CD93 Is Rapidly Shed from the Surface of Human Myeloid Cells and the Soluble Form Is Detected in Human Plasma

Abstract

CD93 is a highly glycosylated transmembrane protein expressed on monocytes, neutrophils, endothelial cells, and stem cells. Antibodies directed at CD93 modulate phagocytosis, and CD93-deficient mice are defective in the clearance of apoptotic cells from the inflamed peritoneum. In this study we observe that CD93, expressed on human monocytes and neutrophils, is susceptible to phorbol dibutyrate-induced protein ectodomain shedding in a time- and dose-dependent manner. The soluble fragment found in culture supernatant retains the N-terminal carbohydrate recognition domain and the epidermal growth factor repeats after ectodomain cleavage. Importantly, a soluble form of the CD93 ectodomain was detected in human plasma, demonstrating that shedding is a physiologically relevant process. Inhibition of metalloproteinases with 1,10-phenanthroline inhibited shedding, but shedding was independent of TNF-α-converting enzyme (a disintegrin and metalloproteinase 17). Phorbol dibutyrate-induced CD93 shedding on monocytes was accompanied by decreased surface expression, whereas neutrophils displayed an increase in surface expression, suggesting that CD93 shed from the neutrophil surface was rapidly replaced by CD93 from intracellular stores. Cross-linking CD93 on human monocytes with immobilized anti-CD93 mAbs triggered shedding, as demonstrated by a decrease in cell-associated, full-length CD93 concomitant with an increase in CD93 intracellular domain-containing cleavage products. In addition, the inflammatory mediators, TNF-α and LPS, stimulated ectodomain cleavage of CD93 from monocytes. These data demonstrate that CD93 is susceptible to ectodomain shedding, identify multiple stimuli that trigger shedding, and identify both a soluble form of CD93 in human plasma and intracellular domain containing cleavage products within cells that may contribute to the physiologic role of CD93.

Regulation of CD93 Cell Surface Expression by Protein Kinase C Isoenzymes

“…More recently, it was reported that CD93 expressed on monocytes and neutrophils is susceptible to phorbol dibutyrate (PDBu)-induced protein ectodomain shedding and that the soluble fragment in human plasma retains the N-terminal carbohydrate recognition domain and the EGF repeats (1). In addition, this report indicated that PDBu-induced CD93 shedding on monocytes was accompanied by a decrease in CD93 cell surface expression, whereas neutrophils displayed an increase in cell surface expression and CD93 shed from the neutrophil surface was rapidly replaced by CD93 from intracellular stores (1). These data, including that from our study, strongly indicate that CD93 has physiological roles in various immune responses.…”

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A

“…For example, thrombomodullin is expressed in endothelial cells and has anticoagulant activity resulting from sequestration of thrombin and activation of protein C (Maruyama et al, 1985;Esmon, 1995). CD93 is expressed in endothelium, cells of myeloid lineage and stem cells (Bohlson et al, 2005). Although not expressed by endothelium (MacFadyen et al, 2005), TEM1/endosialin mediates tumor, but not developmental angiogenesis.…”

Soluble CD93 Induces Differentiation of Monocytes and Enhances TLR Responses

“…This suggested that sCD93 was proteolytically cleaved from the surface-bound form (7). Further studies showed that CD93 was rapidly shed from the surface of human myeloid cells, and sCD93 was detected in human plasma (21,22). Additionally, Greenlee et al (23) demonstrated production of sCD93 in vivo under inflammatory conditions.…”

“…Recently, two separate studies reported the identification of a soluble form of CD93 (sCD93) (7,21). Initially, it was demonstrated that hypoglycosylation of CD93 caused an increase in the levels of sCD93 in culture medium.…”