inhibitors. We provide further evidence implicating ADAM-10 as a metalloproteinase involved in CD40L proteolytic cleavage.