An essential role of interleukin-1beta in mediating NF-kappaB activity and COX-2 transcription in cells of the blood-brain barrier in response to a systemic and localized inflammation but not during endotoxemia

Abstract

When released into the bloodstream, proinflammatory cytokines have the ability to trigger the transcription of different genes in cells of the blood-brain barrier (BBB), including members of the nuclear factor kappa B (NF-kappaB) family and cyclooxygenase-2 (COX-2), the limiting enzyme for the formation of prostaglandins (PGs). The present study investigated the possibility that interleukin-1beta (IL-1beta) plays an essential role in these events during a systemic inflammatory response. Both wild-type and IL-1beta-deficient mice were killed at different times after two different immunogenic stimuli, i.e., intraperitoneal lipopolysaccharide (LPS) injection and intramuscular turpentine injection, used here as a model of systemic localized inflammatory insult. The inhibitory factor kappaBalpha (IkappaBalpha, index of NF-kappaB activity) and COX-2 transcripts were detected throughout the brain by means of in situ hybridization. Systemic LPS injection caused a strong and rapid expression of IkappaBalpha in endothelial cells lining the BBB of large and small blood vessels and thereafter within parenchymal microglia across the brain. This treatment also provoked a transient expression of COX-2 along cells of the vascular system, and the expression pattern and intensity of the signal for both transcripts were essentially the same in wild-type and IL-1beta-deficient animals. In contrast, the induction of these genes that was quite selective to the cells of the BBB in response to intramuscularly turpentine insult was completely abolished in IL-1beta-deficient mice. Indeed, a late and prolonged expression of IkappaBalpha and COX-2 mRNAs was found along the cerebral blood vessels in response to the sterile and localized inflammation in wild-type mice, whereas such induction was absent in the brain of IL-1beta-deficient animals. These results indicate that IL-1beta has an obligatory role in the activation of NF-kappaB molecules and PGs within endothelial cells of the BBB in an experimental model of intramuscularly turpentine-induced inflammation but not during endotoxemia.

Fig. 1.

Time-related influence of endotoxin LPS injection on the expression of the mRNA encoding IκBα in the blood vessels and parenchymal elements of the mouse brain. Animals were killed 0.5, 1.5, 3, and 6 hr after intraperitoneal treatment with LPS (100 μg/100 gm b.w.) or the vehicle (Veh) solution. These dark-field photomicrographs of dipped coronal sections (20 μm) into nuclear emulsion NTB2 exhibit positive signal first within blood vessels and then within small cells scattered across the brain parenchyma. Magnification: left panels, 25×;right panels, 50×.

Fig. 2.

Effects of the bacterial endotoxin LPS or turpentine injection on the expression of IκBα mRNA in the brain of wild-type (WT) and IL-1β-deficient (KO) mice. These dark-field photomicrographs of dipped NTB2 emulsion slides depict IκBα mRNA induction in vascular and nonvascular elements of the brain in mice killed 3 hr after intraperitoneal LPS injection or 7.5 hr after intramuscular turpentine administration. Note that LPS caused induction of the inhibitory factor within blood vessels as well as dispersed small cells in the brain of both mouse groups (middle panels), whereas the systemic and localized inflammatory insult provoked IκBα transcription only in cells associated with the blood–brain barrier in wild-type but not IL-1β knock-out mice (right panels). Magnification, 25×.

Fig. 3.

Representative examples of COX-2 expression in blood vessels of wild-type (WT) and IL-1β-deficient (KO) mice in response to systemic LPS or turpentine injection. Animals were killed 3 hr after intraperitoneal LPS injection (middle panels) and 7.5 hr post-turpentine injection into the left hind paw (right panels). These dark-field photomicrographs show in situ hybridization signals for COX-2 mRNA through the brain microvasculature in wild-type mice receiving either LPS or turpentine challenge and in knock-out animals during endotoxemia. The mRNA encoding COX-2 was undetectable in the cerebral vascular cells of IL-1β knock-out mice in response to the systemic and localized inflammatory insult (right bottom panel). Magnification, 25×.

Fig. 4.

Phenotype of IκBα- and COX-2-expressing cells during endotoxemia. Endothelial cells were labeled by immunohistochemistry using an antisera against the von Willebrand factor (vWF-ir), whereas a COX-2 antibody (COX-2-ir) was used to perform the dual-labeling with IκBα-expressing cells (middle right panel). Cells of myeloid origin were labeled by the immunoperoxidase technique using an antisera directed against ionized calcium binding adapter molecule (iba1-ir, bottom panels). IκBα or COX-2 mRNA was thereafter hybridized on the same sections by means of a radioactive in situ hybridization technique (silver grains). Note the presence of the mRNA encoding COX-2 within endothelial cells (top panels,black arrowheads) but not those positive for iba1 molecule (bottom panels, white arrowheads). Note also the expression of IκBα mRNA in the endothelium of the brain microvasculature and within cells positive for COX-2 protein (middle panels). Black arrowheads, Dual-labeled cells (endothelial–COX-2 mRNA,top panels; endothelial–IκBα mRNA, middle left panel; COX-2-IR–IκBα mRNA, middle right panel); white arrowheads, single iba1-IR cells along the vascular walls (perivascular microglia); white arrows, single iba1-IR cells within the brain parenchyma (parenchymal microglia); black arrows, single COX-2-expressing cells along the cerebral endothelium.bv, Blood vessel. Magnification, 100×.