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Thrombin-induced activation of cultured rodent microglia
T Möller1, U K Hanisch, B R Ransom
1Department of Neurology, University of Washington, Seattle, Washington 98195, USA. moeller@u.washington.edu
Abstract:
Microglia are the resident immune cells of the CNS. Upon brain damage, these cells are rapidly activated and function as tissue macrophages. The first steps in this activation still remain unclear, but it is widely believed that substances released from damaged brain tissue trigger this process. In this article, we describe the effects of the blood coagulation factor thrombin on cultured rodent microglial cells. Thrombin induced a transient Ca(2+) increase in microglial cells, which persisted in Ca(2+)-free media. It was blocked by thapsigargin, indicating that thrombin caused a Ca(2+) release from internal stores. Preincubation with pertussis toxin did not alter the thrombin-induced [Ca(2+)](i) signal, whereas it was blocked by hirudin, a blocker of thrombin's proteolytic activity. Incubation with thrombin led to the production of nitric oxide and the release of the cytokines tumor necrosis factor-alpha, interleukin-6, interleukin-12, the chemokine KC, and the soluble tumor necrosis factor-alpha receptor II and had a significant proliferative effect. Our findings indicate that thrombin, a molecule that enters the brain at sites of injury, rapidly triggered microglial activation.
Insights
Blood coagulation factor thrombin rapidly activates microglia, the brain's immune cells, upon injury. Thrombin triggers calcium release and the production of inflammatory molecules, promoting microglial proliferation.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are the central nervous system's (CNS) resident immune cells, acting as tissue macrophages after brain damage.
- The initial triggers for microglial activation following brain injury are not fully understood.
- Substances released from damaged brain tissue are hypothesized to initiate microglial activation.
Purpose of the Study:
- To investigate the effects of the blood coagulation factor thrombin on cultured rodent microglial cells.
- To elucidate the signaling pathways involved in thrombin-induced microglial activation.
- To determine the functional consequences of thrombin exposure on microglia.
Main Methods:
- Cultured rodent microglial cells were treated with thrombin.
- Intracellular calcium ([Ca(2+)](i)) levels were measured.
- The role of internal calcium stores was assessed using thapsigargin.
- G-protein signaling involvement was tested with pertussis toxin.
- Thrombin's proteolytic activity was blocked using hirudin.
- Production of nitric oxide and release of cytokines (TNF-α, IL-6, IL-12) and chemokines (KC) were quantified.
- Cell proliferation was measured.
Main Results:
- Thrombin induced a transient increase in intracellular calcium ([Ca(2+)](i)) in microglial cells.
- This calcium signal originated from internal stores and was independent of pertussis toxin-sensitive G-proteins.
- The thrombin-induced calcium increase was dependent on its proteolytic activity.
- Thrombin stimulated nitric oxide production, release of pro-inflammatory cytokines (TNF-α, IL-6, IL-12) and chemokines (KC), and soluble TNF-α receptor II.
- Thrombin significantly promoted microglial proliferation.
Conclusions:
- Thrombin, present in the brain at injury sites, acts as a potent trigger for rapid microglial activation.
- Thrombin signaling in microglia involves intracellular calcium release and proteolytic activity.
- Activated microglia release inflammatory mediators and proliferate, suggesting a role in neuroinflammation and tissue repair following injury.