ADAM17 activity during human neutrophil activation and apoptosis

Abstract

Substrates of the metalloprotease ADAM17 (also known as TNF-a converting enzyme or TACE) undergo ectodomain shedding and include various inflammatory modulators. Though polymorphonuclear leukocytes contribute significantly to inflammation, direct analyses of ADAM17 on human neutrophils are very limited. In addition, the current understanding of the processes regulating ADAM17 activity primarily relate to its rapid activation. Therefore, to extend insights into the mechanisms of ADAM17 activity, we examined its surface expression and the shedding of its substrates during extended periods of neutrophil activation and apoptosis. Contrary to studies with immortalized hematopoietic cell lines, we report that surface expression of ADAM17 is maintained by human neutrophils activated with formyl peptides or by FcR/complement receptormediated phagocytosis. Interestingly, bacterial phagocytosis resulted in a significant increase in ADAM17 expression several hours after pathogen engulfment. We provide novel evidence that ADAM17 surface expression is also maintained during spontaneous and anti-Fas-induced neutrophil apoptosis. The well-validated ADAM17 substrates Lselectin and proTNF-a were shed efficiently by neutrophils under each of the conditions tested. Our data thus indicate prolonged ADAM17 expression during neutrophil effector functions. The implications of this may be a role by ADAM17 in both the induction and down-regulation of neutrophil activity. IntroductionParticular members of the a disintegrin and metalloprotease (ADAM) family function as sheddases, and the cell surface proteins that undergo ectodomain proteolysis participate in most cellular processes and various pathologies, such as Alzheimer's disease, cancer, and damaging inflammation [1,2]. At this time, ADAM17[also referred to as TNF-a converting enzyme (TACE)] is the best characterized of the sheddases, and its putative substrates are widespread, including various inflammatory modulators (e.g. TNF-a, IL-1RII, TNFR55, TNFR75, IL-6R, MCSF-R, fractalkine, VCAM-1, and L-selectin) [3][4][5][6][7][8][9][10][11][12]. L-selectin and proTNF-a in particular are wellcharacterized substrates of ADAM17. Gene-targeting of ADAM17 is lethal [5], yet studies have revealed that immature and mature leukocytes as well as cell lines deficient in functional ADAM17 are greatly impaired in their shedding of L-selectin and proTNF-a [4,5,7,12,13], indicating that the primary means of their shedding is not functionally redundant.Ectodomain shedding can be rapid and robust. For instance, shedding of the *1 Â 10 5 L-selectin molecules on the surface of a leukocyte occurs in the first few To that end, we examined the surface expression and activity of ADAM17 on human neutrophils during periods of stimulation and apoptosis. We demonstrate prolonged ADAM17 expression on the surface of human neutrophils under both conditions. We also show that ADAM17 substrates undergo efficient turnover during extended neutrophil activation and apoptosis. Our study thus provides new insights into th...

Different Signaling Pathways Stimulate a Disintegrin and Metalloprotease-17 (ADAM17) in Neutrophils during Apoptosis and Activation

“…These procedures were performed in accordance with protocols approved by the Institutional Review Board: Human Subjects at the University of Minnesota. Neutrophils were enriched as described previously (12,20,21), and cell viabilities were assessed by exclusion of the vital dye Trypan Blue. All Jurkat lines used in this study were cultured in RPMI 1640 complete media supplemented with 8% heat-inactivated FBS and antibiotics (penicillin and streptomycin) at 37°C in a humidified 5% CO 2 incubator.…”

“…Apoptotic neutrophils are eventually engulfed and disposed of by macrophages (9 -11). The catalytic activity of ADAM17 is also induced during neutrophil apoptosis (12)(13)(14). This is significant considering the pleiotropic activity of ADAM17 in regulating inflammation and that an immense number of senescent neutrophils in the blood and recruited neutrophils at sites of inflammation undergo apoptosis (8,15).…”

Acidic Mammalian Chitinase Is Secreted via an ADAM17/Epidermal Growth Factor Receptor-dependent Pathway and Stimulates Chemokine Production by Pulmonary Epithelial Cells

“…A primary anti-EGFR was from Abgent (San Diego, CA). Anti-human ADAM17 monoclonal antibody was purchased from R&D Systems (Minneapolis, MN) and employed as reported previously (20). Secondary Pe-Cy7-and APC-Cy7-labeled antibodies were from Santa Cruz Biotechnology.…”

Attenuated, Oncolytic, but Not Wild-Type Measles Virus Infection Has Pleiotropic Effects on Human Neutrophil Function

“…L-Selectin shedding, a marker of neutrophil activation, was assessed by flow cytometry (22,23). Freshly isolated neutrophils expressed high level of surface L-selectin, indicating that the cells were not already activated by the isolation procedures.…”