Rat area postrema microglial cells act as sensors for the toll-like receptor-4 agonist lipopolysaccharide

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Abstract

The area postrema (AP) represents the sensory circumventricular organ lacking endothelial blood-brain barrier function in direct vicinity to the 4th cerebral ventricle. Administration of lipopolysaccharide (LPS), as opposed to muramyldipeptide (MDP) or fibroblast-stimulating lipopeptide-1 (FSL-1), caused fast transient rises in intracellular calcium concentrations in 10–12% of the microglial cells investigated in a primary microculture of the rat AP, with limited responses of neurons, astrocytes and oligodendrocytes. In addition, a marked release of the pro-inflammatory cytokines TNF-α and IL-6 was determined in LPS-treated AP microcultures. Pre-incubation of AP microcultures with LPS for 18 h suppressed LPS-induced calcium signaling and attenuated cytokine secretion. Evidently, the AP can act as a sensor for circulating LPS and has the capacity to develop endotoxin-tolerance.

Introduction

Circumventricular organs (CVOs) represent brain structures endowed with dense vascularization, cells in direct contact with the cerebrovascular system and an absence of classical blood-brain barrier function (Johnson and Gross, 1993, McKinley et al., 1990). Structural and functional similarities are observed in a subgroup of these specialized brain structures named sensory CVOs: the vascular organ of the lamina terminalis (OVLT), the subfornical organ (SFO) and the area postrema (AP). These sensory CVOs possess capillaries with a fenestrated endothelium surrounded by perivascular spaces, and the parenchyma of these structures is composed of glial cells and of neuronal soma, dendritic/axonal processes and terminals revealing multiple reciprocal connectivities to other (extra-)hypothalamic nuclei (McKinley et al., 2003). Due to these properties, the SFO, OVLT and AP can act as sensors for chemical messengers circulating in the bloodstream. In addition to their function as structures involved in the maintenance of various vital homeostatic systems (Fry and Ferguson, 2007, Johnson and Gross, 1993, McKinley et al., 2003), sensory CVOs are regarded as important players within the multiple immune-to-brain communication pathways (Quan and Banks, 2007, Roth et al., 2004).

The AP is a component of the dorsal vagal complex, a major viscerosensory and autonomic center of the medulla oblongata, which acts as a brain monitor and integrator of systemic autonomic state (Price et al., 2008). Lesions of the AP or inactivation of the dorsal vagal complex including the AP attenuate the immune-mediated activation of the hypothalamo-pituitary-adrenal (HPA) axis (Lee et al., 1988) and parts of the inflammation-induced sickness behavior (Marvel et al., 2004). Systemic treatment of experimental animals with bacterial lipopolysaccharide (LPS) induces the expression of pro-inflammatory cytokines within the AP (Breder et al., 1994, Goehler et al., 2006, Quan et al., 1999). The explanation for the LPS-mediated localized formation of cytokines at the level of the AP was provided by studies demonstrating the existence of Toll-like receptor-4 (TLR4) and CD14 in the AP, both of which are employed by LPS for intracellular signal transduction (Laflamme and Rivest, 2001, Rivest, 2003). Due to the constitutive expression of TLR4 and CD14 within the AP, a pathogen-associated molecular pattern (PAMP) such as LPS can be sensed by cellular elements located in this specialized brain structure and is thus able to trigger brain-intrinsic responses even prior to the appearance of pro-inflammatory cytokines in the blood.

In this context, changes in intracellular calcium concentration ([Ca2+]i) have been measured in brain cells to elucidate LPS-mediated Ca2+-signaling in these cells (Bader et al., 1994, Choi et al., 2002, Hoffman et al., 2003, Kann et al., 2004). This approach might thus be a useful tool to demonstrate possible activation of cells within the AP by LPS as a putative and rapid component of the immune-to-brain communication pathways in this specific brain structure. The objectives of the experiments reported here can be summarized as follows. (1) Using a primary microculture of the rat AP, we aimed to investigate whether stimulation with the TLR4-agonist LPS, or for comparison with the TLR2-agonist muramyldipeptide (MDP) and the TLR2/6-agonist fibroblast-stimulating lipopeptide 1 (FSL-1), might result in measurable changes of [Ca2+]i in AP cells. (2) The phenotypes of investigated cells were determined immunocytochemically with antisera directed against cell-specific marker proteins for neurons, astrocytes, microglial cells and oligodendrocytes. (3) Concentrations of the bioactive cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured in the supernatants of the AP microcultures. (4) We finally investigated, at the level of Ca2+-signaling and of cytokine formation, whether refractoriness to the LPS-induced responses (“endotoxin-tolerance”) might develop in the AP primary microcultures after pre-incubation with LPS, MDP or FSL-1.

The results of these experiments should increase and improve the knowledge and understanding about the putative role of the AP as a true sensor for selected circulating PAMPs, i.e. for LPS, MDP or FSL-1.

Section snippets

Animals

Wistar rat pups of both sexes obtained from an in-house breeding colony were used for all experiments, with parent animals originating from Charles River WIGA (Sulzfeld, Germany). Animal care, breeding and experimental procedures have been conducted according to the guidelines approved by the Hessian Ethical Committee. Adult rats had free access to drinking water and standard lab chow; the pups were reared by their mothers in large M4-size cages. Room temperature was controlled at 24 ± 1 °C and

LPS induces calcium signaling in cells of AP microcultures

Primary rat AP microcultures after 5–6 days of in vitro differentiation contained small-sized bi- or tripolar neurons expressing microtubule-associated protein 2a + b, squamous or stellate astrocytes richly endowed with cytoskeletal GFAP, mature oligodendrocytes expressing CNPase and quiescent as well as some activated microglial cells staining for IB4, OX42 (both not shown) and/or ED-1 (Fig. 1). Endothelial cells and fibroblasts proved not to be present as indicated by negative immunolabeling

Area postema cells as potential sensors for circulating lipopolysaccharide

TLRs have been identified as a family of receptors of the innate immune system for a limited number of pathogen-associated molecular patterns (PAMPs), that are conserved across numerous types of pathogens (Kopp and Medzhitov, 2003). The PAMPs used in the present study are recognized by TLR4 (LPS, Chow et al., 1998), TLR2 (MDP, Takeuchi et al., 1999), or the functional pair TLR2/6 (FSL-1, Okusawa et al., 2004). Activation of these TLRs triggers signaling pathways, which finally lead to a

Acknowledgement

This study was supported by the Deutsche Forschungsgemeinschaft (DFG project GE 649/6-1).

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