Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro

Abstract

We have previously reported that neutrophilic granulocytes rapidly release part of their Fc gamma RIII from the plasma membrane upon in vitro activation, probably by proteolytic cleavage. In plasma and other body fluids, released or soluble Fc gamma RIII has been found in considerable amounts. In the present study, neutrophils were kept in maintenance culture for 18 to 24 hours. Forty percent of the neutrophils completely lost Fc gamma RIII, and the remainder of the cells showed a 60% decrease in Fc gamma RIII expression on their surface. Released Fc gamma RIII was detected in the culture supernatant. Nevertheless, more than 90% of the cells was viable as judged by hydrolysis of fluorescein diacetate. The presence of interferon gamma, granulocyte colony-stimulating factor, or granulocyte- macrophage colony-stimulating factor, but not interleukin-3 (IL-3), IL- 6, or IL-8, in the culture medium increased the number of cells that still expressed Fc gamma RIII. We found that this loss of Fc gamma RIII was not the result of cell activation but correlated strongly with apoptosis. The Fc gamma RIII-negative subpopulation exhibited typical morphologic changes, such as nuclear condensation and DNA fragmentation. Furthermore, this subpopulation appeared to have acquired the property of binding Annexin V, a calcium-dependent, phospholipid-binding protein with high affinity for phosphatidylserine. The external exposure of this phospholipid by cells has been reported to occur during apoptosis. The property of Annexin V binding was not shared by the nonapoptotic, Fc gamma RIII-positive subpopulation. In this respect, we identified binding of Annexin V as an convenient marker for apoptotic cells. Our results indicate that soluble Fc gamma RIII in body fluids might be derived for a large part from neutrophils undergoing apoptosis in the tissues.

Association of Ceramide Accumulation with Photodynamic Treatment-Induced Cell Death

“…2 and 3). A time overlap between nuclear changes (DNA fragmentation, chromatin condensation) and PS exposure has been observed by others (37)(38)(39). The simultaneous appearance of AV( +)/PI( +) and AV( +)/PI( -) cell subpopulations in U937 cells has been shown by Fabisiak et al (40) in murine cell lines, suggesting that cells are undergoing apoptosis, as well as necrosis.…”

Clearance of apoptotic neutrophils and resolution of inflammation

“…Neutrophils downregulate a number of membrane proteins as they age, including CD16 (FcγRIII), CD31 (PECAM-1), CD32, CD35 (Complement receptor 1), CD43, CD45, CD44, CD47 (IAP), CD50 (ICAM-3), CD55 (Complement regulatory protein DAF), CD62L (L-selectin), CD63, CD66b, CD87 (uPAR), CD88 (C5a receptor), and CD120a (tumor necrosis factor receptor 1), thus significantly changing the apoptotic neutrophil surface. [35][36][37][38][39][40] Some of these proteins, including CD31 and ICAM-3, are also functionally modified, 41,42 whereas other proteins, including ICAM-1, are upregulated on apoptotic neutrophils. [41][42][43] Finally, new antigens are expressed on the surface of apoptotic neu-…”

p38 Mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase activities have opposite effects on human neutrophil apoptosis

“…As an indicator of neutrophil apoptosis, we analyzed the exposure of phosphatidylserine (PS) on the surface of apoptotic cells by staining with FITC-conjugated annexin V, a calcium-dependent phospholipid-binding protein with high affinity for PS (21). We performed specific binding of annexin V by incubating neutrophils (2 × 10 5 cells) for 30 min at 4 û C in the dark in 60 µl of binding buffer (150 mM NaCl, 2.5 mM CaCl 2 , 10 mM HEPES; pH 7.4) containing a saturating concentration of annexin V. We determined nonspecific binding by using calcium-free binding buffer (150 mM NaCl; 10 mM EDTA; 10 mM HEPES, pH 7.4).…”