A method for the selective depletion of perivascular and meningeal macrophages in the central nervous system

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Abstract

The perivascular (PVM) and meningeal (MM) macrophages form a distinct population of resident CNS cells, selectively expressing the mature macrophage marker ED2 in the rat. In order to elucidate the role of the PVM and MM in rats during normal functioning of the brain and pathology, we have developed a strategy employing a single intraventricular injection of clodronate liposomes. This resulted in a complete depletion of the PVM and MM. Clodronate liposomes did not deplete the microglial cells. In other parts of the body, a temporal and mild depletion effect was observed, which was restored within 1 week. Detailed analysis of the elimination and repopulation kinetics of the PVM and MM revealed a slow repopulation of the CNS, starting at 14 days post depletion. This selective depletion method of the PVM and MM will enable us to get direct insight in their functions during normal and pathologic conditions of the CNS.

Introduction

In the past, the brain was characterized immunologically as a site of limited reactivity Wekerle et al., 1986, Cserr and Knopf, 1992. As quoted by Leslie Brent: “It is beneficial to the organism not to turn the brain into an inflammatory battlefield” (Brent, 1990). This so-called immune privilege of the central nervous system (CNS) was based on different observations implicating an isolation of the brain from the immune system (reviewed by Cserr and Knopf, 1992). In the mean time, evidence for the communication between the CNS and the immune system has become available (Weller et al., 1996), and it has become clear that CNS-endogenous cells may themselves initiate, regulate and sustain an immune response (Becher et al., 2000).

One of the cells that may actively regulate CNS immunoreactivity is the perivascular macrophage (PVM). The PVM, as a component of the blood–brain barrier, is considered to form the first line of defence once the endothelial integrity of the barrier is damaged or circumvented. Unlike the pericyte, the PVM is never completely enclosed within the basal lamina of the blood vessels (Angelov et al., 1998a). Among the resident macrophages in the CNS, i.e. microglial cells, perivascular and meningeal macrophages (MM) Graeber and Streit, 1990, Lassmann et al., 1991, Lassmann et al., 1993, Kida et al., 1993, Mato et al., 1996, the PVM forms a distinct population of cells. They are bone marrow derived (Hickey et al., 1992), express the ED2 antigen (Graeber et al., 1989), scavenger receptors and are actively phagocytic (Mato et al., 1996). In response to CNS injury, the PVM upregulate MHC Class II (Streit et al., 1989) and produce several inflammatory mediators Angelov et al., 1998b, Bauer et al., 1995, Elmquist et al., 1997. In spite of this extensive morphological and phenotypical characterization of the PVM, essentially nothing is known about their biological function. The role of the meningeal macrophages that line the vasculature of the meninges in the CNS and are therefore considered to be a subset of PVM, is even less well characterized.

Selective depletion of macrophages is a widely accepted approach to investigate their functional aspects in vivo. Selective elimination of macrophages by intravenous injection of clodronate liposomes has been described (van Rooijen and Sanders, 1994). Macrophages ingest the liposomes by phagocytosis, the phospholipid bilayers of the liposomes are disrupted under influence of lysosomal phospholipases, and the intracellular released clodronate induces apoptosis van Rooijen et al., 1996, van Rooijen and Sanders, 1997. Free clodronate has an extremely short half-life, explaining the fact that nonphagocytic cells are not affected (van Rooijen and Sanders, 1996). Since macrophages are present in nearly every organ of the body and have important immunoregulatory functions, the liposome depletion method is widely used. Organ-specific macrophage depletion is possible, depending on the administration route of the liposomes. For example, selective in vivo elimination of alveolar macrophages can be achieved after intratracheal injection of clodronate liposomes Thepen et al., 1989, Wijburg et al., 1997. Another example is the in vivo manipulation of testicular macrophages by local injection, to investigate their regulatory capacity on the Leydig cell steroidogenesis (Gaytan et al., 1996). The effect of macrophage depletion by clodronate liposomes has also been reported in different CNS pathological conditions, and has demonstrated the macrophage participation during endotoxin-induced uveitis (Pouvreau et al., 1998). We have previously demonstrated the role of macrophages during experimental allergic encephalomyelitis (EAE) by using systemic mannosylated clodronate liposome application. This liposome treatment resulted in a suppression of clinical signs of EAE and a marked reduction of infiltrating macrophages in the CNS, indicating that these macrophages play an important role during EAE pathogenesis (Huitinga et al., 1990). In contrary to the infiltrating macrophages, the perivascular macrophages were not depleted by this treatment, probably because liposomes do not pass the intact blood–brain barrier (Bauer et al., 1995). More definite conclusions on the role of the PVM can be obtained once selective elimination of these cells is possible. In rabbits, a method for the depletion of MM has been described and used to study their role during meningitis (Trostdorf et al., 1999). In this study, unfortunately, the liposome treatment only resulted in a partial depletion of the meningeal macrophages, with no further information about: (1) the effects on the perivascular macrophages, (2) the effects in various parts of the CNS and (3) the periphery, (4) the turnover kinetics and (5) the selectivity of the elimination protocol.

Here, we describe a method for the selective depletion of the perivascular and meningeal macrophages in rats. The depletion and repopulation kinetics, selectivity and effect on the periphery have been studied in detail.

Section snippets

Animals

Male Wistar rats were obtained from CPB-Harlan (Zeist, Netherlands) and weighed 300–350 g at the time of the experiment. Animals were kept under routine laboratory conditions and were allowed free access to food and water. Microbiological status of the animals was according to the FELASA recommendations.

Preparation of liposomes

Multilamellar mannosylated liposomes were prepared as described before (van Rooijen and Sanders, 1989). Briefly, 178 mg phosphatidylcholine and 27 mg cholesterol were dissolved in 8 ml

Complete depletion of the perivascular and meningeal macrophages

In order to achieve local depletion of the perivascular and meningeal macrophages in the CNS, we tried local injection into the CNS of clodronate mannosylated liposomes at various doses and time points. After evaluation, treatment with a single intraventricular injection of 50 μl mannosylated clodronate liposomes gave the best result. This dose comprises 1/10 of the dose that is generally used for systemic application Huitinga et al., 1990, Bauer et al., 1993. ED2 immunohistochemistry 4 days

Discussion

There are three populations of resident macrophages in the CNS: microglial cells Graeber and Streit, 1990, Lassmann et al., 1991, Lassmann et al., 1993, and perivascular- and meningeal-macrophages Kida et al., 1993, Mato et al., 1996. Although the perivascular (PVM) and meningeal (MM) macrophages have been characterized morphologically and phenotypically Angelov et al., 1998a, Hickey and Kimura, 1988, Graeber et al., 1989, Mato et al., 1996, Streit et al., 1989, Bauer et al., 1993, essentially

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