Research ReportBrain microvessel endothelial cell responses to tumor necrosis factor-alpha involve a nuclear factor kappa B (NF-κB) signal transduction pathway
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).
References (38)
- et al.
Intracarotid tumor necrosis factor-alpha administration increases the blood–brain barrier permeability in cerebral cortex of the newborn pig: quantitative aspects of double-labeling studies and confocal laser scanning analysis
Neurosci. Lett.
(1996) - et al.
Expression of tumor necrosis factor alpha after focal cerebral ischemia in the rat
Neuroscience
(1996) - et al.
Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death
J. Biol. Chem.
(2004) - et al.
Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells
J. Biol. Chem.
(2001) - et al.
The influence of cytokines on the integrity of the blood–brain barrier in vitro
J. Neuroimmunol.
(1996) - et al.
Mechanistic studies on the inactivation of the proteasome by lactacystin
J. Biol. Chem.
(1996) - et al.
NF-kappaB activation suppresses host cell apoptosis during Rickettsia rickettsii infection via regulatory effects on intracellular localization or levels of apoptogenic and anti-apoptotic proteins
FEMS Microbiol. Lett.
(2004) - et al.
NF-κB and Rel proteins in innate immunity
Adv. Immunol.
(1995) - et al.
Increased permeability of primary cultured brain microvessel endothelial cell monolayers following TNF-α exposure
Life Sci.
(1999) Cellular mechanisms by which tumor necrosis factor-a produces disruption of the blood–brain barrier
Brain Res.
(2002)
Novel inhibitors of cytokine-induced IκB-alpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo
J. Biol. Chem.
Early increases in TNF-alpha, IL-6 and IL-1 beta levels following transient cerebral ischemia in gerbil brain
Neurosci. Lett.
Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling
Int. J. Biochem. Cell. Biol.
Cytokine-mediated induction of cyclo-oxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide
Br. J. Pharmacol.
Endothelial apoptosis induced by oxidative stress through activation of NF-kappaB: antiapoptic effect of antioxidant agents on endothelial cells
Hypertension
Inhibitory action of nitric oxide on circulating tumor necrosis factor-induced NF-kappaB activity and COX-2 transcription in the endothelium of the brain capillaries
J. Neuropathol. Exp. Neurol.
Tumor necrosis factor-α-induced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IκB kinase 1/2 in human alveolar epithelial cells
Mol. Pharmacol.
TNF-α-induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C-γ2, protein kinase C-α, tyrosine kinase, NF-κB-inducing kinase, and IκB Kinase 1/2 pathway
J. Immunol.
Conjugated polyhydroxybenzene derivatives block tumor necrosis factor-alpha-mediated nuclear factor-kappaB kinase activation and cyclooxygenase-2 gene transcription by targeting IkappaB kinase activity
Mol. Pharmacol.
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