Purified complement C3b triggers phagocytosis and activation of human neutrophils via complement receptor 1

Abstract

The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo

“…Opsonization of pathogens by the complement system greatly enhances phagocytosis ( 39 , 40 , 41 ). The experiments presented above were conducted using bacterial particles that were not opsonized before the experiment.…”

Reactivating the complement system using peptidic bacterial labeling tag

“…This fusion protein, referred to as FH-FC, demonstrated the ability to mediate complement-dependent killing in vitro and exhibited promising efficacy in animal models of both gonorrhea and meningococcal bacteremia. In a conceptually related study, Rooijakkers and colleagues employed a click chemistry reaction to conjugate purified complement C3b onto E. coli bacteria [35]. They reacted the thioester of purified C3b with DBCO-PEG4maleimide, resulting in C3b-DBCO, which they reacted with bacterial LPS decorated with Keto-deoxyoctulosonate-N3, leading to the creation of C3b-E. coli conjugates.…”