Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding

Abstract

A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response.

The recruitment of leukocytes into the inflammatory foci is a cardinal event in the inflammatory response. This cell accumulation is preceded by a highly coordinated sequence of interactions between leukocytes and endothelial cells, a process termed adhesion cascade (1, 2). Neutrophils are, as part of the innate immune response against pathogens, the first immune cells to be recruited at inflammatory foci, exerting their function via the phagocytosis of foreign bodies, the releasing of several antimicrobial polypeptides stored in their intracellular granules (3), and the generation of oxygen-free radicals. Human neutrophils contain three major different types of granules: azurophilic or primary granules, specific or secondary granules, and gelatinase-rich or tertiary granules, as well as an apparently releasable organelle named secretory vesicle (3, 4). Specific and tertiary granules, together with secretory vesicles, constitute a reservoir of a wide array of plasma membrane proteins involved in the adhesion cascade, that are translocated to the cell surface in response to neutrophil activation. Azurophilic granules are mainly involved in the phagocytic and microbicidal function of neutrophils and thereby contain a large number of lytic enzymes (3, 4).

The effective regulation of neutrophil influx to tissue is important to minimize the destructive effect that neutrophil cytotoxic granule contents may produce in the normal tissue during the inflammatory response (5). Several adhesion molecules, including members of the selectin family, participate coordinately in the migration of flowing neutrophils through vascular endothelium (1, 2). Neutrophils use L-selectin to roll along endothelium in the initial phase of the adhesion cascade. In seconds, L-selectin is shed from the surface of rolling neutrophils and both rolling speed and neutrophil accumulation are increased when in vitro shedding is blocked (6). Thus, a correct regulation of the L-selectin ectodomain processing appears to be necessary for an efficient transendothelial migration of neutrophils (7). A member of the a disintegrin and metalloprotease domain (ADAM)4 family of membrane metalloproteinases, ADAM-17 (CD156b), also known as TNF-α-converting enzyme (TACE), has shown proteolytic activity on L-selectin (8). Although it is well-established that ADAM-17 is required for efficient L-selectin shedding, the fact that TACE-deficient cells generate a significant shedding of L-selectin (9), in addition to other evidence (7), strongly suggests that there may be other sheddase and/or additional factors involved in this process.

The ADAM protein family consists of a number of widely distributed transmembrane glycoproteins that play key roles in sperm-egg binding and fusion, muscle cell fusion, neurogenesis, susceptibility to type I hypersensitivity, and proteolysis of several membrane proteins (10, 11, 12). The basic structure of an ADAM family protein is well-conserved and comprises a cysteine-rich domain, a disintegrin domain, with adhesive properties, and a metalloproteinase domain responsible for the ectodomain shedding of membrane proteins and for the cleavage of extracellular matrix components (11). Catalytically active ADAMs are usually activated by furin-catalyzed removal of the prodomain. However, some ADAM members do not seem to be catalyzed by furin-like proteases, including ADAM-8 (CD156a), whose prodomain is removed by a metalloproteinase (13). ADAM-8, originally cloned from monocytic cells (14), is expressed mainly in cells of the immune system, such as monocytes, granulocytes, and B cells. Although mice lacking the ADAM-8 gene presents no major structural defects (15), different reports have suggested a role for ADAM-8 in a number of pathological processes (16, 17). Recently, ADAM-8 has been involved in osteoclast (OCL) fusion (18). Its expression is increased in OCLs compared with OCL precursors and stimulates OCL formation in murine bone marrow cultures. ADAM-8 has also shown to act as a sheddase. In particular, it is able to convert membrane-bound CD23 (19) and the neural cell adhesion molecule CHL1 into soluble forms (20). However, the regulation of the expression and/or function of ADAM-8 in cells of the immune system, where it is preferentially expressed, is still poorly understood. The recent implication of ADAM-8 in the allergic response (17), in addition to its potential role in the process of leukocyte infiltration shown by ADAM-8-transgenic mice (21) have increased the interest for this surface metalloproteinase in the regulation of the inflammatory response.

In this study, we describe the expression profile, localization, and regulation of ADAM-8 in human neutrophils. We show that ADAM-8 is localized both on the cell membrane and in the cytoplasmic granules of resting neutrophils. Upon cell activation, both in vitro and in vivo, ADAM-8 was mobilized from the granules to the cell membrane and finally released to the extracellular milieu by a metalloprotease-dependent proteolysis. The physiological relevance of these observations was supported by the finding that the presence of ADAM-8 in the knee joints of patients diagnosed with rheumatoid arthritis (RA) correlated with the intensity of the local inflammatory response. In addition, we show that both the membrane-bound and the active soluble form of ADAM-8 (sADAM-8) were able to cleave L-selectin from the plasma membrane, further supporting a key role for this metalloproteinase in neutrophil function at inflammatory sites.