Year: 2012
Type: article
Abstract: FcγRIIA, a transmembrane receptor for immunoglobulin G (IgG), triggers phagocytosis, endocytosis and oxidative burst in leukocytes.Signal transduction via FcγRIIA leading to these outcomes has been relatively well studied.To identify additional signaling partners, a yeast twohybrid analysis employing the cytoplasmic domain of FcγRIIA was performed.Interestingly, the SH2 domain of signal transducer and activator of transcription 6 (STAT6) appeared in our analysis.STAT6 traditionally interacts with interleukin-4 (IL-4) receptors to induce expression of IL-4 and IL-13 in T cells.Macrophages have also been observed to produce IL-4 in response to pharmaceutical and pathogenic stimuli and to respond to IL-4.However, production of IL-4 or IL-13 subsequent to FcγR ligation has not been observed.Therefore, to better define the specific interaction of FcγRIIA with STAT6, we engineered Chinese hamster ovary cells to express FcγRIIA and RFP-STAT6 to determine if specific ligation of FcγRIIA induces STAT6 nuclear translocation.This resulted in lack of consistent expression of the RFP-STAT6 causing us to perform our subsequent experiments in the human macrophage-like cell line, THP-1.To iii further elucidate this interaction, FcγRIIA immunoprecipitation followed by western blotting of FcγRIIA and the whole cell lysates from THP-1 macrophages, was performed.We observed the constitutive interaction of non-phosphorylated STAT6 with FcγRIIA.We subsequently sought to examine STAT6 activation after stimulation of THP-1 macrophages with various FcγR ligands.Using IL-4 as a positive control, we observed heat-aggregated IgG induces significant amounts of STAT6 translocation compared to untreated cells (p<0.05).We then sought to determine if IL-4 was produced from THP-1 macrophages after FcγR ligation.We were unable to detect IL-4 from THP-1 macrophages, but were able to detect IL-4 from Jurkat cells, a human T cell line, that were stimulated with ionomycin and phorbol myristal acetate.We then examined the effects of IL-4 on FcγR-mediated activities.We observed that transient exposure of THP-1 macrophages to IL-4 had no significant effect on endocytosis or phagocytosis.Our current observations indicate an interaction exists between FcγRIIA and STAT6 through the SH2 domain of STAT6 producing nuclear translocation of non-phosphorylated STAT6 without induction of IL-4 gene expression.This observance may signal an important avenue of research in understanding the mechanisms of phosphorylation of STAT6 and STAT6 induced gene expression.ix 4-5 Quantification of phagocytosis using epifluorescence microscopy analysis of THP-1 macrophages stimulated with 50ng/mL IL-4………………………………41 4-6 Trend of endocytosis analyzed through flow cytometric analysis of THP-1 macrophages stimulated with 50ng/mL IL-4………………………………………42 x List of Abbreviations Ab…………………Antibody CHO………………Chinese Hamster Ovary Cells CHO-IIA………….Chinese Hamster Ovary Cells transfected with human FcγRIIA CHO-IIAGFP……..Chinese Hamster Ovary cells transfected with FcγRIIA-GFP CXCL8……………Chemokine Interleukin-8 EA…………………IgG-opsonized erythrocytes FcR………………...Fc Receptor FcαR……………….Fc Receptor for IgA FcδR……………….Fc Receptor for IgD FcεR……………….Fc Receptor for IgE FcγR……………….Fc Receptor for IgG FcµR………………Fc Receptor for IgM FITC……………….Fluorescein Isothiocyanate FITC-haIgG……….FITC-conjugated haIgG γc………………….Common Gamma Chain H 131 ……………….High-responder FcγRIIA isoform HaIgG……………..Heat-Aggregated IgG Complexes IC…………………..Immune Complex IFN………………...Interferon IL…………………..Interleukin IL-4R………………Interleukin-4 Receptor Ig…………………...Immunoglobulin IgA…………………Immunoglobulin A IgE………………….Immunoglobulin E IgG………………….Immunoglobulin G IP 3 ………………….Inositol triphosphate ITAM……………….Immunoreceptor Tyrosine-based Activation Motif ITIM………………..Immunoreceptor Tyrosine-based Inhibitory Motif kDa…………………Kilodalton M1………………….Classically Activated Macrophages M2…………………
Author: Elizabeth P. Beil
Institution: University of Toledo
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W99098322