The circumventricular organs participate in the immunopathogenesis of experimental autoimmune encephalomyelitis

In multiple sclerosis and in its animal model, experimental autoimmune encephalomyelitis (EAE), inflammatory cells obtain access to the central nervous system (CNS) parenchyma and the cerebrospinal fluid (CSF) and initiate the events leading to signs of paralysis. The endothelial blood-brain barrier (BBB) has been considered the obvious place for entry for circulating lymphocytes into the CNS. Therefore most investigations have focused on defining the molecular mechanisms involved in leukocyte recruitment from the circulating blood across the endothelial BBB. The adhesion molecules, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), both members of the immunoglobulin superfamily, are upregulated on the endothelial cells of cerebral vessels during EAE and actively involved in the recruitment of inflammatory cells across the BBB (summarized in [1]).

Trafficking pathways for the entry of immune cells into the CSF remain unknown to date. The CSF of healthy individuals contains between 150,000 cells and 500,000 cells. During multiple sclerosis this number increases dramatically. Neither in the healthy individual nor during multiple sclerosis does the cellular composition of the CSF reflect that of the peripheral blood, suggesting a stringent control for leukocyte entry into the CSF at all times [2]. Recently it was considered that leukocytes enter the CSF using a direct pathway through the choroid plexus. The microvessels within the choroid plexus are different to those in brain parenchyma, the most significant of which is that the endothelial cells allow free movement of molecules via fenestrations and intercellular gaps (reviewed in [3]). Instead, the barrier is located at the level of the choroid plexus epithelial cells, which form tight junctions inhibiting paracellular diffusion of water soluble molecules [4].

Migration of leukocytes through the choroid plexus into the CSF has been suggested by the finding that fluorescently labeled splenocytes are present in the choroid plexus stroma two hours after intravenous injection in mice [5]. The adhesion molecules ICAM-1 and VCAM-1, which are required for leukocyte entry into the CNS, are expressed on the choroid plexus epithelium [6], become upregulated during EAE, and can mediate lymphocyte binding in vitro [7]. These observations suggest that the choroid plexus is involved in the communication of the immune system with the CNS probably by allowing the entry of immune cells directly into the CSF spaces.

Besides the choroid plexus there are additional structures in the CNS of mammals lacking an endothelial BBB. These areas fulfill neurohemal and neurosecretory functions, in that the neurons monitor hormonal stimuli and other substances within the blood or secrete neuroendocrines into the blood, and are commonly referred to as the circumventricular organs (CVOs; reviewed in [8, 9]). CVOs are localized at strategic points close to the midline of the brain within the ependymal walls lining the 3^rd and the 4^th ventricle. Because they lack an endothelial BBB they lie within the blood milieu and thus form a blood-CSF barrier. At the cellular level the barrier between the CVOs and the neuropil is established by specialized epithelial cells called tanycytes. The median eminence (ME) belongs to the purely neuroendocrine CVOs also including the pineal gland and the neurohypophysis. The ME is localized between the stem of the infundibular or pituitary stalk of the hypophysis and the hypothalamus at the base of the 3^rd ventricle. The ME contains the terminations of axons from hypothalamic neurons and specialized glial cells. Neuroendocrine functions of the ME include the release of gonadotropin-releasing hormone by which the ME influences reproductive functions [10].

The sensory CVOs such as the subfornical organ (SFO), the organum vasculosum of the lamina terminalis (OVLT) and the area prostrema (AP) are characterized by a high density of small neurons, astrocytes and microglial cells [11]. The SFO and the OVLT are structures in the anterior wall of the 3^rd cerebral ventricle. The SFO bulges from the roof of the 3^rd ventricle into its lumen at the level of the interventricular foramina, whereas the OVLT is localized in the lamina terminalis between the chiasma opticum and the anterior commissure. The AP can be found at the caudal end of the fossa rhomboidea in the floor of the 4^th ventricle. The sensory CVOs monitor the changes in osmotic, ionic and hormonal composition of the blood and are therefore involved in regulating thirst and fluid metabolism [12].

The strategic localization of the CVOs in the wall of the ventricles prompted us to ask whether the CVOs might, similar to the choroid plexus, be involved in the recruitment of immune cells into the brain during EAE. We have performed an extensive immunohistological study localizing adhesion molecules, MHC class I and II antigens and leukocyte subpopulations within the subfornical organ (SFO), the median eminence (ME), the organum vasculosum of the lamina terminalis (OVLT) and the area postrema (AP) of the SJL/N mouse. During EAE similar changes were observed within all four CVOs included in this study. Specifically, the presence of significantly increased numbers of CD45⁺ leukocytes in the CVOs suggested the recruitment of inflammatory cells into the the parenchyma of the CVOs probably mediated via endothelial ICAM-1 and VCAM-1 which were both upregulated on the microvessels within the CVOs. These changes were accompanied by a significant upregulation of MHC class I throughout the CVOs and induction of MHC class II on perivascular cells. Our observations indicate an active communication between the CVOs and the CNS parenchyma and thus the involvement of the CVOs in the inflammatory process of the CNS during EAE.

In the mouse the panendothelial antigen MECA-32 becomes specifically downregulated in brain endothelia during the maturation of the blood-brain barrier [15]. As a consequence, the MECA-32 antigen was absent on the mature cerebral endothelium, whereas it remained present on vessels outside of the CNS and the microvessels within the choroid plexus and the circumventricular organs (CVOs, Figures 1, 2, 3, 4, 5, 6, 7, 8).

In the present study we investigated four CVOs, namely the subfornical organ (SFO), the area postrema (AP), the median eminence (ME) and the organum vasculosum of the lamina terminalis (OVLT), which all lack an endothelial blood-brain barrier (BBB) and demonstrate fenestrated MECA-32⁺ microvessels.

In the present study we provide evidence that immune cell invasion into the CNS during EAE is accompanied by a prominent immune activation of the CVOs. Changes within the CVOs are characterized by the presence of an increased number of CD45⁺ cells, the considerable induction of MHC class I and MHC class II molecules within the CVOs as well as microglial activation documented by increased expression of Mac-1. Upregulated expression of adhesion molecules on the CVO microvessels suggest their involvement in the recruitment of macrophages, T- and B cells into the CVOs during EAE. Taken together, these observations indicate an active participation of the CVOs in the immunopathogenesis of EAE.

The involvement of CVOs in the communication of the immune system with the nervous system has been considered before. Due to their fenestrated capillaires the CVOs are often referred to as "windows of the brain" and have been thought to serve as entry points for pro-inflammatory cytokines into the CNS [16]. In fact, receptors for inflammatory cytokines and bacterial fragments are constitutively expressed in cells within the sensory CVOs and the release of pro-inflammatory cytokines such as interleukin-1 or TNF-α outside the CNS has been demonstrated to deliver signals into the CNS via the CVOs leading to neuroendocrine responses such as elevations in adrenocorticotropic hormone (ACTH) in the plasma, development of fever and the activation of the hypothalamic-pituitary-adrenal (HPA) axis [17]. Additionally, CVOs play an active role in the development of brain dysfunction during sepsis. Induction of experimental endotoxin shock by injection of lipopolysaccaride induces the sequential expression of molecules involved in innate immune responses such as CD14 or Toll-like receptors first in the CVOs and subsequently in the brain parencyhma (reviewed by [18]). Furthermore, involvement of CVOs in the establishment of an acute phase response during peripheral immune stimuli was demonstrated [19]. Finally, a role for CVOs in sending signals into the brain during systemic inflammation is supported by observations that lesions of CVOs block several components of brain-controlled illness responses (i.e. fever or neuroendocrine modifications) [20].

An active participation of CVOs in the auto-immunopathogenesis in EAE has not been reported to date. In the present study, we found that CVOs in the healthy SJL/N mouse presented a low number of perivascular CD45⁺ cells, probably macrophages, similar to other regions of the CNS. We only occasionally detected expression of MHC class I or MHC class II on individual perivascular cells suggesting that MHC-positive cells rarely reside within CVOs of healthy mice. Adhesion molecules associated with inflammation could not be detected. In contrast, during clinical EAE at the time when immune cells have penetrated the BBB and perivascular infiltration of T cells and macrophages is observed within the CNS parenchyma, the CVOs demonstrated a dramatic increase in the presence of CD45⁺ cells. As CD45 is upregulated on activated microglial cells, which reside in high numbers in CVOs [21], the increased number of CD45⁺ cells detected within the CVOs during EAE is most probably due to both the recruitment of circulating CD45⁺ immune cells from the periphery into these areas and additionally to the upregulation of CD45 on microglial cells residing within the CVOs.

Most of the CD45⁺ cells also expressed the macrophage marker F4/80 or Mac-1, which can be expressed on macrophages or activated microglial cells. Based on the immunological and morphological criteria, these cells most probably can be classified as perivascular macrophages or ramified microglial cells, as classical parenchymal microglial cells, as well as scattered rounded macrophage-like cells. Activation of microglial cells by upregulated expression of CD45 and induction of Mac-1 therefore parallels the events observed within the CNS parenchyma protected by the BBB during EAE.

Microglial activation within the CVOs during EAE is further supported by our observation that MHC class I antigens were induced throughout the BBB-deficient parenchyma in all CVOs. In addition, MHC class II molecules could now regularly be detected on perivascular microglial cells or macrophages within the CVOs, which may function as a cellular barrier against blood-borne pathogens. Thus, increased expression of MHC class I and II molecules on microglial cells and perivascular macrophages, which are known characteristic immunopathological changes observed in the CNS parenchyma during EAE, are similarly observed in the BBB-deficient areas of the CVOs [22].

Besides the immune activation of the CVOs, recruitment of a low number of CD3⁺ T cells and B220⁺ B cells was detected in the parencyhma of CVOs during EAE, demonstrating that immune cells are invading these areas during EAE. It has been shown that endothelial VCAM-1 and ICAM-1 are involved in lymphocyte recruitment across brain endothelium in vitro and in vivo [13, 23‐25]. The upregulated expression of ICAM-1 and VCAM-1 on the microvessels of the CVOs during EAE suggests that the same molecules mediating immune cell entry across the BBB might be involved in guiding inflammatory cells into the CVOs. This notion was confirmed by our findings that both molecules are available on the luminal surface of CVO microvessels, with the exception of the median eminence, where luminal VCAM-1 could not be detected. Inflammatory cells within the CVOs were found to stain positive for Mac-1⁺ and LFA-1⁺, therefore resemling those, present in the inflammatory cuffs surrounding brain venules forming a barrier [26]. In contrast, whereas during EAE inflammatory cells within the brain express high levels of α4-integrins [26] only few α4-integrin⁺ leukocytes were found within the CVOs. These observations indicate that inflammatory cell recruitment into the CVOs is not necessarily mediated by the same mechanisms as those regulating leukocyte recruitment across the inflamed BBB, which during EAE was shown to be mainly dependent on α4-integrin and VCAM-1[1, 25].

Whether immune cells enter the brain or CSF via the CVOs cannot be determined by this study. It is, however, tempting to speculate that CVOs have an active role in determining inflammatory cell recruitment into the brain as we observed a significantly increased number of inflammatory cuffs and infiltrating cells in close vicinity to the CVOs as compared to regions of the brain with a greater distance to the CVOs. These observations suggest that during EAE the inflamed CVOs deliver cellular or molecular signals into the CNS, which may influence immune cell entry into the CNS and CSF.

Taken together our observations demonstrate that the CVOs are involved in the immunopathogenesis of EAE. Considering their strategic localization within the walls of the ventricles, they may be involved in immune cell entry from the blood into the CNS parenchmyma and also from the blood into the CSF. Therefore, the development of neuroprotective strategies will require consideration of the molecular changes in the CVOs during CNS inflammation.