Live Cell Imaging of Outward and Inward Vesiculation Induced by the Complement C5b-9 Complex

Oren Moskovich,Zvi Fishelson

doi:10.1074/jbc.m703742200

Abstract

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.

Highlights

Activation cascade may occur through the classical, alternative, or lectin pathways
The three initiation pathways lead to activation of the terminal complement pathway and to assembly of the membrane attack complexes (MACs)2 that are composed of the complement components C5b, C6, C7, C8, and C9
To test the capacity of C9-AF488 to induce lysis of K562 cells, the cells were treated with anti-K562 antibodies, followed by C9D-NHS or HI-C9D-NHS supplemented with C9-AF488 for 60 min at 37 °C

Summary

Introduction

Activation cascade may occur through the classical, alternative, or lectin pathways. The three initiation pathways lead to activation of the terminal complement pathway and to assembly of the membrane attack complexes (MACs) that are composed of the complement components C5b, C6, C7, C8, and C9 ( known as the C5b-9 complexes). To avoid accidental killing by complement, cells utilize several protective strategies, including expression of membrane complement regulatory proteins, such as CD46, CD55, and CD59 that block the activation cascades at specific stages Vesicles shed directly from the plasma membrane by a process of outward vesiculation were named ectosomes or microparticles. Data shown here demonstrate rapid shedding of C5b-9 complexes in small vesicles (ectosomes) directly from the cell surface. This is followed by removal of the C5b-9 via an endocytic pathway and its targeting to a late endosomal compartment, probably for degradation and/or exocytosis via MVB

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