The online version of this article (https://doi.org/10.1186/s12974-017-1050-z) contains supplementary material, which is available to authorized users.
Exposure of the developing brain to immune mediators, including antibodies, is postulated to increase risk for neurodevelopmental disorders and neurodegenerative disease. It has been suggested that immunoglobulin G-immune complexes (IgG-IC) activate Fc gamma receptors (FcγR) expressed on neurons to modify signaling events in these cells. However, testing this hypothesis is hindered by a paucity of data regarding neuronal FcγR expression and function.
FcγR transcript expression in the hippocampus, cortex, and cerebellum of neonatal male and female rats was investigated ex vivo and in mixed cultures of primary hippocampal and cortical neurons and astrocytes using quantitative PCR analyses. Expression at the protein level in mixed cultures of primary hippocampal and cortical neurons and astrocytes was determined by immunocytochemistry, western blotting, proteotype analysis, and flow cytometry. The functionality of these receptors was assessed by measuring changes in intracellular calcium levels, Erk phosphorylation, and IgG internalization following stimulation with IgG-immune complexes.
FcgrIa, FcgrIIa, FcgrIIb, FcgrIIIa, and Fcgrt transcripts were detectable in the cortex, hippocampus, and cerebellum at postnatal days 1 and 7. These transcripts were also present in primary hippocampal and cortical cell cultures, where their expression was modulated by IFNγ. Expression of FcγRIa, FcγRIIb, and FcγRIIIa, but not FcγRIIa or FcRn proteins, was confirmed in cultured hippocampal and cortical neurons and astrocytes at the single cell level. A subpopulation of these cells co-expressed the activating FcγRIa and the inhibitory FcγRIIb. Functional analyses demonstrated that exposure of hippocampal and cortical cell cultures to IgG-IC increases intracellular calcium and Erk phosphorylation and triggers FcγR-mediated internalization of IgG.
Our data demonstrate that developing neurons and astrocytes in the hippocampus and the cortex express signaling competent FcγR. These findings suggest that IgG antibodies may influence normal neurodevelopment or function via direct interactions with FcγR on non-immune cells in the brain.
Additional file 1: Developmental changes in Fcgr expression in the brain. Relative changes in Fcgr expression in the hippocampus, cortex, and cerebellum of postnatal day (PND) 7 Sprague Dawley rats compared to their PND1 littermates. (XLSX 10 kb)12974_2017_1050_MOESM1_ESM.xlsx
Additional file 2: Developmental changes in Fcgr expression in the liver and spleen. Relative changes in Fcgr expression in the liver and spleen of postnatal day (PND) 7 Sprague Dawley rats compared to their PND1 littermates. (XLSX 10 kb)12974_2017_1050_MOESM2_ESM.xlsx
Additional file 3: CD11b immunoreactivity in hippocampal and cortical cultures at DIV 0 and DIV 7. Hippocampal and cortical cells on DIV0 and DIV7 were immunolabeled for MAP2b (magenta), GFAP (green), and CD11b (orange). Almost no staining was observed for CD11b across both culture types and DIV. Quantification of CD11b staining on DIV 0 in hippocampal cells showed that fewer than 0.6% of cells were positive for CD11b (representative images of CD11b positive cells are shown here). (JPEG 1340 kb)12974_2017_1050_MOESM3_ESM.xlsx
Additional file 4: Percentage of MAP2b and GFAP, and CD11b cells in hippocampi cell cultures at DIV 0. Dissociated hippocampal cell cultures were plated and fixed at DIV 0 to immunostain for MAP2b, GFAP, and CD11b. Immunoreactivity was imaged using the ImageXpress high-content imaging system; and the number of immunoreactive cells was quantified using the Custom Module Editor in the MetaXpress Software (Molecular Devices). (JPEG 219 kb)12974_2017_1050_MOESM4_ESM.jpg
Additional file 5: IFNγ does not affect Erk phosphorylation in primary neuronal cell cultures. DIV 7 hippocampal and cortical cell cultures were exposed for 24 h to varying concentrations of IgG-IC (10 or 100 μg/ml) or rat anti-mouse IgG (10 or 100 μg/ml) in the presence or absence of 30 ng/ml IFNγ. Cell lysates were separated by SDS PAGE and immunoblotted for pErk, total Erk, and GAPDH. The optical density of bands immunoreactive for pErk and total Erk was normalized to the optical density of GAPDH immunoreactive bands from the same sample. The ratio of pErk to Erk is plotted as a percentage of vehicle controls. Data from a single replicate per condition in one experiment. r@m: rat anti-mouse IgG; IC: IgG-IC immune complex. (PDF 403 kb)12974_2017_1050_MOESM5_ESM.jpg
Additional file 6: Summary of the published literature documenting FcγR expression in neurons and macroglia. Tabulated summary of evidence from the published literature for expression of FcγR in neurons and macroglia in the central and peripheral nervous system in rodents and humans. (XLSX 13 kb)12974_2017_1050_MOESM6_ESM.pdf
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- Fc gamma receptors are expressed in the developing rat brain and activate downstream signaling molecules upon cross-linking with immune complex
Ana Cristina Grodzki
Marc van Oostrum
Pamela J. Lein
- BioMed Central