Effect of different tumor necrosis factor (TNF) reactive agents on reverse signaling of membrane integrated TNF in monocytes
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).
Section snippets
All three TNF antagonists can induce reverse signaling of mTNF and thus suppress the apoptosis inducing capacity in the supernatant of LPS-stimulated MO
All experiments presented have been performed with the monocytic cell line Mono Mac 6 (MM6) [39]. Here we tested in comparison with the previously [13], [40] used anti-TNF antibody 195F two additional TNF antagonists that are currently under clinical investigation, for their potency to induce reverse signaling in MO, i.e. Ifx (a chimeric monoclonal antibody with murine variable regions and human IgG1 and κ constant region [30]) and Eta (a fusion protein with extracellular domain of the p75 TNF
Discussion
TNF is believed to play a key role in the pathogenesis of acute and chronic inflammatory disorders such as CD [41] or RA [42], and in transplant related complications, including acute graft versus host disease (GvHD) [43], [44], following allogeneic stem cell transplantation.
Ifx and Eta are able to neutralize TNF–TNF receptor (TNFR) interactions in the “classical” direction, and both the TNF reactive agents have shown efficacy in RA [26], [34], [45], but only Ifx is efficacious in CD [23], [35]
Cell culture and reagents
The monocytic cell line Mono Mac 6 (MM6) was kindly provided by Prof. Dr. H.W.L. Ziegler-Heitbrock (University of Leicester, U.K.) [39]. The MM6 cells were cultured in RPMI, supplemented with 10% fetal calf serum (FCS), l-glutamine (2 mmol/l), MEM non-essential amino acids (1−2x), vitamins (1x), antibiotics, MEM Sodium Pyruvate (1 mM) and OPI-supplement (Sigma, Deisenhofen, Germany). Human umbilical vein endothelial cells (HUVEC) were prepared according to the method of Jaffe et al. [53].
Acknowledgments
This work is supported by grants no. Ei68/2-3 and Ei68/3-3 from the Deutsche Forschungsgemeinschaft (DFG) to G.E.
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