Elsevier

Brain Research

Volume 1048, Issues 1–2, 28 June 2005, Pages 24-31
Brain Research

Research Report
Brain microvessel endothelial cell responses to tumor necrosis factor-alpha involve a nuclear factor kappa B (NF-κB) signal transduction pathway

https://doi.org/10.1016/j.brainres.2005.04.028Get rights and content

Abstract

The involvement of nuclear factor kappa B (NF-κB) in TNF-induced increases in cerebral microvascular permeability was evaluated both in vitro, using primary cultured bovine brain microvessel endothelial cells (BBMEC), and in vivo, using the rat cranial window model. In primary cultured BBMEC, TNF exposure resulted in an increased appearance of the Rel A subunit of NF-κB in immunoblots of cell lysates. Increases in the Rel A subunit of NF-κB were observed as early as 30-min after administration of TNF. The increased permeability and the secretion of prostaglandin E2 in response to TNF exposure in BBMEC monolayers were significantly reduced by several different NF-κB inhibitors, including PDTC, CAPE, BAY 11-7085, and lactacystin. Similar results were also obtained in the rat cranial window model where treatment with the COX-2 inhibitor, NS-398 (0.1 μM), or the NF-κB inhibitor, PDTC (10μM), significantly reduced the permeability increases produced by TNF. These studies suggest that the increases in BBB permeability following TNF exposure are attributable to activation of an NF-κB-mediated signaling pathway in the cerebral microvasculature.

Introduction

Tumor necrosis factor-alpha (TNFα) is a proinflammatory cytokine released in response to viral and bacterial infections and tissue trauma. A major site of action for TNFα is the microvasculature where alterations in cytokine expression, adhesion molecule expression, and permeability are produced [15], [23], [32]. Within the cerebral microvasculature, TNFα-induced increases in permeability have been reported [1], [10], [11], [13], [18], [23], [24], [26]. Increases in circulating levels of TNFα are found in pathological conditions such as bacterial meningitis, acquired immune deficiency syndrome (AIDS)-related dementia, multiple sclerosis and stroke [5], [33]. Thus, understanding the cellular mechanisms responsible for TNFα-induced increases in cerebral microvascular permeability could provide further insight into the alterations in BBB integrity that occur during inflammatory conditions of the central nervous system.

Previous studies indicated that increases in brain microvessel endothelial cell permeability produced by TNFα were both time- and concentration-dependent and were correlated with alterations in cytoskeletal structure of the cells [11], [23]. Studies by Mark et al. [24] demonstrated the permeability and cytoskeletal changes observed in cultured brain microvessel endothelial cells following TNF exposure were associated with the induction of cyclooxygenase 2 (COX-2) and the subsequent release of PGE2. The lag time between TNFα exposure and TNFα-mediated effects on microvasculature permeability in both in vitro and in vivo models of the BBB suggest alterations at the gene transcription level. However, the potential intracellular signal transduction pathways involved are not well understood.

Intracellular signal transduction pathways activated by TNFα can be grouped into three general categories. TNF-mediated apoptotic cell death, which is mediated through a caspase cascade [21], [30], TNFα-induced mitogenic, or TNFα-induced inflammatory responses that are mediated through transcription factors like Activator Protein one (AP-1) and nuclear factor kappa B (NF-κB) [21], [38]. The current study focuses on the involvement of NF-κB in TNFα-induced increases in cerebral microvasculature permeability using both in vitro (primary cultured bovine brain microvessel endothelial cells) and in vivo (rat cranial window) models. These studies show that TNFα exposure in primary bovine brain microvessel endothelial cells (BBMEC) causes activation of NF-κB. Furthermore, treatment of BBMEC with NF-κB inhibitors were able to abolish TNF-induced increases in both permeability and prostaglandin release. Similar reductions in brain microvessel permeability were also observed in TNFα-treated rats following NF-κB inhibition. Together, these studies indicate that the increases in prostaglandin release and resulting increases in brain microvessel endothelial cell permeability following TNFα exposure occur through an NF-κB-dependent signaling process.

Section snippets

Cell isolation and culturing

Primary cultured bovine brain microvessel endothelial cells (BBMEC) were isolated from the gray matter of fresh bovine cerebral cortices through a combination of mechanical and enzymatic digestions and centrifugal separations [28]. Freshly isolated BBMEC were plated at a seeding density of 50,000 cells/cm2 on collagen-coated, fibronectin-treated 6-well culture plates or Transwell™ polycarbonate membrane inserts (24 mm; 0.4 μm pore size; Costar, Cambridge, MA). The BBMEC were cultured in a

Effects of NF-κB inhibitors on TNFα-mediated release of PGE2 from BBMEC

BBMEC monolayers treated with various doses of human recombinant TNFα (0.1 ng/ml to 100 ng/ml) exhibited a dose- and time-dependent increase in PGE2 release when compared to control monolayers receiving culture media alone. Significant increases in PGE2 release were observed within 4 h of TNFα exposure (1 ng/ml to 100 ng/ml) and remained elevated throughout the 12-h study (Fig. 1). However, at later time points, increased PGE2 release was observed with all TNF concentrations examined. To

Discussion

Tumor necrosis factor-α is a proinflammatory cytokine that is released in response to tissue injury and bacterial and viral infections. The effects of TNFα on cerebrovascular permeability have been well-documented with both in vitro [11], [13], [23], [24] and in vivo [1], [10], [18], [26] studies reporting increased permeability following TNFα exposure. In cultured brain microvessel endothelial cells, the permeability and cytoskeletal changes observed following TNFα exposure are associated with

Acknowledgments

This work was supported by PHS grant R29-NS-36831 (DWM) and NIH grant HL-40781 (WGM).

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