Short communicationEctodomain shedding by ADAM17 (a disintegrin and metalloproteinase 17) in canine neutrophils
Introduction
ADAM17 (a disintegrin and metalloproteinase 17) is a transmembrane protease that is constitutively expressed by most cells in the body (Lambrecht et al., 2018). It cleaves its substrates on the cell surface typically in a cis manner at a specific extracellular site, a process referred to as ectodomain shedding. Its catalytic activity is highly inducible upon cell activation by a broad range of stimuli. This process occurs very rapidly and involves ADAM17 transitioning from a low to high activity state through a conformational change and intermolecular interactions (Mishra et al., 2017). ADAM17 substrates have a key role in regulating inflammatory and immune processes and include various cytokines, their receptors, and adhesion molecules (Lambrecht et al., 2018; Mishra et al., 2017; Zunke and Rose-John, 2017). ADAM17 has been well-characterized in human and mouse neutrophils where it directly regulates various effector functions, including their activation, migration, and phagocytosis of antibody-opsonized targets (Mishra et al., 2017). Aberrant ADAM17 induction, however, has pathological consequences (Lambrecht et al., 2018; Mishra et al., 2017; Zunke and Rose-John, 2017). Using models of sepsis, we have reported that mice with ADAM17-null leukocytes have significantly reduced mortality (Long et al., 2012, 2010; Mishra et al., 2016). This was associated with increased neutrophil recruitment, reduced bacteria levels at sites of infection, and markedly reduced systemic levels of proinflammatory cytokines, establishing a central role of ADAM17 in sepsis pathogenesis (Long et al., 2012, 2010; Mishra et al., 2016). Moreover, in human patients, ADAM17 activity and a functional polymorphism of its gene corresponded with sepsis progression (Kermarrec et al., 2005; Shao et al., 2016).
One of the best studied substrates of mouse and human ADAM17 is the leukocyte adhesion protein CD62L (L-selectin) (Ivetic, 2018; Mishra et al., 2017). Canine CD62L has been characterized at the protein, cellular, and functional levels and is very similar to human CD62L (Abbassi et al., 1991; Crockett-Torabi and Fantone, 1997). Similar to human and murine CD62L (Jutila et al., 1990; Kishimoto et al., 1989, 1990), canine CD62L also undergoes a rapid downregulation in expression upon neutrophil activation, suggesting it is similarly regulated by ectodomain shedding (Abbassi et al., 1991). At this time, the function of ADAM17 in canine neutrophils has not been reported. We demonstrate its expression and that blocking its activity disrupts the downregulation of CD62L expression. Given that ADAM17 has a broad immunomodulatory role in humans (Lambrecht et al., 2018; Mishra et al., 2017; Zunke and Rose-John, 2017), this sheddase may be a key therapeutic target in dogs for different inflammatory disorders.
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Animals
Peripheral blood was collected from 15 healthy pet dogs with owner consent. All animals received routine veterinary examinations and vaccines. The dog breeds represented in this study include four mixed breed dogs, three Labrador retrievers, one beagle, one Weimaraner, and one German shorthaired pointer. Breed information was not readily available for five donors due to client de-identification at time of collection. Blood collection was carried out in strict accordance with the recommendations
Effects of selective ADAM17 inhibitors on CD62 L downregulation by activated canine neutrophils
The mAb CL2/6 recognizes canine CD62L and it has been reported that canine neutrophils rapidly downregulate CD62L upon their activation (Abbassi et al., 1991). We observed that neutrophil staining with this mAb rapidly and efficiently decreased upon their treatment with various stimuli, as determined by flow cytometry (Fig. 1A,B). To investigate the involvement of ADAM17 in the downregulation of canine CD62 L, we utilized several small molecule hydroxamate-based metalloproteinase inhibitors.
Declaration of Competing Interest
None.
Acknowledgments
We would like to thank Kathy Stuebner and Amber Winter from the University of Minnesota, College of Veterinary Medicine, Clinical Investigation Center for assistance in acquiring canine peripheral blood samples, Taylor DePauw with flow cytometry assistance, Dr. Jianming Wu for his assistance with the figures and copy editing the manuscript. This work was supported by the National Institutes of Health [grant number HL128580]. KS was supported by a Howard Hughes Medical Institute and Burroughs
References (39)
- et al.
Ectodomain shedding of the cell adhesion molecule Nectin-4 in ovarian cancer is mediated by ADAM10 and ADAM17
J. Biol. Chem.
(2017) - et al.
Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme
J. Biol. Chem.
(2000) - et al.
The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion
Blood
(2003) - et al.
Regulation and lectin activity of the human neutrophil peripheral lymph node homing receptor
Blood
(1990) - et al.
Potent, exceptionally selective, orally bioavailable inhibitors of TNF-alpha Converting Enzyme (TACE): novel 2-substituted-1H-benzo[d]imidazol-1-yl)methyl)benzamide P1’ substituents
Bioorg. Med. Chem. Lett.
(2008) - et al.
Cloning, sequencing and expression in the dog of the main amyloid precursor protein isoforms and some of the enzymes related with their processing
Neuroscience
(2010) - et al.
ADAM17 cleaves CD16b (FcgammaRIIIb) in human neutrophils
Biochim. Biophys. Acta
(2013) - et al.
Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer
Cancer Cell
(2006) - et al.
The shedding protease ADAM17: physiology and pathophysiology
Biochim. Biophys. Acta Mol. Cell. Res.
(2017) - et al.
Canine neutrophil margination mediated by lectin adhesion molecule-1 in vitro
J. Immunol.
(1991)