Monocyte chemoattractant protein-1 in the choroid plexus: a potential link between vascular pro-inflammatory mediators and the CNS during peripheral tissue inflammation

Abstract

During peripheral tissue inflammation, inflammatory processes in the CNS can be initiated by blood-borne pro-inflammatory mediators. The choroid plexus, the site of cerebrospinal fluid (CSF) production, is a highly specialized interface between the vascular system and CNS, and thus, this structure may be an important element in communication between the vascular compartment and the CNS during peripheral tissue inflammation. We investigated the potential participation of the choroid plexus in this process during peripheral tissue inflammation by examining expression of the small inducible cytokine A2 (SCYA2) gene which codes for monocyte chemoattractant protein-1 (MCP-1). MCP-1 protein was previously reported to be induced in a variety of cells during peripheral tissue inflammation. In the basal state, SCYA2 is highly expressed in the choroid plexus as compared with other rat CNS tissues. During hind paw inflammation, SCYA2 expression was significantly elevated in choroid plexus, whereas it remained unchanged in a variety of brain regions. The SCYA2-expressing cells were strongly associated with the choroid plexus as vascular depletion of blood cells by whole-body saline flush did not significantly alter SCYA2 expression in the choroid plexus. In situ hybridization suggested that the SCYA2-expressing cells were localized to the choroid plexus stroma. To elucidate potential molecular mechanisms of SCYA2 increase, we examined genes in the nuclear factor-kappa B (NF-kappaB) signaling cascade including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and inhibitor of kappa B alpha (IkappaBalpha) in choroid tissue. Given that we also detected increased levels of MCP-1 protein by ELISA, we sought to identify potential downstream targets of MCP-1 and observed altered expression levels of mRNAs encoding tight junction proteins TJP2 and claudin 5. Finally, we detected a substantial up-regulation of the transcript encoding endothelial leukocyte adhesion molecule 1 (E-selectin), a molecule which could participate in leukocyte recruitment to the choroid plexus along with MCP-1. Together, these results suggest that profound changes occur in the choroid plexus during peripheral tissue inflammation, likely initiated by blood-borne inflammatory mediators, which may modify events in CNS.

Fig. 1

Distribution of SCYA2 mRNA in choroid plexus and in selected brain regions. Total RNA preparations from tissues were subjected to semi quantitative RT-PCR analysis using specific primers for SCYA2. Tissues were taken from non-perfused animals (A) or animals that were perfused with saline (B) to flush blood leukocytes out of the vascular tree. The levels of SCYA2 mRNA were normalized to a housekeeping gene (see Materials and Methods). All graphs are presented as mean ± SEM.

Fig. 2

Examination of the effects of hind paw inflammation on SCYA2 mRNA levels in choroid plexus and in selected brain regions. Tissues were taken from non-perfused animals (A) or animals that were perfused with saline (B) to flush blood leukocytes out of the vascular tree. Samples are from control and hind paw inflamed animals as indicated in the figures. The levels of SCYA2 transcripts in hind paw inflamed versus control animals were compared for each tissue. For each tissue, SCYA2 expression from control animals was set to a value of one (control expression/control expression). Graphed values for SCYA2 expression in 24h hind paw inflamed animals were determined by dividing average expression following inflammation by the average obtained in control animals. Graphs are presented as mean ± SEM. *P < 0.05 and **P < 0.01 as determined by Student's t-test.

Fig. 3

Effects of hind paw inflammation on SCYA2/MCP-1 levels in rat choroid plexus. (A) Time course of SCYA2 expression as determined by RT-PCR in choroid plexus of hind paw inflamed animals. Mean ± SEM are presented. ***P < 0.001 as determined by a one-way ANOVA followed by a Bonferroni correction. (B) Representative gel images showing bands corresponding to SCYA2 and GAPDH. At 1 h, no differences could be detected in SCYA2 levels as compared to control samples. A marked increased could be detected by 24 h. (C) Increase in MCP-1 protein 24 h after hind paw inflammation. Supernatant was obtained from homogenized choroid plexus taken from saline perfused animals. MCP-1 levels were determined by ELISA which was normalized to total protein. Graph is presented as mean ± SEM. *P < 0.05 as determined by Student's t-test.

Fig. 4

In situ hybridization of SCYA2 transcript expression in choroid plexus after hind paw inflammation. Representative microphotographs showing SCYA2 expression in the choroid plexus in a control animal (A) or in a hind paw inflamed animal (B). Higher magnification of SCYA2-expressing cells demonstrates that they are not epipthelial cells (black arrow) or cells located on the apical side (*) of the epithelium such as epiplexus cells. In contrast, the SCYA2 expressing cells appear on the basolateral side (white arrow) of the epithelium where they could be in the stroma.

Fig. 5

Effects of hind paw inflammation on cytokine transcript levels in rat choroid plexus. Total RNA preparations from tissues were subjected to semi quantitative RT-PCR analysis using specific primers for TNF-α (A), IL-1β (B), caspase 1 (C), IL-1R2 (D), IκBα (E) and Adam 17 (F). Tissues were taken from animals that were perfused with saline. Graphs shows relative levels of mRNAs and are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by a one-way ANOVA followed by a Bonferroni correction.

Fig. 6

Effects of hind paw inflammation on cell adhesion molecule transcript levels in rat choroid plexus. Total RNA preparations from tissues were subjected to semi quantitative RT-PCR analysis using specific primers for various cell adhesion molecules (A). Tissues were taken from animals that were perfused with saline. Graphs shows relative levels of mRNAs and are presented as mean ± SEM. *** P < 0.001 as determined by Student's t-test. Insert shows gel image of E-selectin in choroid plexus from control and 24 h hind paw inflamed animals. (B) Time course of E-selectin transcript induction following hind paw inflammation. Tissues were taken from animals that were perfused with saline. Graphs shows relative levels of E-selectin mRNAs and are presented as mean ± SEM. *P < 0.05 as determined by a one-way ANOVA followed by a Bonferroni correction.

Fig. 7

Effects of hind paw inflammation on tight junction protein transcript levels in rat choroid plexus. Total RNA preparations from tissues were subjected to semi quantitative RT-PCR analysis using specific primers for TJP2 and claudin 5 (A), TJP1 and occludin (B). Tissues were taken from animals that were perfused with saline. Graphs shows relative levels of mRNAs and are presented as mean ± SEM. Graphs are presented as mean ± SEM. *P < 0.05 and **P < 0.01, and ***P < 0.001 as determined by a one-way ANOVA followed by a Bonferroni correction.