The interaction between tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 promotes the recruitment of neutrophils into the ischemic brain

Abstract

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are expressed in endothelial cells and perivascular astrocytes. Here, we show that TWEAK induces a dose-dependent increase in the expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in astrocytes, and that this effect is mediated by its interaction with Fn14 via nuclear factor-kappaB pathway activation. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in wild-type (Wt) astrocytic cultures. Likewise, incubation under OGD conditions induces the expression of MCP-1 in Wt astrocytes but not in astrocytes deficient on either TWEAK (TWEAK(-/-)) or Fn14 (Fn14(-/-)). We also found that TWEAK induces the passage of neutrophils to the abluminal side of an in vitro model of the blood-brain barrier. Our earlier studies indicate that cerebral ischemia increases the expression of TWEAK and Fn14 in the endothelial cell-basement membrane-astrocyte interface. Here, we report that middle cerebral artery occlusion increases the expression of MCP-1 and the recruitment of neutrophils into the ischemic tissue in Wt but not in TWEAK(-/-) or Fn14(-/-) mice. These novel results indicate that during cerebral ischemia, the interaction between TWEAK and Fn14 leads to the recruitment of leukocytes into the ischemic tissue.

Figure 1

The TWEAK induces the passage of neutrophils through the endothelial cell-basement membrane-astrocyte interface. Average number of neutrophils in the lower compartment of an in vitro model of the BBB assembled with either Wt or Fn14^−/− astrocytes after 24 h of incubation with either vehicle (−, white bars) or rTWEAK 100 ng (+, black bars) and the addition of 5 × 10⁵ neutrophils to the upper chamber of the system containing bovine brain microvascular endothelial cells. n=6. Lines denote s.d.

Figure 2

The binding of TWEAK to Fn14 induces the expression of MCP-1 in astrocytes via NF-κB pathway activation. (A) Mean MCP-1 protein concentration in Wt astrocytes incubated for 24 h with rTWEAK 0 to 1000 ng. Lines denote s.d. n=6 observations per time-point. (B) Mean MCP-1 protein concentration in Wt astrocytes pretreated with either TWEAK alone (black bar,−) or with the NF-κB inhibitor BAY 11-7085 and TWEAK 100 ng (white bar, +). Lines denote s.d. n=6. (C) Mean MCP-1 protein concentration in wild-type (Wt) and Fn14^−/− astrocytes incubated with either vehicle control (black bars, −) or rTWEAK 100 ng (white bars, +) for 24 h. Lines denote s.d. n=6.

Figure 3

The interaction between endogenous TWEAK and Fn14 mediates hypoxia-induced astrocytic MCP-1 expression. (A) Real-time quantitative PCR analysis of TWEAK and Fn14 mRNA expression in Wt astrocytes exposed to OGD conditions for 6 h. n=6; ^*P<0.005 compared with controls kept under normoxic conditions. (B) Mean MCP-1 protein concentration in Wt, TWEAK^−/−, and Fn14^−/− astrocytes exposed to normoxic (N, black bars) or hypoxic (H, white bars) conditions for 6 h followed by 18 h of incubation under normoxic conditions. A subset of TWEAK^−/− astrocytes was incubated under hypoxic conditions with rTWEAK (gray bar; ^**P<0.005 compared with TWEAK^−/− astrocytes maintained under OGD conditions in the absence of rTWEAK). Lines denote s.d. n=6 observations per time-point.

Figure 4

Effect of genetic deficiency of TWEAK and Fn14 on cerebral ischemia-induced MCP-1 expression. (A) Real-time quantitative PCR analysis of MCP-1 in brain extracts from wild-type (Wt, white bar), TWEAK^−/− (black bar), and Fn14^−/− (gray bar) mice 6 h after MCAO. Lines denote s.d. n=6 observations per time-point. ^*P<0.001 compared with the other experimental groups. (B) Mean MCP-1 protein concentration in the ischemic (Ipsi., black bars) and nonischemic (Con., white bars) hemispheres of Wt, TWEAK^−/−, and Fn14^−/− mice 24 h after MCAO. Lines denote s.d. n=6 observations per time-point. NS, not significant.

Figure 5

Effect of genetic deficiency of TWEAK and Fn14 on the recruitment of neutrophils into the ischemic tissue after MCAO. (A) Representative immunohistochemical staining for neutrophils in each AOI of wild-type (A), TWEAK^−/− (B), and Fn14^−/− mice (C) mice 24 h after MCAO. Green is a 40-kDa antigen expressed only by polymorphonuclear cells and blue is DAPI. Magnification × 40. (B) Mean number of infiltrating neutrophils in AOI 1-3 in wild-type (Wt), TWEAK^−/−, and Fn14^−/− mice 24 h after MCAO. Lines depict s.e.m. n=6. ^*P<0.001 compared with TWEAK^−/− and Fn14^−/− mice. (C) Myeloperoxidase activity in the ischemic brain tissue of Wt, TWEAK^−/−, and Fn14^−/− mice 24 h after MCAO. Lines depict s.e.m. n=6. ^*P<0.001 compared with TWEAK^−/− and Fn14^−/− mice.