Figure 2

Neutrophils bind to astrocytes in vitro. A. FACS-sorted resting or LPS-primed neutrophils were labeled with CFSE and then were co-cultured with resting or poly I:C-primed astrocyte monolayer at a ratio of 5:1 in a 96-well plate for 1.5 hr (left) or for the indicated periods (right). CFSE intensity was analyzed (**p < 0.01 vs resting neutrophil alone; ++p < 0.01, LPS-primed neutrophils plus poly I:C-primed astrocytes vs resting neutrophils plus resting astrocytes. n = 4 - 6/bar). B. After the procedure described in A, cells were fixed with methanol and immunoreacted for GFAP (red) followed by fluorescent microscopy. Binding of resting neutrophils (green) to resting astrocytes (left), and LPS-primed neutrophils to poly I:C-primed astrocytes (right). C. Resting (PMN-U), LPS-primed neutrophils (PMN-L) or fMLP-primed neutrophils (PMN-F) were co-cultured with CFSE-labeled and resting (astrocyte-U) or Poly I:C primed astrocytes (astrocyte-P) at a ratio of 5:1. After incubation for indicated periods, neutrophil-astrocyte clusters were identified as CFSE⁺Gr1⁺ events. Percentage of astrocytes binding to neutrophils was quantified by flow cytometry. D. Representative flow cytometry graphs of C. Numbers in the quadrants show the percentages of astrocytes binding to neutrophils, respectively. E. Flow cytometry analysis indicates that astrocyte size is critical for binding capacity of neutrophils. CFSE⁺ astrocytes were gated into four groups based on their forward scatter intensity: small (a), median (b), large (c) and largest (d). The numbers show the percentages of astrocytes binding to neutrophils.