Transmembrane TNF-alpha reverse signaling leading to TGF-beta production is selectively activated by TNF targeting molecules: Therapeutic implications

Abstract

Tumor necrosis factor (TNF)-α is a potent pro-inflammatory cytokine exerting pleiotropic effects on various cell types. It is synthesized in a precursor form called transmembrane TNF-α (mTNF-α) which, after being processed by metalloproteinases, is released in a soluble form to mediate its biological activities through Type 1 and 2 TNF receptors in TNF receptor expressing cells. In addition to acting in soluble form, TNF-α also acts in the transmembrane form both as a ligand by activating TNF receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into mTNF-α bearing cells. Since the discovery that TNF-α plays a determining role in the pathogenesis of several chronic inflammatory diseases, anti-TNF agents are increasingly being used in the treatment of a rapidly expanding number of rheumatic and systemic autoimmune diseases, such as rheumatoid arthritis, Crohn’s disease, psoriasis, psoriatic arthritis, ankyloting spondylitis, Wegener granulomatosis and sarcoidosis. There are 5 TNF antagonists currently available: etanercept, a soluble TNF receptor construct; infliximab, a chimeric monoclonal antibody; adalimumab and golimumab, fully human antibodies; and certolizumab pegol, an Fab’ fragment of a humanized anti-TNF-α antibody. Though each compound can efficiently neutralize TNF-α, increasing evidence suggests that they show different efficacy in the treatment of these diseases. These observations indicate that in addition to neutralizing TNF-α, other biological effects induced by TNF-α targeting molecules dictate the success of the therapy. Recently, we found that mTNF-α reverse signaling leads to transforming growth factor (TGF)-β production in macrophages and anti-TNF agents selectively trigger this pathway. In this review we will focus on the potential contribution of the activation of the mTNF-α signaling pathway to the success of the anti-TNF therapy.

Introduction

Tumor necrosis factor (TNF)-α is a powerful pro-inflammatory cytokine which plays a critical role in the pathogenesis of various chronic inflammatory diseases, such as rheumatoid arthritis [1]. Though TNF-α is a pleiotropic cytokine, it is produced primarily by cells of the monocytic lineage − such as macrophages, Langerhans cells, microglia, astroglia, Kupffer cells, and alveolar macrophages [2], [3]. In addition, other cells and tissues including lymphoid cells, endothelial cells, mast cells, fibroblasts, and neuronal tissue were also reported to express TNF-α [4]⋅ TNF-α is barely expressed by quiescent cells. Its production in macrophages can be induced by a wide variety of stimuli, including bacteria, tumor cells, immune complexes, viruses, complement factors, cytokines, irradiation, ischemia/hypoxia and trauma. The biosynthesis of TNF-α is strictly controlled. Its gene expression is regulated at the transcriptional level by several transcription factors, such as nuclear factor kappa B (NFκB) and nuclear factor activated T cells (NF-AT). The TNF-α mRNA appears within 30 min in response to most of the stimuli, but the TNF protein expression is controlled mostly post-transcriptionally. Thus, TNF-α production is also regulated at the translational level via the UA-rich sequence in the 3′ untranslated region of human TNF-α mRNA [5]. Translation of TNF mRNA leads to the intracellular production of trimeric pro-TNF protein, which matures and is inserted into the plasma membrane as transmembrane TNF-α (mTNF-α).

Transmembrane TNF-α is expressed as a cell surface II polypeptide consisting of 233 amino acid residues [6], [7], [8]. After being processed by metalloproteinases such as TNF-α-converting enzyme [9], [10], the soluble form of TNF-α (s TNF-α) of 157 amino acid residues is released and mediates its biological activities by triggering Type 1 and 2 TNF receptors (TNF-R), which also function as receptor trimers [11]. However, increasing evidences suggest that not only soluble TNF-α, but also its precursor form, mTNF-α, is involved in the regulation of the inflammatory response acting primarily locally.

TNF-R1 is constitutively expressed in most tissues, whereas protein levels of TNF-R2 are highly regulated, and the receptor typically appears in cells of the immune system. Binding of TNF-α onto TNF-R1 is considered to be irreversible, whereas binding of TNF-α onto TNF-R2 has both rapid on and off kinetics. Therefore, it is believed that TNF-R2 might act as a “ligand passer” to TNF-R1 in some cells by elevating the local concentration of TNF-α at the cell surface [12]. In the vast majority of cells, TNF-R1 is the key component in mediating TNF signaling, whereas in the lymphoid system TNF-R2 plays the determining role [11]. Because TNF-R2 can only be fully activated by the transmembrane, but not by the soluble form of TNF, it is very likely that its real contribution to the immune responses is generally underestimated [13]. The reason for this difference in binding is not fully explained yet, but the different stabilities of the individual ligand/receptor complexes very likely contribute to the phenomenon [13], [14]. Though TNF-R1 activation generally triggers pro-inflammatory responses, apoptosis-related signaling events can also be induced, whereas the TNF-R2 signal, especially in activated T cells, induces cell survival pathways that can contribute to the initiation of cell proliferation [15].

In response to an inflammatory signal, similar to mTNF-α, the extracellular domains of both TNF-Rs can also be proteolytically cleaved by matrix metalloproteinase family of enzymes, yielding soluble receptor fragments (sTNF-Rs) [16]. sTNF-Rs retain the ability to bind TNF-α, and thus function as endogenous inhibitors of it [17]. Due to the lack of cooperativity in ligand binding, however, the affinities of sTNF-Rs are low compared to their membrane-bound forms (mTNF-Rs). TACE was suggested to act as a processing enzyme for TNF-R1 cleavage as well [18], and during inflammation this cleavage plays a determining role in controlling cellular TNF responsiveness, as cleavage-resistant TNF-R1 mutations are linked with dominantly inherited autoinflammatory syndromes named TNF-R1-associated periodic syndromes [19] (Fig. 1).

Even though TNF-α participates in various normal homeostatic mechanisms including host defense or wound healing [20], dysregulated TNF-α production has been detected in a wide variety of inflammatory diseases. In addition, TNF-α-activated macrophages are well known to contribute to the immunopathology of many autoimmune diseases [21]. Thus, macrophage-derived TNF-α has been implicated in the pathomechanism of rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Wegener granulomatosis (WG), sarcoidosis (S), psoriasis (P), psoriatic arthritis, ankylosing spondylitis (AS), juvenile chronic arthritis, atherosclerosis, and sepsis [22], [23], [24], [25], [26], [27].

Nowadays five TNF-α blocking agents are currently licensed for treating TNF-α-driven diseases. These five drugs are: 1) etanercept, a recombinant human soluble fusion protein of TNF-R2 coupled to the Fc portion of IgG [28]; 2) infliximab, an anti-TNF human-murine chimeric IgG1 monoclonal antibody [29]; 3) adalimumab, a human anti-human TNF-α antibody that was produced by phage display [30]; (4); golimumab, a human anti-TNF-α IgG1κ monoclonal antibody that can be administered by the patient [31]; and 5) certolizumab pegol, an Fab’ fragment of a humanized anti-TNF-α antibody [32]. Though the pharmacokinetic properties of Fab’ in vitro are usually poor, attachment of a 40 kDa polyethylene glycol (PEG) moiety markedly increased the half-life time of certolizumab to a value comparable with that of a whole antibody. These drugs are almost equal in neutralizing of both soluble and membrane-bound TNF-α [33], however, significant differences can be found as to the disease they are effective. Thus, infliximab, golimumab, adalimumab and certolizumab pegol are effective, while etanercep failed in the trials of Crohn’s disease, Wegener granulomatosis and sarcoidosis and is less effective in the treatment of skin psoriasis [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49] (Table 1). These observations suggest that in addition to the neutralization of TNF-α, other effects induced by anti-TNF targeting molecules dictate the therapeutic results.