Phagocytosis is a central event in the innate immune responses that are triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Particularly, complement-mediated phagocytosis is accomplished by specific recognition of bound complement components by the corresponding complement receptors on the phagocytes. Pathogen invasion leads to the generation of C3 convertase which yields C3b. The final product C3bi binds to three other complement receptors CR1, CR3 (CD11b/CD18, integrin αMβ2) and CR4 (CD11c/CD18, integrin αXβ2). Binding of C3bi to CR3 induces phagocytosis most strongly after phagocyte activation.
To clarify the molecular mechanism of phagocytosis, it is essential to establish a reproducible experimental system of phagocytosis in vitro. As for Fcg receptor mediated phagocytosis, sheep erythrocytes or zymosan (Saccharomyces cerevisiae) are used as particles to be incorporated and pretreated with specific antibodies prior to phagocytosis assays. In the case of complement-mediated phagocytosis, the complement activation cascade in the serum is effectively utilized. C3b is spontaneously produced and destroyed, and when non-self microbes such as zymosan are exposed to the serum at 37℃, C3b binds to zymosan covalently and becomes C3 convertase by the action of cofactor proteins. Cleavage of C3 to C3b and finally to C3bi is amplified, and zymosan is surrounded by C3bi and incorporated effectively by phagocytes which express CR3.
Regulation of complement receptor mediated phagocytosis is more complex than simple activation of adhesion. This has been clear since the work of Silverstein almost 30 years ago, when he showed that peritoneal macrophages can bind complement opsonized targets but cannot phagocytose them without an additional signal. Thus,activation of integrins to be adhesive is not sufficient to allow them to interact with the cellular machinery that mediated phagocytosis. Work by Griffin demonstrated that while complement receptors are not normally mobile in the plane of the memebrane of unactivated cells, such mobility is required for phagocytosis. Pharmacologic agents and cytokines that activate complement receptor mobility also activate complement-mediated phagocytosis. Based on modern understanding of integrin function, these data suggest that the resting macrophages used in these experiments likely expressed complement receptors that were of sufficient affinity to bind opsonized particles but were not able to cluster. Cell activation allowed the engaged receptors to cluster, a function required for phagocytosis. Among the characterized cytokines able to mediate this change in complement receptor function are IL-4,M-CSF,TNF-α,and GM-CSF.
Subsequently,several groups showed that activating signals could increase the diffusion of integrin receptors by releasing them from cytoskeletal constraint. Based on these data,it is likely that affinity modulation of macrophage integrins is sufficient to allow binding of opsonized particles, but increased diffusion to allow clustering is required as well in order to activate ingestion. Thus,for complement-mediated phagocytosis, the molecular mechanisms that control integrin clustering represent a critical control point.
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