Meningothelial Cells (MTC) of the Arachnoid Membrane Respond to Neutrophils: A Model for Delayed Cerebral Injury (DCI) After Aneurysmal Subarachnoid Hemorrhage (aSAH)
Vijay Chhabra1, Aminata Coulibaly2, Jackson Narrett3, J. Javier Provencio4
1Swarthmore College, 2Neuroscience, University of West Virginia, 3Yale New Haven Hospital, 4University of Virginia Health System
Objective:
To understand how inflammation signals from the meninges to the brain parenchyma in aSAH.
Background:
DCI is a cause of poor outcome after aSAH. Neutrophils in the meninges are implicated in inflammation that impacts outcome in murine aSAH. Myeloperoxidase (MPO) from neutrophils is critical for brain dysfunction after aSAH. How inflammation in the meninges bridges the subarachnoid space is not understood. Here, we investigate whether cells of the arachnoid membrane, meningothelial cells (MTC, which traverse the subarachnoid space) are signaling links between meninges and brain. We hypothesize that MPO drives cytokine production and behavioral changes in MTCs.
Design/Methods:
MTCs (BenMen1, hTERT immortalized human MTC line) were co-cultured with MPO or neutrophils (separate experiments). Cells were exposed to experimental conditions for 4hrs: Experiment 1, control, MPO, MPO + H2O2, H2O2, and LPS (positive control); Experiment 2, control, LPS, and neutrophils (± LPS) for cytokine analysis. Also, MTCs were incubated with MPO (+H2O2), stained after 1hr. Significance defined as p<0.01 on 1-way INOVA and p<0.05 pairwise comparisons (Student's t-test/Welch correction).
Results:
MTC make a limited repertoire of innate cytokines. Contrary to our hypothesis, IL-8, MCP-1 and IL-6 production in the setting of MPO (±H2O2) are suppressed in MTC. Conversely, neutrophil co-culture with MTC increases IL-8 (IL-6 is increased with LPS-activated neutrophils). MPO (+ H2O2)-treated MTC become enlarged with limited processes and show phagocytosis of adjacent TMC cells.
Conclusions:
Neutrophils, not MPO, are the stimulus for release of the cytokines IL-8 and IL-6 (MPO alone appears suppressive) from MTC, but MPO-treated BenMen1 cells expand their cell bodies, retract processes and phagocytose adjacent cells. This data suggests that neutrophils cause MTC to release inflammatory cytokines using an MPO-independent mechanism that may signal the brain. In addition, MPO stimulates morphological and behavioral changes in MTC which may also play a role in brain injury independent of inflammatory cytokine release.