Reverse signalling of membrane TNF in human natural killer cells: a comparison of the effect of certolizumab pegol and other anti-TNF agents

Abstract

Background: Certolizumab pegol (CZP) has a different structure compared with the other anti-TNF agents in that it is the only PEGylated Fab' anti-TNF and it lacks an Fc region. Membrane TNF-α (mTNF-α) can mediate 'reverse signalling' into the cells on which it is expressed, and differences have been seen among the anti-TNFs in mediating this effect. Natural killer (NK) cells are the only lymphocyte subpopulation that constitutively expresses high levels of mTNF-α, and it has been suggested that they may be involved in rheumatoid arthritis (RA) pathogenesis. Objectives: To further understand the role of mTNF-α-expressing NK cells during anti-TNF treatment, we examined the effect of CZP and the other anti-TNFs, adalimumab (ADA), etanercept (ETA) and infliximab (IFX), by reverse signalling via mTNF-α on cellular activities of peripheral blood NK cells. Methods: Peripheral blood mononuclear cells from healthy volunteers were isolated and incubated in the presence of 100 U/mL recombinant human (rh) interleukin-2 (IL-2) for 20 h. Subsequently, 10 μg/mL anti-TNF agents or isotypic controls (human IgG1k and Fab' PEG) were added for a further 4 h. NK cells were identified by flow cytometry via CD3 and CD56 markers, and antibody-dependent cellular cytotoxicity (ADCC) was measured by loss of cell membrane integrity, by binding of 7-aminoactinomycin D (7-AAD) to DNA. For supernatant analysis of soluble cytokine production and β-hexosaminidase release as an index of cell degranulation, NK cells were isolated by positive selection with magnetic separation (Miltenyi Biotec) and then incubated with rhIL-2 and anti-TNF agents. The concentration of the soluble cytokine interferon-γ (IFN-γ) was determined by enzyme-linked immunosorbent assay. β-hexosaminidase release was quantified upon enzymatic cleavage of 4-methylumbelliferyl N-acetyl-β-D-glucosaminide in citrate buffer (0.1 M, pH 4.5) by spectrophotometric analysis using 360-nm excitation and 465-nm emission filters. Results: When isolated NK cells were incubated with anti-TNF agents in the presence of rhIL-2, IFN-γ production was significantly increased from the control level of 84 pg/mL to 1.1 ng/mL. All 4 anti-TNF agents stimulated NK cell degranulation, as measured by β-hexosaminidase levels, to a level of 27% degranulation compared with a control level of 3%. However, ADCC measured in NK cells was detectable only with ADA, ETA and IFX (44.3%, 46.4% and 47.9%, respectively) and not with CZP (1%) due its lack of an Fc region. Conclusions: Our observations suggest that anti-TNF agents may result in increased NK cellmediated cytotoxicity by promoting the release of multiple cytotoxic effector molecules and inflammatory cytokines via reverse signalling through constitutively expressed mTNF-α. CZP can activate NK cells but, in contrast to conventional anti-TNFs, does not mediate ADCC due to its unique structure (lacking an Fc region).