Differential and time-dependent expression of monocyte chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain: effects of ischemia and peripheral lipopolysaccharide administration

doi:10.1016/S0165-5728(96)00203-2

Journal of Neuroimmunology

Volume 74, Issues 1–2, April 1997, Pages 35-44

https://doi.org/10.1016/S0165-5728(96)00203-2 Get rights and content

Abstract

Increasing evidence indicates a key role of chemoattractant cytokines in the accumulation of leukocytes in the central nervous system (CNS) during the course of inflammatory processes. Monocyte chemoattractant protein (MCP-1/JE), a member of the β-chemokine (C–C chemokine) family, functions as a potent chemoattractant and activator for monocytes. We have investigated the induction of MCP-1 mRNA using in situ hybridization histochemistry (ISH) and characterized its cellular source by combination of ISH and immunocytochemistry in ischemic rat brains as well as in brains of endotoxin-treated rats. Our results show that 6 h–2 d after middle cerebral artery occlusion (MCAO), MCP-1 mRNA is present in astrocytes surrounding the ischemic tissue (penumbra). At later time points (after 4 d), MCP-1 mRNA is found in macrophages and reactive microglia in the infarcted tissue. Peripheral administration of the bacterial lipopolysaccharide (LPS) induced MCP-1 mRNA throughout the brain in a time-dependent manner (1 h–1 d, peak of expression 6–8 h) and was found in astrocytes. In summary, we have found expression of MCP-1 in (a) astrocytes and to a lesser extent in macrophages/reactive microglia after MCA-occlusion and in (b) astrocytes after peripheral administration of LPS. These findings support that MCP-1 is involved in the CNS response to acute trauma or infection and thus may play a key role in inflammatory processes of the brain.

Introduction

JE/MCP-1 was originally identified as a growth factor-inducible early-response gene in murine fibroblasts (Cochran et al., 1983). More recent studies have shown that mature secreted MCP-1, a 76 amino-acid protein, has cytokine-like properties (Rollins et al., 1988) and attracts monocytes and macrophages to infectious and inflammatory sites. Indeed, MCP-1 (reviewed in Rollins, 1991; Leonard and Yoshimura, 1990) has been closely associated with inflammatory pathology both in human diseases as well as in animal models. Expression of MCP-1 has been described in the periphery during renal ischemia (Safirstein et al., 1991), during liver and lung injury (Czaja et al., 1994; Warren et al., 1993; Sakanashi et al., 1994), and rheumatoid arthritis (Akahoshi et al., 1993). Recently, several reports have also shown that MCP-1 plays a role in the central nervous system (CNS). Its expression has been reported in human gliomas (Kuratsu et al., 1993; Desbaillets et al., 1994), during experimental autoimmune encephalomyelitis (EAE) (Ransohoff et al., 1993) and in brain ischemia models (Kim et al., 1995; Wang et al., 1995). The cellular source of MCP-1 in brain, however, remains controversial. MCP-1 in rat brain was detected in endothelial and macrophage like cells after acute trauma and EAE (Kim et al., 1995; Berman et al., 1996). Astrocytes, however, have also been suggested as a possible source of MCP-1. This has been shown in vivo after EAE and mechanical trauma in the mouse brain (Ransohoff et al., 1993; Glabinski et al., 1996) as well as in vitro, in primary astrocyte cultures (Hurwitz et al., 1995; Hayashi et al., 1995).

Permanent occlusion of the middle cerebral artery (MCAO) in rats is a commonly used model of focal stroke resulting from thromboembolic events in humans (Tamura et al., 1981). In two recent studies (Kim et al., 1995; Wang et al., 1995), induction of MCP-1 mRNA after MCAO was shown using Northern analysis. However, no clear-cut cellular source for MCP-1 was provided. In addition, the bacterial lipopolysaccharide (LPS) has been shown to induce the expression of MCP-1 in murine peritoneal macrophages (Introna et al., 1987) and in astrocyte cultures (Hayashi et al., 1995). However, induction of MCP-1 expression by LPS has not yet been demonstrated in vivo in rat brain. Therefore, we have investigated the effect of intravenously injected LPS on MCP-1 mRNA induction in rat brain.

In the present study, we have used radioactive in situ hybridization (ISH) with specific MCP-1 mRNA antisense oligonucleotide probes and immunocytochemistry to determine the temporal induction profile and the cellular source of MCP-1 mRNA in the inflamed rat brain following ischemia and peripheral LPS administration.

Section snippets

Animal treatments

Middle cerebral artery occlusion (MCAO) was performed in male spontaneously-hypertensive rats (SHR), weighing approximately 250 g. Under isoflurane anesthesia, the left middle cerebral artery (MCA) was permanently occluded with a bipolator, as described previously (Sauter and Rudin, 1986; Tamura et al., 1981). Sham-operated animals were treated the same way as the operated rats except for closure of the MCA. SHR animals were used since in these rats MCAO induces a highly reproducible infarct

Middle cerebral artery occlusion

Using in situ hybridization histochemistry, a slight signal of MCP-1 mRNA was detected in the brains of sham-operated rats at the site of experimental intervention (data not shown). No signal was detected in brains of non-operated rats (Fig. 1A) or in the contralateral hemispheres of MCA-occluded rats as shown in Fig. 1. Induction of MCP-1 mRNA was detected 6 h after the onset of occlusion (Fig. 1B) and the highest signal density was observed 24 h after occlusion in the area medial to the

Discussion

The present study shows differences in the temporal and spatial pattern of expression of MCP-1 mRNA in rat brain following MCA-occlusion and peripheral administration of LPS. After MCAO, MCP-1 gene expression was induced initially in astrocytes in the penumbra zone (6 h–2 d) and then progressively appeared in macrophages/reactive microglia in the infarcted area (4–6 d). In contrast to the local expression after MCAO, MCP-1 mRNA was induced in astrocytes throughout the brain following peripheral

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