Spatiotemporal induction patterns of cytokine and related immune signal molecule mRNAs in response to intrastriatal injection of lipopolysaccharide

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Abstract

The brain’s response to a direct immune challenge was examined by in situ hybridization histochemistry. Lipopolysaccharide (bacterial endotoxin) injected acutely into rat striatum induced mRNA expression for inhibitory factor κBα, interleukin (IL)-1β, tumor necrosis factor-α, IL-6, IL-12 p35, inducible nitric oxide synthase, IL-1 receptor antagonist, and the type 1 IL-1 receptor. Expression patterns were evaluated at select time points ranging from 15 min to 3 days post-injection. Rats injected with vehicle alone were used to control for mechanical effects. Following lipopolysaccharide administration, a wave of mRNA induction within brain parenchyma radiated outward from the injection site, generally peaking in intensity at the 16-h time point. The individual profiles of cytokine mRNA induction patterns reveal that the brain’s immune response to local inflammatory stimulation is quite elaborate and in many ways resembles the progression of cytokine induction customary of localized inflammation in peripheral tissues.

Introduction

Basic knowledge of the response of the central nervous system (CNS) to immune challenges is evolving. CNS studies are revealing active and unique forms of immune regulation comprised of selective passage of immune-derived cells across the blood–brain barrier (BBB) and local mediation and elaboration of inflammatory processes (reviewed by Fabry et al., 1994, Xiao and Link, 1998). In particular, resident brain cells produce their own cytokines that serve as mediators in response to both peripheral immune challenges (Hansen et al., 1998, Quan et al., 1998) and central invasion of infectious agents (Higgins and Olschowka, 1991, Wesselingh et al., 1994).

Acting in a pleotrophic cascade of autoinduction, synergism, and inhibition, cytokines appear able to function both protectively (Strijbos and Rothwell, 1995, Bruce et al., 1996, Klein et al., 1997) and destructively (Campbell, 1995), though the circumstances dictating these differential effects are not well understood. Knowing more about the unique regulation of immune responses in the brain may aid in developing therapeutic strategies for ischemia, traumatic lesions, infection, and chronic inflammatory or neurodegenerative diseases associated with an upregulation of inflammatory cytokines.

One commonly studied model of immune challenge is local administration of lipopolysaccharide (LPS), an endotoxin derived from the cell wall of gram-negative bacteria. LPS is a potent proinflammagen in peripheral tissue, where it triggers an acute inflammatory reaction characterized by cytokine release, recruitment of inflammatory cells, activation of the complement cascade, and accumulation of clotting factors (Kirkpatrick, 1997). There is strong evidence that the pathophysiological effects of LPS in non-CNS tissue are mediated by the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Intratracheal injection of IL-1β or TNF-α mimics the inflammatory response to LPS in rat (Tracey et al., 1986, Ulich et al., 1991b). A significant reduction in endotoxin-induced mortality occurs in animals administered TNF-α antibodies (Beutler et al., 1985) or receptor antagonists for TNF-α (Ashkenazi et al., 1991, Lesslauer et al., 1991) or IL-1β (Ohlsson et al., 1990, Wakabayashi et al., 1991).

Injection of LPS directly into the brain has been employed to investigate the progression of brain inflammation. Intraparenchymal LPS induces strong local glial activation (Andersson et al., 1992, Bell et al., 1996), and it may also induce a brain cytokine response, though the evidence is indirect. An intracranial injection of LPS plus interferon-γ (IFNγ) elevates IL-1β mRNA levels at 2, 3 and 5 days after injection (Higgins and Olschowka, 1991). Intracranial LPS induces an upregulation of macrophage scavenger receptor — a candidate adhesion molecule receptor — on resident microglia 24 h after injection (Bell et al., 1994) and the adhesion molecules VCAM and ICAM-1 in brain endothelia 6 h after injection (Bell and Perry, 1995). This upregulation may be due to local cytokine activity because cytokines such as IL-1β and TNF-α are known to induce synthesis and secretion of certain endothelial adhesion molecules (Wong and Dorovini-Zis, 1992, Wong and Dorovini-Zis, 1995, McCarron et al., 1993). Finally, elevations in levels of inducible nitric oxide synthase (iNOS) mRNA (the cytokine-inducible form of NOS), iNOS immunoreactivity, and nitric oxide metabolites have been reported following intrahippocampal LPS administration (Garcion et al., 1998, Yamada et al., 1999).

No direct experiment, however, has detailed the acute brain cytokine response to intracranial LPS. An anatomical examination, using in situ hybridization histochemistry, allows us to track the spatiotemporal progression of brain cytokine signaling and compare it to that of peripheral tissues following local induction of inflammation. Cytokine production and release in response to LPS application in vitro has been demonstrated for brain microglia and astrocytes (Frei et al., 1987, Robbins et al., 1987, Lieberman et al., 1989, Righi et al., 1989, Sawada et al., 1989), but whether these cells behave similarly in vivo is not known.

Section snippets

Animals and LPS injection

Forty male Sprague-Dawley rats (200–300 g; Taconic Farms, Germantown, NY, USA) were group housed in a 12:12 h L:D cycle with access to food and water ad libitum. Rats were anesthetized (isoflurane gas) and injected unilaterally in the caudate-putamen (striatum) through a 32 ga. needle connected to a micro-injection pump releasing 1 μl over 5 min. Injections were stereotaxically made 0.5 mm rostral and 2.8 mm lateral relative to bregma and 4.0 mm ventral from dura. Dose-dependency tests were

General features of all mRNA transcripts

LPS injected into the caudate-putamen caused significant induction of mRNA of IκBα, IL-1β, TNF-α, IL-6, IL-12 p35, IL-1R1, IL-1ra, and iNOS in the striatum and more distant sites over the surveyed time course of 15 min to 3 days (all times are post-injection). There was no detectable transcript expression of IL-4, IL-10, and IFNγ observed at any location in this time span. Increasing doses of LPS (0.05, 0.5, or 5.0 μg) evaluated at 3 h resulted in dose-dependent increases in mRNA expression

Discussion

Previous studies have established an upregulation of pro-inflammatory cytokine and IκBα mRNA expression levels in the brain in response to a peripheral administration of LPS (Breder et al., 1994, Buttini and Boddeke, 1995, Quan et al., 1997, Quan et al., 1998, Vallières and Rivest, 1997, Nadeau and Rivest, 1999). Here we display a uniquely vigorous brain cytokine mRNA induction following centrally administered LPS. LPS injected directly into the caudate-putamen triggered a coordinated induction

Acknowledgements

Dr. Nancy Tresser analyzed and interpreted H&E-stained material. Some preliminary studies were performed in the Laboratory of Neurotoxicology, NIMH, by MGP.

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    Present address: Department of Oral Biology, The Ohio State University, Columbus, OH 43210, USA.

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