Abstract
Macrophages are professional phagocytes that are uniquely situated between the innate and adaptive arms of immunity with a high capacity for phagocytosis and proinflammatory cytokine production as well as antigen presentation. Phagocytosis is a critical process to eliminate microbes, apoptotic cells and other foreign particles and is accelerated by host-generated opsonins, such as antibodies and complement. Early phagocytosis studies established the paradigm that FcγR-mediated phagocytosis was more proinflammatory than Complement Receptor (CR)-mediated uptake in macrophages. Using qPCR, cytokine antibody arrays and ELISA, we revisited this research question in primary macrophages. Using qPCR we determined that CR-mediated phagocytosis increases levels of TNF-α, IL-1β, IL-6, and MMP-9, compared to FcγR-mediated phagocytosis and control unstimulated cells. We confirmed these findings at the protein level using cytokine antibody arrays and ELISAs. We next investigated the mechanism behind upregulated cytokine production during CR-mediated phagocytosis. IκBα protein levels were reduced after phagocytosis of both IgG- and C3bi-sRBCs indicating proteolytic degradation and implicating NF-κB activation. Inhibition of NF-κB activation impacted IL-6 production during phagocytosis in macrophages. Due to the roles of calpain in IκBα and integrin degradation, we hypothesized that CR-mediated phagocytosis may utilize calpain for proinflammatory mediator enhancement. Using qPCR and cytokine antibody array analysis, we saw significant reduction of cytokine expression during CR-mediated phagocytosis following the addition of the calpain inhibitor, PD150606, compared to untreated cells. These results suggest that the upregulation of proinflammatory mediators during CR-mediated phagocytosis is potentially dependent upon calpain-mediated activation of NF-κB.
Highlights
Macrophages, meaning large phagocytes, are a heterogeneous population of cells that are important for maintaining homeostasis, surveillance and killing of pathogens through phagocytosis [1]
We investigated mRNA levels of TNF-α, IL-1β, IL-6, and MMP-9 in bone marrow-derived murine macrophages (BMDMs) after phagocytosis of immunoglobin G (IgG)- or C3bi-opsonized targets
A long-standing belief in the scientific community is that phagocytosis mediated by Fcγ Receptors (FcγRs) is potent at inducing inflammation whereas Complement Receptor (CR)-mediated phagocytosis in macrophages is non-inflammatory
Summary
Macrophages, meaning large phagocytes, are a heterogeneous population of cells that are important for maintaining homeostasis, surveillance and killing of pathogens through phagocytosis [1]. Phagocytosis is an evolutionarily conserved defense mechanism by which macrophages capture and kill pathogens and remove apoptotic cells into specialized intracellular compartments. Cytokine Induction During Opsonic Phagocytosis is mediated by scavenger receptors, Fcγ Receptors (FcγRs), and Complement Receptors (CRs) [2]. The intracellular domains of Fcγ receptors contain the immunoreceptor tyrosine-based activation motif (ITAM) that activates downstream signaling cascades [6, 7]. The ligation of IgG-opsonized particles to FcγRs leads to receptor crosslinking which activates Src tyrosine kinase resulting in tyrosine phosphorylation of the ITAM motif [8]. ITAM phosphorylation is important for the recruitment and activation of Syk [9]. The inhibitory FcγRIIB activates signaling cascades by recruiting inositol polyphosphate-5phosphatase SHIP1 to counteract signaling from activating FcγRs [7]
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