The immunoglobulin family plays a crucial role in the body's immune defense mechanisms. IgG is the most abundant subclass of antibody in serum and is comprised of several subtypes in both humans and mice: IgG1, IgG2 (IgG2 is subdivided into IgG2a and IgG2b in mice), IgG3, and IgG4 (Vidarsson et al., 2014). The functions of IgG subtypes include opsonization, neutralization, agglutination, complement activation, phagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) (Napodano et al., 2021). IgG facilitates immune responses primarily by binding to the Fcγ receptor family on various cell types via its constant region, the fragment crystallizable (Fc) region. The mouse Fcγ receptor family is comprised of 5 subclasses: FcγRІ, FcγRІІ, FcγRІІІ, FcγRІV, and FcRN (Bruhns, 2012). These Fcγ receptors are functionally divided into activating and inhibitory receptors. FcγRІ (CD64), FcγRІІІ (CD16), and FcγRІV are activating receptors, whereas FcγRІІ (CD32) is an inhibitory receptor. The ultimate consequences of engaging Fcγ receptors depend on whether the activating or inhibitory signal predominates (Levin et al., 2015). Fcγ receptor activation is initiated after the clustering and forming of IgG-containing immune complexes. When IgG complexes bind to these receptors, they initiate an intracellular signaling cascade through the γ chain, which contains immunoreceptor tyrosine-based activation motifs (ITAM) (Brandsma et al., 2016). This activation and subsequent phosphorylation of ITAM further activates downstream signaling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway (Abram and Lowell, 2007). Activation of Fcγ receptors is essential for mediating various cellular responses including immune-complex-triggered ITAM activation, cytokine production, phagocytosis, endocytosis, and ADCC (Okun et al., 2010).

There are limited in vitro assays available that allow for assessment of antibody-triggered signaling in innate cells. For instance, the US National Toxicology Program established a comprehensive in vitro screening protocol for the identification of immunotoxic compounds. This protocol consists of a three-tier phase system; tier 1 includes evaluation of myelotoxicity; tier 2 includes evaluation of leukocytes and tier 3 includes innate and adaptive immune function assays (Corsini and Roggen, 2017). Standard assays for the activation of innate cells frequently use toll-like receptor (TLR) agonists (Hume et al., 2001; Jones et al., 2001; Dobrovolskaia et al., 2003), but there are no assays that activate the innate cells through Fcγ receptors. Although studies use Fcγ receptor knockout mice, which lack functional Fcγ receptors, to gain an understanding of immune complex engagement and downstream signaling (Whtimer et al., 1997; McKenzie et al., 1999; Keeler and Fox, 2021), utilizing an IgG1 immune complex will more accurately mimic in vivo signaling mechanisms as an alternative method for studying the immunotoxic effects of drugs and chemicals on innate cells through Fcγ receptor-mediated signaling.

Thus, the purpose of this study was to investigate the immune response after the activation of innate cells with an immune complex that is effective in triggering Fcγ receptors. We hypothesized that the IgG1 immune complex would activate innate cells, resulting in increased cytokine production and complement system activation. We accumulated several pieces of evidence to demonstrate successful Fcγ receptor signaling by performing a systematic comparison of the efficacy of different stimulators. This in vitro assay can help in the identification of immunotoxicants that alter Fcγ receptor signaling. For instance, for those drugs and chemicals that affect antibody production, this system can be utilized to determine if drug- or chemical-mediated alteration of antibody production leads to disruptions in antibody-triggered signaling in innate cells.