Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death

Abstract

Background: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. Methods: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. Results: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content .2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. Conclusions: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology. Key terms: lactadherin; phosphatidylserine; apoptosis; leukemia; annexin V Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death (1-3) and marks those cells for clearance by macrophages (1). Transient exposure of PS on viable cells accompanies sperm capacitation (4), phagocytosis of apoptotic cells (5), myotube formation (6), and neutrophil (7) and platelet stimulation (8). On stimulated platelets, exposed PS supports procoagulant reactions, and on lymphocytes PS externalization appears mechanistically coupled to the function of ion channels and the multidrug resistance protein in lymphocytes (9). However, in spite of the essential relationship of PS exposure to cellular function, the topology and kinetics of PS exposure remain only partially characterized.PS exposure is frequently detected through binding of fluorescence-labeled annexin V (3). The extent to which annexin V binds to a membrane is a complex function related to the free annexin V concentration, membrane PS content, phosphatidylethanolamine content, and ambient Ca 11 concentration (10-15). Indeed, recent reports indicate that the cooperative interaction of annexin V on the plasma membrane drives a mechanism whereby invagination of the plasma membrane is followed by internal vesiculation (16).The complexity of annexin V binding and the internalization of annexin V by stressed cells leaves uncertainty about the extent to which annexin V fluorescence represents plasma membrane PS exposure in early apoptosis.Lactadherin is also a PS-binding protein and has a domain structure that includes two epithelial growth factorlike domains and two le...

Bovine Lactadherin as a Calcium-independent Imaging Agent of Phosphatidylserine Expressed on the Surface of Apoptotic HeLa Cells

“…Finally, the involvement of PS binding was confirmed, inasmuch as admittance of a polyclonal antibody directed against the C2 PS binding domain of lactadherin quenched the staining capability of lactadherin. Collectively these experiments provide direct evidence that lactadherin cell attachment is mediated by PS binding alone and is not the result of specific or nonspecific interactions with other plasma membrane components, thus confirming earlier studies characterizing these binding properties (Andersen et al 2000;Shi et al 2004Shi et al ,2007.…”

“…However, recent flow cytometric and confocal microscopic studies suggest that lactadherin enables detection of surface exposure of PS in apoptotic suspension cells at an earlier time point than does annexin V (Dasgupta et al 2006;Shi et al 2007). In addition, lactadherin and annexin V are known to diverge in their specificity toward PS.…”

Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells

“…However, the studies of lactadherin binding have been very limited and the cell features recognized by the two proteins have not been compared. Our results of preferential internalization of annexin V by cells undergoing apoptosis agree with previous reports (3,29).…”

Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase

“…Virtually identical results were obtained when extracellular PS was detected with lactadherin ( Figure 4B), excluding that annexin V staining was of limited value to detect relevant procoagulant PS. 32 Importantly, there was no correlation between TF PCA and PS exposure on ATG-treated cells (r 5 20.03; Figure 4C). …”