ADAM17 cleaves CD16b (FcγRIIIb) in human neutrophils

Abstract

CD16b (FcγRIIIb) is exclusively expressed by human neutrophils and binds IgG in immune complexes. Cell surface CD16b undergoes efficient ectodomain shedding upon neutrophil activation and apoptosis. Indeed, soluble CD16b is present at high levels in the plasma of healthy individuals, which appears to be maintained by the daily turnover of apoptotic neutrophils. At this time, the principal protease responsible for CD16b shedding is not known. We show that CD16b plasma levels were significantly decreased in patients administered a selective inhibitor targeting the metalloproteases ADAM10 and ADAM17. Additional analysis with inhibitors selective for ADAM10 or ADAM17 revealed that only inhibition of ADAM17 significantly blocked the cleavage of CD16b following neutrophil activation and apoptosis. CD16b shedding by ADAM17 was further demonstrated using a unique ADAM17 function-blocking mAb and a cell-based ADAM17 reconstitution assay. Unlike human CD16, however, mouse CD16 did not undergo efficient ectodomain shedding upon neutrophil stimulation or apoptosis, indicating that this mechanism cannot be modeled in normal mice. Taken together, our findings are the first to directly demonstrate that ADAM17 cleaves CD16 in human leukocytes.

NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17)

“…This mechanism has been difficult to study in the mouse because CD16 is not shed and is therefore human specific. 32 Although little is known how ADAM17 directly interacts with its various substrates in resting (constitutive shedding) and activated cells, our data suggests a role for ADAM17 in the cleavage of CD16 and regulating NK cell function mediated through ADCC.…”

“…To further establish the role of ADAM17 in CD16 and CD62L shedding after IL-12 and IL-18 stimulation, we used a specific ADAM17 inhibitory monoclonal antibody generated by phage display that contains individual antibody variable domains to 2 distinct ADAM17-specific epitopes. 25,32 This antibody effectively blocked CD16 down-regulation ( Figure 4D), further demonstrating that ADAM17 cleaves CD16 on the cell surface of NK cells. Moreover, the level of inhibition of CD16 shedding by the ADAM17 inhibitor and antibody were equivalent, indicating that the inhibitor did not block additional sheddases ( Figure 4D).…”

“…We have determined that CD16B shedding from neutrophils also requires ADAM17. 32 The cleaved portion of the receptor may be involved in removing excess free antibody during antibody-mediated responses. In neutrophils, cleaved CD16B can bind to: complement receptors (CR3 and CR4) to induce monocyte production of IL-6 and IL-8, 40 B cells to suppress antibody production, 41 and dendritic cells to induce maturation.…”

“…Cells were stained with antibodies against extracellular antigens and were then fixed and permeabilized before intracellular staining to detect IFN-g. A highly selective ADAM17 inhibitor (BMS566394) from Bristol-Myers Squibb (referred to as inhibitor 32) 23 was used at a 10 mM concentration, as previously described, 24 and was added to 1 3 10 6 /mL purified NK cells 30 minutes before testing. We also used a specific ADAM17 inhibitory monoclonal antibody (D1[A12], 6 mg/mL) generated by phage display that contains individual antibody variable domains to 2 distinct ADAM17-specific epitopes 25 to confirm ADAM17 specificity (kindly provided by Dr Gillian Murphy, University of Cambridge, UK).…”

A double-edged sword - the role of human ADAM17 in NK cell activity

“…Mouse models for ADAM17 deficiency are partial since knock-out of ADAM17 in mice is embryonic lethal [6] . In addition, mouse CD16 was shown not to be cleaved by ADAM17 [7] . Furthermore, when a single amino acid in the RESEARCH HIGHLIGHT human CD16 is replaced by the corresponding amino acid in mice, the majority of the cleavage is lost [8] .…”