Effect of different tumor necrosis factor (TNF) reactive agents on reverse signaling of membrane integrated TNF in monocytes

Abstract

Reverse signaling of transmembrane TNF (mTNF) contributes to the versatility of this cytokine superfamily. Previously, we could demonstrate that mTNF acting as receptor confers resistance to bacterial lipopolysaccharide in monocytes and macrophages (MO/MΦ). Reverse signaling can be induced by incubation with the monoclonal anti-TNF antibody 195F and other TNF antagonists, such as the humanized monoclonal antibody infliximab and the humanized soluble TNF receptor construct etanercept, respectively, all in former or present clinical use. Here, we addressed the question whether there are differences in modulating the LPS response in MO/MΦ among these three antagonists. Whereas 195F and infliximab suppress both, the release of an LPS-induced endothelial cell apoptotic factor and proinflammatory cytokines, etanercept only protected against the LPS-triggered apoptosis activity, but left the LPS-induced cytokine release unchanged. These data could have clinical impact with regard to TNF neutralization strategies.

Introduction

Tumor necrosis factor (TNF) is a multifunctional cytokine produced by activated monocytes (MO) and macrophages (MΦ) as well other cell types [1], [2], [3]. TNF plays a critical role in several inflammatory disorders, including endothelial cell (EC) damage following allogeneic stem cell transplantation [4], inflammatory bowel diseases, such as Crohn's disease (CD) and autoimmune diseases, including rheumatoid arthritis (RA) [5], [6]. It is known that the transmembrane form of TNF (mTNF) is able to induce apoptosis in EC [7], whereas, importantly, the soluble form is ineffective in that respect. In several other inflammatory settings mTNF is known to elicit signals distinct from the soluble form of this cytokine [8], [9], [10], [11]. In addition, LPS activated MO or MΦ can induce apoptosis in EC by cell–cell contact via mTNF or by their cell-free supernatants [12], containing a yet unidentified soluble apoptosis factor, whose identification is in work. The LPS-induced apoptotic activity and cytokine release can be suppressed by preincubation of MO/MΦ with mTNF binding agents. This suppression is a result of reverse signaling by mTNF [13]. The phenomenon of reverse signaling has been described for several members of the TNF superfamily, e.g. for CD30L [14], [15], CD40L [16], Ox40L [17], CD95L (FasL) [18], CD137L [19], [20] and mTNF [13], [21], [22] itself.

To inhibit TNF activity in RA and CD, two distinct TNF antagonists, infliximab (Ifx) [23], [24], [25] and etanercept (Eta) [26], [27], [28] are currently evaluated in clinical trials. Although both are potent neutralizers of TNF bioactivity, there are fundamental differences in immune complex formation and binding specificities to TNF [29]. Ifx, a chimeric monoclonal antibody with murine variable regions and human immunoglobulin (Ig) G1 constant regions [30], is highly effective in the treatment of CD [31]. Eta, a dimeric fusion protein consisting of the extracellular portion of the p75 TNF receptor linked to the Fc domains of a type 1 human immunoglobulin (IgG1) [32], has comparable effects to those observed with Ifx in rheumatoid disease [33], [34]. But in CD, Eta is less effective than Ifx [35]. This observation might be explained with additional data showing that Ifx can induce apoptosis in MO [36] and T-lymphocytes [37]. These authors concluded, that Ifx and not Eta binds to the mTNF on lamina propria T cells or on peripheral MO from patients with CD, and thus only Ifx is effective in inducing programmed cell death. Another possibility could be that Eta also binds to mTNF, but with less avidity as compared to Ifx, and this type of signal may not be strong enough to trigger a cell death program [29], [38].

We hypothesized that reverse signaling of mTNF might be responsible for the distinct efficacy of these two TNF neutralizing agents in CD. The present study compared the published effects of reverse signaling induced by the anti-TNF antibody 195F [13] with that of Ifx and Eta. As the relevant cell system we have used the monocytic cell line Mono Mac 6 (MM6).