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

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 m) 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.The complement system is a major component of the innate immune system. It efficiently protects the host from pathogenic microorganisms, is involved in immune complex regulation, and serves an important link between innate and adaptive immune responses. Complement comprises a group of more than 30 proteins; most of them participate in the cascade-like activation process, whereas others serve as control proteins or act as cellular receptors (1, 2). Initiation of the complement 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 (also known as the C5b-9 complexes). Upon formation of C5b-7, C5b-8, and finally C5b-9 complexes, they adhere and then insert into the target cell membrane (3). The C5b-9 complexes may contain 1-18 C9 molecules attached to a C5b-8 complex. When the number of C9 molecules per C5b-8 exceeds 12, they self-polymerize and form a cylinder-shaped transmembrane structure (4). Upon its assembly, the MAC expresses neoantigens that can be detected by specific monoclonal antibodies (5). Although red blood cells require only one effective MAC to lyse, nucleated cells are killed by complement only after a combined action of several MACs (6). Although larger C5b-9 channels containing several C9 a...

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

“…Complement activation has previously been linked with the generation of microparticles, 8,9 but the results of the current studies also demonstrate that microparticles themselves can directly activate the complement system. Although immunoglobulin and complement proteins have been detected on the surface of microparticles from apoptotic cells 14 and microparticles isolated from patients with inflammatory diseases, [15][16][17] these previous studies did not Microparticles from the supernatant of unmanipulated or CsA-treated cells were exposed to serum, and C3 deposition on the microparticles was assessed by Western blot analysis using a polyclonal antibody to C3 and a monoclonal antibody to C3d.…”

“…6,7 Complement activation on the cell membrane can induce cells to release microparticles. 8,9 Although complement proteins can be detected on the surface of microparticles released from apoptotic and injured cells, 10 less is known regarding whether microparticles from specific cell types themselves can cause complement activation. We hypothesized that injury of endothelial cells induces the release of complement-activating microparticles into the circulation.…”

Intracellular Ca2+ operates a switch between repair and lysis of streptolysin O-perforated cells

“…They, most likely, encompass plasma membrane regions situated in the vicinity of SLO-induced pores. 14 The annexin A1-positive Ca 2 þ 'hot spots' were restricted to small regions within the plasma membrane, which suggested that the diffusion between these structures and the cytoplasm was effectively limited. Annexins are able to promote fusion of adjacent membranes in the presence of Ca 2 þ .…”

Complement Activation on B Lymphocytes Opsonized with Rituximab or Ofatumumab Produces Substantial Changes in Membrane Structure Preceding Cell Lysis

“…For example, a considerable body of evidence indicates that one of the ways that cancer cells inhibit C-mediated killing is by expressing increased levels of C control proteins (11, 49 -53). Moreover, when sublytic amounts of C are used to attack nucleated cells, the cells can respond by implementing strategies to enhance their resistance to subsequent assaults by C (46,48,53).…”

“…Reaction of nucleated cells with sublytic amounts of C can lead to the rapid release of vesicles that are enriched in the MAC, and this process appears to be mediated by the mitochondrial heatshock protein 70, also called mortalin (45)(46)(47)(48). It is important to determine whether the streamer production we have seen could be related to, or is simply another manifestation of, the vesicle release process described by Morgan, Fishelson, and their colleagues (45)(46)(47).…”