Release of cytokines by brain endothelial cells: A polarized response to lipopolysaccharide

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Abstract

Brain endothelial cells (BECs) comprise the blood–brain barrier (BBB) and are an active part of the neuroimmune system, responding to and transporting cytokines. BECs also have the ability to secrete neuroimmune substances, including cytokines. A unique feature of the BEC is its polarization, with its luminal (blood-facing) and abluminal (brain-facing) cell membranes differing in their lipid, receptor, and transporter compositions. This polarization could have functional consequences for neuroimmune communication. We postulated (i) that cytokine secretion from the luminal or abluminal membranes could differ under baseline or stimulated conditions and (ii) that an immune challenge from one side of the BBB could result in cytokine release from the other. We used an in vitro BBB model of mouse BECs cultured as monolayers to investigate cytokine secretion into luminal and abluminal chambers. Our major findings in these studies were: (i) the first demonstration that interleukin (IL)-1α, IL-10, and granulocyte-macrophage colony-stimulating factor are secreted from BECs and confirmation of the secretions of IL-6 and tumor necrosis factor-α, (ii) that constitutive and lipopolysaccharide (LPS)-stimulated secretion of cytokines is polarized in favor of luminal secretion, and (iii) that response to neuroimmune stimulation is also polarized as exemplified by the finding that abluminal LPS more robustly induced secretion of IL-6 than did luminal LPS. Overall, these findings support the BBB as an important source of cytokines. Furthermore, the BBB can respond to immune challenges received from one side of the neuroimmune axis by releasing cytokines into the other.

Introduction

Brain endothelial cells (BECs) constitute an important part of the neuroimmune axis. They are able to both transport and secrete neuroimmune substances, including cytokines. BECs are exposed to endogenous and exogenous neuromodulatory substances, immune cells, neurotransmitters and hormones, environmental stresses and cues, and potentially toxic compounds at both their brain (abluminal) and blood (luminal) sides. Many of these substances, including lipopolysaccharide (LPS) and the endogenous peptide β-amyloid, have been shown or postulated to alter BBB functions (Banks and Morley, 2003, Blanc et al., 1997, Dorheim et al., 1994, Fiala et al., 1998, Jancso et al., 1998, Persidsky et al., 1997, Reyes et al., 1999, Xaio et al., 2001). Systemically, LPS activates macrophages and monocytes through binding to CD14 and toll-like receptor-4 (TLR4) and leads to release of nitric oxide, prostaglandins, and a repertoire of cytokines such as IL-1, TNF-α, and IL-6 (Cao et al., 1997, Chow et al., 1999, Wright et al., 1991). LPS can evidently have similar effects on BECs, inducing the release of IL-6 (Reyes et al., 1999). Other factors also induce the release from BECs of endothelin, IL-8, IL-6, MCP-1, TNF, RANTES, nitric oxide, and prostaglandins (Didier et al., 2002, Dorheim et al., 1994, Hofman et al., 1999, Lee et al., 2001, MCGuire et al., 2003, Simpson et al., 1998, Vadeboncoeur et al., 2003).

BECs are specially modified and positioned to perform BBB functions such as preventing harmful substances from entering the brain and rigorously controlling the passage of constituents in the blood-to-brain or brain-to-blood direction. Collectively, these functions of BBB are aided by a polarization of BBB properties; that is, lipid composition and transporter and receptor protein populations are distributed unevenly and, in some cases, uniquely between the luminal or abluminal membranes of the BEC (Banks and Broadwell, 1994, Betz and Goldstein, 1978, Davson and Segal, 1996, Taylor, 2002, Vorbrodt, 1994, Zambenedetti et al., 1996). Thus, BECs have the potential to receive signals from one compartment (e.g., blood) and to secrete into the other (e.g., brain). For example, BEC cultured as monolayers (MBEC) and exposed to luminal gp120, the viral coat of HIV-1, secrete the cytokine endothelin-1 into the abluminal chamber (Didier et al., 2002).

The MBEC model has been used for over 20 years to study BBB functions (see Fig. 1 schematic). Cells polarize with luminal (blood-side) and abluminal (brain-side) properties reminiscent of in vivo conditions, including the presence and function of tight junctions and polarized transporters (Deli et al., 2005, Joo, 1993, Minokoshi et al., 2002). Anatomical studies of the in vivo BBB show that BECs are very closely associated with neighboring cells such as astrocytes, pericytes, and microglia. These cells help to induce and maintain the BBB by secreting soluble factors capable of acting on the BEC, thus providing the local conditions required for the proper induction, maintenance, and functioning of the BBB. For this particular reason, many BMEC models co-culture BEC with astrocytes, macrophages, or glial cells. However, these models would fail to shed light on the specific abilities of BECs to release cytokines. Keeping that in mind, we used an MBEC model culturing BECs alone to (i) inventory which of 10 common cytokines BECs spontaneously secrete, (ii) determine whether the spontaneous secretion was polarized, (iii) determine the effect of LPS on BEC cytokine secretion, and (iv) determine whether the effect of LPS itself was polarized.

Section snippets

Reagents

Mouse β-amyloid 1–42 and 1–40 were obtained from American Peptide. Dnase I, LPS, fibroblast growth factor, and fibronectin were obtained from Sigma (St. Louis, MO). ProteoPlex murine cytokine array kits detecting 10 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, IFN-γ, and TNF-α) were provided by EMD Biosciences (St. Louis, MO).

Isolation and culture of brain endothelial cells

The protocol for isolating mouse BECs and growing them into monolayers (MBEC) was modified as previously reported by our laboratory (Banks et al., 2004

Spontaneous polarized secretion of cytokines by MBEC

We first examined the baseline level of cytokine secretion by MBEC. Since MBEC presents a luminal (facing blood) and an abluminal (facing brain) side, we measured the levels of cytokines in the luminal and abluminal chambers (n = 2/group). Under our experimental conditions, IL-6, GM-CSF, and TNF-α were detected in the fluids from both the luminal and abluminal chambers, while IL-1α and IL-10 were detected in the luminal fluid only (Fig. 2). The concentrations for the other cytokines (IL-1β, IL-2,

Discussion

BECs constitute a distinct category of endothelial cells because of their separation of the CNS and blood and the polarized nature of the luminal and abluminal membranes. BECs have been added to the list of cells which secrete neuroimmune substances into the brain and blood (Didier et al., 2002, Dorheim et al., 1994, Hofman et al., 1999, Lee et al., 2001, MCGuire et al., 2003, Simpson et al., 1998, Vadeboncoeur et al., 2003). Here, we took advantage of a well-established in vitro monolayer

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