Figure 7Figure 7
Microglia are required for pathogenic AQP4-IgG to upregulate CXCL1 in cultured mouse astrocytes. ( A ) Experimental design of the IgG binding to AQP4 on astrocytes in primary glial cultures established from wild-type and Aqp4 –/– pup brains. Microglia were depleted by treating Clodrosome (100 μg/mL) before subculture. Two days after adding IgG or cytokines, CXCL1 production was assessed by immunostaining or ELISA. ( B ) Immunoblot analysis of WT, Aqp4 –/– primary glial cells using IgGs specific for AQP4 and actin (loading control). ( C ) CXCL1 immunoreactivity was assessed in wild-type and AQP4 ± cells exposed to a control nonpathogenic monoclonal mouse IgG specific for the AQP4 cytoplasmic C-terminal domain (CCD-AQP4-IgG, m5) or a pathogenic extracellular domain–reactive IgG (ECD-AQP4-IgG, m21) or to IFN-γ plus TNF-α cytokines. Astrocytes are identified by AQP4 and GFAP immunoreactivities; microglia by IBA1; DNA is blue (DAPI). ( D ) Cellular CXCL1 (upper) was quantified from flu