Peripheral macrophage recruitment in cuprizone-induced CNS demyelination despite an intact blood–brain barrier

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Abstract

The contribution of peripheral macrophage was assessed in cuprizone intoxication, a model of demyelination and remyelination in which the blood–brain barrier remains intact. Flow cytometry of brain cells isolated from cuprizone-treated mice revealed an increase in the percentage of Mac-1+/CD45hi peripheral macrophage. To confirm these results in situ, C57BL/6 mice were lethally irradiated, transplanted with bone marrow from GFP-transgenic mice, and exposed to cuprizone. GFP+ peripheral macrophages were seen in the CNS after 2 weeks of treatment, and infiltration continued through 6 weeks. While the peripheral macrophages were far outnumbered by the resident microglia, their recruitment across the blood–brain barrier alludes to a potentially important role.

Introduction

The blood–brain barrier (BBB) was originally thought to limit access of blood-borne immune cells into the healthy CNS, while its disregulation was concomitant with disease Perry et al., 1997, Miller, 1999. Indeed, in the CNS demyelinating diseases, multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), a compromise in the BBB and subsequent leukocyte infiltration precedes the appearance of clinical symptoms and correlates with the severity of disease Kermode et al., 1990, Sharief et al., 1993, Bourrie et al., 1999. Furthermore, a recent study has shown that local disruption of the BBB through the ablation of astrocytes leads to a sustained influx of peripheral leukocytes, illustrating the importance of this barrier in regulating immune cell trafficking into the CNS (Bush et al., 1999).

Nevertheless, a number of reports have now nullified the notion that the BBB is an impenetrable obstacle to leukocytes. Activated T cells can migrate across the intact BBB into the non-inflamed CNS, regardless of specificity, and can be found surveying the CNS for harmful pathogens or neoplasm (Hickey, 2001). In addition, microglia, the parenchymal brain resident macrophage, migrate out of the bone marrow, cross the intact BBB, and colonize the CNS shortly after birth with a low level of turnover throughout adulthood (Male and Rezaie, 2001). The resident meningeal and perivascular macrophage similarly populate the CNS, and their higher rates of turnover indicate they routinely cross the intact BBB Lawson et al., 1992, Kennedy and Abkowitz, 1997, Hickey, 2001. In addition, leukocyte infiltration into the CNS has also been noted in diseases in which BBB breakdown is negligible, such as in the twitcher mice (Wu et al., 2000) and in the more chronic disease, scrapie Williams et al., 1995, Betmouni et al., 1996.

In the cuprizone intoxication model of CNS demyelination, no BBB disruption has been detected by horseradish peroxidase (HRP) tracing or immunohistochemical staining for serum proteins Bakker and Ludwin, 1987, Kondo et al., 1987, despite a robust inflammatory response. In C57BL/6 mice fed low levels of the copper chelator, cuprizone, oligodendrocyte apoptosis precedes region-specific demyelination, which first becomes evident after 2 weeks of exposure and reaches completion in the corpus callosum by 5 weeks Hiremath et al., 1998, Morell et al., 1998, Mason et al., 2000. In addition to astrogliosis and the upregulation of numerous inflammatory cytokines, a massive activation and accumulation of Mac-1+ or RCA-1+ microglia/macrophage accompanies demyelination Blakemore, 1973, Ludwin, 1978, Hiremath et al., 1998, Morell et al., 1998, Matsushima and Morell, 2001. While many of these microglia/macrophage are undoubtedly serving to clear myelin debris, our laboratory and others have also shown that they can secrete molecules that both exacerbate disease (Hiremath et al., in preparation; Arnett et al., 2001) and, more importantly, protect or promote remyelination Arnett et al., 2001, Arnett et al., 2002, Mason et al., 2001b. However, it was not known if these Mac-1+ cells accumulating in the demyelinating lesions were exclusively comprised of brain resident microglia, proliferating locally or possibly being recruited from different regions of the CNS, or if blood-borne monocytes/macrophage were extravasating across the intact BBB.

In murine models, identification of resident microglia and peripheral macrophage is particularly difficult due to similar morphology after activation as well as shared cell surface markers. In the past, many groups have used bone marrow-chimeric animals to selectively track cells infiltrating into the CNS. Several different methods have been employed to distinguish donor bone marrow-derived cells versus host recipient cells including MHC allotypes Hoogerbrugge et al., 1988, Popovich and Hickey, 2001, the Y chromosome Williams et al., 1995, Eglitis and Mezey, 1997, transduced retroviral vectors (Eglitis and Mezey, 1997), and a β-galactosidase transgene (Kennedy and Abkowitz, 1997). All these methods, however, require further staining to detect bone marrow-derived cells, and the sensitivity for some may be as low as 60% (Williams et al., 1995). An alternative method of delineating these two populations is by flow cytometric analysis of CD45 expression in which resident microglia have low expression levels of CD45 (Mac-1+/CD45lo) and infiltrating macrophage have high (Mac-1+/CD45hi) Sedgwick et al., 1991, Ford et al., 1995. The cell yields from the isolation procedure from whole brain are extremely low, however, and in situ localization of either population is difficult.

In this report, we use both flow cytometry and green fluorescent protein (GFP)-positive, bone marrow-transplanted mice to demonstrate that peripheral macrophage are indeed migrating into the brain during cuprizone-induced demyelination despite the presence of an intact BBB. While flow cytometric analysis revealed a significant increase in the Mac-1+/CD45hi populations, the use of GFP transgenic mice as donors for the bone marrow transplants allowed us to reliably track GFP+ peripheral cell as they infiltrated into the brain parenchyma and localized to areas of demyelination.

Section snippets

Animals, bone marrow transplant, and cuprizone treatment

All animals were housed in a pathogen-free facility and were maintained in accordance with NIH guidelines and approved protocols by the University of North Carolina Institutional Animal Care and Use Committee. C57BL/6 (B6) mice were either purchased from Jackson Laboratories (Bar Harbor, ME) or bred and maintained in the UNC animal facility. C57BL/6 mice hemizygous for the enhanced GFP gene, under the control of the chicken β-actin promoter and CMV enhancer, were originally purchased through

The percent of Mac-1+/CD45hi cells was significantly increased in the brains of BBB-intact cuprizone-treated mice

To determine if peripheral cells were migrating into the brains upon cuprizone-induced demyelination, leukocytes were isolated from the brains of untreated and 6-week-treated B6 mice and analyzed for Mac-1 and CD45 expression by flow cytometry. Intracranially, LPS-injected mice served as a positive control for peripheral macrophage CNS infiltration (Perry and Andersson, 1992), and cells from these mice were used to define the Mac-1+/CD45+ populations (Fig. 1A). Five percent (±2.9) of the Mac-1+

Discussion

Cuprizone-induced demyelination is characterized by a robust microglia/macrophage response (Hiremath et al., 1998). At 4, 5 and 6 weeks of cuprizone exposure, the Mac-1+ or RCA-1+ population is so densely packed within the demyelinating lesions that individual cells are difficult to discern. Yet, it was not known if peripheral macrophage migrate across the intact BBB and contribute to this cellular accumulation or if the Mac-1+ or RCA-1+ population was exclusively comprised of brain-resident

Acknowledgements

We would like to thank Kathy Deschesne for invaluable help with mouse irradiations, Julie Farnsworth for flow cytometry assistance, and Dr. Robert Bagnell and Victoria J. Madden for helping with microscopy. We also thank Elise Cash and the Neurodevelopmental Disorder Research Center Morphology Core Facility (funded by the Mental Retardation Research Center core grant, HD03110) for access to histology equipment and Kasturi Puranam and Julie Jones for critical reading of the manuscript. This work

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