Selective localization of bone marrow-derived ramified cells in the brain adjacent to the attachments of choroid plexus

Abstract

Although the immune system modulates higher functions of the brain under non-inflammatory conditions, how immune cells interact with brain parenchymal cells remains to be determined. Using bone marrow chimeric mice in which the recipients’ immune system was reconstituted by marrow cells derived from GFP-transgenic mice by syngeneic intra-bone marrow-bone marrow transplantation (IBM-BMT) and by intravenous (IV)-BMT, we examined the distribution, density and differentiation of donor-derived marrow cells in the brain parenchyma 2 weeks and 1, 4 and 8 months after BMT. Marrow-derived cells started to populate discrete brain regions from 1 to 4 months after BMT, exhibited ramified morphology and expressed Iba-1. The ramified marrow-derived cells were distributed in more brain regions and for a longer time after IBM-BMT than IV-BMT. Most of these discrete regions were adjacent to the attachments of choroid plexus that comprised thinned brain parenchyma consisting of astroglial processes in the narrow channel between the ependyma and pia. These specific portions of astroglial processes expressed fractalkine. In the choroid plexus stroma, not only Iba-1+ myeloid cells but also non-myeloid CXCL12-expressing cells were of bone marrow-origin. Transcripts of fractalkine, CXCL12 and their related molecules such as CX3CR1, ADAM10 and CXCR4 were detected in the tissue consisting of the choroid plexus, the attachments and adjacent brain parenchyma. Thus, bone marrow cells selectively enter the discrete brain regions adjacent to the attachments of choroid plexus and differentiate into ramified myeloid cells. Fractalkine in the attachments of choroid plexus and CXCL12 in the choroid plexus stroma may be involved in these brain–immune interactions.

Introduction

It has been established that the immune system modulates the functional and behavioral processes of the central nervous system (CNS) (Yirmiya and Goshen, 2011). Exposure to pathogens stimulates the peripheral immune system and induces inflammatory responses, including the elevated expression of pro-inflammatory cytokines such as interleukin (IL)-1 (Derijk et al., 1991, Oppenheim et al., 1986), IL-6, and tumor necrosis factor (TNF)-α (Roth et al., 1993). Increased levels of pro-inflammatory cytokines in peripheral tissues induce the local synthesis of pro-inflammatory cytokines within the brain parenchyma (Ban et al., 1992, Fontana et al., 1984, Hagan et al., 1993), leading to behavioral alteration such as sickness behavior (Hart, 1988, Kent et al., 1992, Rothwell, 1991) and impaired learning (Yirmiya et al., 2002).

The immune system also plays an important role in maintaining the higher functions of the brain under non-inflammatory conditions. For example, severe combined immune deficiency (SCID) mice and nude mice that are deficient in mature T cells exhibit cognitive deficits and behavioral abnormalities that are remedied by T cell restoration (Kipnis et al., 2004). In addition, systemic depletion of CD4-positive T cells leads to reduced hippocampal neurogenesis and impaired learning in the Morris water maze (Wolf et al., 2009). T cells are not normally present in the brain parenchyma under non-inflammatory conditions. But during cognitive task performance, it is necessary that T cells accumulate in the meninges and express high levels of IL-4, which skews meningeal myeloid cells toward an anti-inflammatory phenotype (Derecki et al., 2010, Derecki et al., 2011). However, the question of whether peripheral immune cells located in the meningeal spaces interact with cells within the brain parenchyma via cell-to-cell contact and, if so, what is the site of interaction, remains.

One of the most powerful tools for addressing these questions is the bone marrow chimera in which the recipients’ immune system is reconstituted with donors’ labeled bone marrow cells (BMCs) by transplantation following irradiation, since this allows us to trace the distribution of labeled immune cells entering any organ. Studies using bone marrow chimeras to examine the brain distribution of bone marrow-derived cells have been reported since the late 80s’ (Davoust et al., 2008). These studies have consistently shown that bone marrow-derived cells under non-inflammatory conditions are primarily located in the leptomeninges, choroid plexus and perivascular spaces (Anandasabapathy et al., 2011, Chinnery et al., 2010, Hess et al., 2004, Hickey and Kimura, 1988, Soulas et al., 2009, Vallieres and Sawchenko, 2003). Bone marrow-derived cells also enter the circumventricular organs (CVOs), which contain a high density of capillaries lacking blood brain barrier (BBB) (Vallieres and Sawchenko, 2003). These bone marrow-derived cells preserve hematopoietic identity or express markers of the myeloid lineage. However, there has been some controversy over whether bone marrow-derived cells infiltrate the brain parenchyma protected by the complete BBB under non-inflammatory conditions. Some investigators have stated that, after bone marrow transplantation (BMT) by intravenous (IV) injection of donor cells, donor-derived cells were widely distributed throughout the brain parenchyma, and differentiated into microglia with a highly ramified morphology (Asheuer et al., 2004, Simard and Rivest, 2004). Others have claimed that the donors’ bone marrow-derived cells were found chiefly in association with blood vessels, and only rarely in the parenchyma of the brain (Hickey and Kimura, 1988, Soulas et al., 2009, Vallieres and Sawchenko, 2003). A recent study indicated that postnatal hematopoietic progenitors did not significantly contribute to microglia renewal or homeostasis in the adult brain (Ginhoux et al., 2010).

Meanwhile, a novel BMT procedure, namely intra-bone marrow (IBM)-BMT has been developed. In this procedure BMCs are collected from the marrow of the donors’ long bones by perfusion, and whole BMCs are injected directly into the bone marrow cavity of the recipients instead of being injected IV (Ikehara, 2003, Ikehara, 2011). The IBM procedure facilitates the efficacious and speedy transfer of not only hematopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs) from donor into recipient. When IBM-BMT is performed at the same time as the transplantation of heterotopic organs, it facilitates the induction of persistent donor-specific tolerance without rejection and reduces the incidence of graft versus host disease to far below the incidence level when the IV-BMT method is used (Esumi et al., 2003, Guo et al., 2008, Kaneda et al., 2005, Okazaki et al., 2008, Taira et al., 2005). IBM-BMT has also been successful in treating a wide variety of diseases in animal models, including autoimmune disease (Kushida et al., 2001), hematotologic malignant neoplasms (Suzuki et al., 2005), diabetes mellitus (Taira et al., 2005), emphysema (Adachi et al., 2006), senile osteoporosis (Takada et al., 2006) and dementia (Li et al., 2009).

In the present study, we investigated the distribution and time-dependent changes in the density of bone marrow-derived cells as well as their differentiation in the brain parenchyma in a non-inflammatory condition in bone marrow chimeric mice produced by the IBM procedure, and then compared the findings with those by the IV procedure. Based on the pattern of marrow-derived cell distribution, we focused on the anatomical, histological and cytokine expression features of the choroid plexus stroma, the attachments of choroid plexus and adjacent brain parenchyma.