Thrombin induces release of proinflammatory chemokines interleukin-8 and interferon-γ-induced protein-10 from cultured human fetal astrocytes

Abstract

Thrombin is a multifunctional serine proteinase that induces a variety of responses from neural cells by cleavage of proteinase-activated receptors (PARs) including PAR1 and PAR4. Thrombin/PAR signaling has been implicated in the neuroinflammatory response that occurs in the brain following stroke and other central nervous system pathologies. The neuroinflammatory response involves astrocytes and results in induction of proinflammatory chemokines including interleukin-8 (IL-8 or CXCL8) and interferon-γ-induced protein-10 (IP-10 or CXCL10) in these cells. Astroctyes are known to express PARs, however the effect of thrombin on astrocytic chemokine secretion is unknown. Here we characterize the ability of thrombin to induce proliferation/metabolic activity and chemokine secretion in primary human fetal astrocytes. Thrombin induces dose-dependent astrocyte proliferation as well as release of both IL-8 and IP-10, but not IL-6 or the chemokine regulated and normal T cell expressed and secreted (RANTES). The chemokine responses were mimicked by PAR1, but not PAR4, activating peptides. Our data indicate that astrocytic chemokine release is part of the neuroinflammatory response triggered by the exposure of the central nervous system to thrombin.

Figure 1:. Thrombin-induced proliferation/metabolic activity.

Human fetal astrocytes were stimulated with increasing concentrations of thrombin ± PPACK (10 μg/ml) as indicated. Proliferation/metabolic activity was assessed 24 h later using the WST-1 assay. Data are normalized to unstimulated control and expressed as mean ± S.E.M. n≥9, exp≥3, ** indicates p< 0.01.

Figure 2:. Thrombin-induced chemokine release.

Human fetal astrocytes were stimulated with increasing concentrations of thrombin ± PPACK (10 μg/ml) as indicated. IL-8 (A) and IP-10 (B) release was assessed 24 h later in conditioned medium as described in Methods. Data are expressed as mean ± S.E.M. n≥6, exp≥2, * indicates p< 0.05, ** indicates p< 0.01.

Figure 3:. Effect of PAR activating peptides on chemokine release.

Human fetal astrocytes were stimulated with indicated concentrations of PAR₁ agonist peptide (PAR1 AP), PAR₁ scrambled peptide (PAR1ScrP) or PAR₄ agonist peptide (PAR4 AP). IL-8 (A) and IP-10 (B) release was assessed 24 h later in conditioned medium as described in Methods. Data are expressed as mean ± S.E.M. percent response relative to thrombin plus DMSO (vehicle) control. n≥6, exp≥2, * indicates p< 0.05, ** indicates p< 0.01.