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Published online before print August 2, 2006
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,1
* Center for Anatomy, Institute of Cell Biology and Neurobiology, Department of Experimental Neuroimmunology, Charité, Universitätsmedizin Berlin, Berlin, Germany; and
Institute of Clinical Neuroanatomy, J. W. Goethe-University, Frankfurt/Main, Germany
1 Correspondence: Institute of Clinical Neuroanatomy, J. W. Goethe-University, Theodor-Stern-Kai 7, Frankfurt/Main 60 590, Germany, E-mail: Bechmann{at}med.uni-frankfurt.de
ABSTRACT
Although drainage pathways of soluble antigens from brain to cervical lymph nodes have been well established, there is no direct evidence for similar routes of leukocytes leaving the central nervous system. We developed a protocol allowing the cross-sectioning of an entire head-neck preparation while preserving the signal of the GFP. We monitored how GFP-expressing CD4 T lymphocytes injected into the entorhinal cortex after lesion or the lateral ventricle of unlesioned C57/bl6 mice reach cervical lymph nodes. Irrespective of the injection site, we demonstrate their passage through the cribroid plate, appearance in the nasal mucosa, and specific accumulation in one of the cervical lymph nodes.
Key Words: CNS • cell trafficking • immune tolerance
INTRODUCTION
The brain was long believed to be widely ignored by the immune system. This concept was designed to explain its "immunologically privileged status" [1 ] (i.e., the relative tolerance to various grafts [2 ]) and based on two morphological peculiarities of the CNS: the absence of classical lymph vessels and the blood-brain barrier (BBB) [3 ]. However, the BBB originally described a mechanical barrier for hydrophilic molecules [4 , 5 ], and specialized tight junctions were identified as its morphologic correlate [6 , 7 ]. These "belts of tight junctions" are dominant at the capillary segment of the vascular arbor, and leukocyte recruitment takes place in postcapillary venules [8 ]. In fact, the continuous influx of T cells [9 , 10 ] and monocytes [11 12 13 14 ] into the CNS has been demonstrated, and several routes of their entry have been characterized [15 , 16 ]. Thus, the BBB in its original sense is not designed to keep leukocytes out of the brain [3 , 17 ].
Similarly, the idea that brain antigens are trapped within the skull as a result of the absence of lymph vessels has also been defeated. In their seminal work, Cserr and Knopf [18 ] described how soluble molecules drain along the olfactory nerves through the cribroid plate to eventually reach the cervical lymph nodes, and many others [19 , 20 ] have confirmed this pathway. It is, however, not clear whether leukocytes also use this route to leave the CNS. Using an adapted protocol of decalcification, which allows us to cut whole head/neck cross-sections and to preserve the fluorescence of GFP-expressing cells, we have here addressed this issue. We demonstrate that regardless of whether unspecifically activated CD4+ T cells are injected into a lesion site or the lateral ventricle, they uniformly pass the cribroid plate and reach the nasal mucosa and eventually target specific cervical lymph nodes.
MATERIALS AND METHODS
Principal approach
Two sets of experiments were performed. In the first set, the time window of the arrival of intrathecally injected T cells in isolated lymphoid organs was established. In the second set, whole head/neck sections of animals were performed to identify the T cell route from brain to cervical lymph nodes. To test whether the specific accumulation in one cervical lymph node (see 
Fig. 5
) was dependent on injection site and/or concomitant brain lesion, cells were injected into the ventricles or into the lesioned entorhinal cortex at any time-point.
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Ex vivo isolation and reinjection of T cells
Cervical, inguinal, axial, and abdominal lymph nodes as well as the spleen were collected from the GFP-expressing mice and homogenized through a nylon mash (70 µm, from Becton Dickinson, Franklin Lakes, NY). The spleen and lymph node cell suspension were washed, and erythrocytes were lysed by incubation in 0.83% NH4Cl for 5 min at room temperature followed by washing in RPMI 1640 (Gibco Life Technologies, Gaithersburg, MD). Cells were activated with 5 ng/ml PMA (1:2000) and 1 µg/ml ionomycin (1:1000; both from Sigma Chemical Co., St. Louis, MO) for 24 h. After a resting time of 48 h, positive selection of CD4+ T cells was performed using a MACS column and CD4 microbeads (both from Miltenyi Biotec, Auburn, CA). Lively cells were counted with Trypan blue, and for injection purposes, 5 million cells were suspended in 4 µl phosphate buffer (PB). Cells were injected at the lesion site, immediately after entorhinal cortex lesion (no longer than 5 min after ECL) or intraventricularly (see below) with a 5-µl Hamilton pipette.
ECL and ventricular injection [22 ]
Stereotactic surgery was performed under deep ketamine anesthesia. For ECL, the left entorhinal cortex was lesioned using a 2-mm broad knife. The animals were fixed in a stereotactic apparatus (Kopf Instruments, Tujunga, CA), and the following coordinates measured from 
(where the longitudinal and the suture meet) were used for lesions: anteroposterior, 0.4 mm; lateral, 1.6 mm; and dorsoventral, down to the base of the skull.
For right ventricular injection, the animals were fixed in a stereotactic apparatus (Kopf Instruments), and the following coordinates measured from were used for injection: anterior, 3 mm; lateral, 1 mm; and dorsoventral, 2 mm into the brain parenchyma. Aspiration of cerebrospinal fluid was performed to confirm proper needle placement.
Histology
All animals were deeply anesthetized and perfused transcardially with 100 ml NaCl followed by 150 ml of a fixative containing 4% paraformaldehyde in 0.1 M PB, pH 7.4.
In the first set of experiments, 5 x 106 activated, CD4-positive-selected T cells were injected into the lesion site immediately after ECL, and animals were killed at 5 min, 1 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 72 h, and 7 days (n=4 per time) after injection. In another group of animals (n=3 per time), the same number of nonactivated, CD4-positive-selected T cells was injected into the lesion site, and animals were killed at 12, 24, and 36 h postinjection (hpi). In a third group, activated and CD4-positive-selected T cells were injected into the ventricles of nonlesioned mice (n=3), and animals were killed at 12, 24, 36, and 48 hpi.
After perfusion, brains, deep and superficial cervical and inguinal lymph nodes, and spleens were harvested and postfixed overnight at 4°C in 4% paraformaldehyde. Following three washes in PB, 50 µm sections were cut on a vibratome. For native fluorescence microscopy, sections were embedded in Immu-Mount (Shandon, Pittsburgh, PA) and immediately studied under an Olympus BX-50 microscope using narrow-band filters. After taking pictures, selected sections were dried overnight and stained with H&E to analyze the morphology of the injected cells in the lymph nodes. GFP cells were confirmed to be T lymphocytes using a monoclonal anti-CD4 antibody RM4-5 (Becton Dickinson) visualized with the secondary antibody Alexa Fluor 568 (Molecular Probes, Eugene, OR).
When the time-frame of T cell appearance in lymph nodes had been established, the second set of experiments was performed to assess the migratory route. Lesioned and unlesioned animals were injected with 5 x 106 activated T cells into the lesion site or the ventricle, respectively. These mice were killed at 12 h, 18 h, 24 h, 36 h, 48 h, and 7 days (n=at least three in each group) postinjection and perfused with 4% paraformaldehyde in 0.1 M PB as described above. Whole heads and necks were dissected from the torso, and the skin was removed. For decalcification of the specimen, a solution of 1000 ml 0.1 M PB, pH 7.4, containing 160 g EDTA and 10 g sucrose was prepared. Heads and necks were incubated at 50°C for 2 days. Subsequently, they were put into a solution of 20% sucrose in 0.1 M PB for 1 day and 30% sucrose for 2 days. The specimens were then frozen at 80°C for at least 1 day. Finally, frozen sections were cut on a cryostat and embedded immediately in Immu-Mount and stored at 4°C until analysis and thereafter.
Flow cytometry
FACS analysis was performed using a FACSCalibur and revealed the population of injected cells to be 93% CD4-positive and 6% CD8-positive. Monoclonal R-PE-conjugated rat anti-mouse antibodies were obtained from Becton Dickinson (Clone RM4-5 for CD4 and Clone 53-6.7 for CD8).
Diamidino-2-phenylindole (DAPI) staining
To show the viability of the cells observed in the cervical lymph nodes, 2 HCl DAPI (Sigma Chemical Co.) staining was performed on slices from animals allowed to survive 7 days. Slices were incubated for 5 min in DAPI solution, washed four times with PB, embedded in Immu-Mount, and studied under a fluorescent microscope immediately afterward to demonstrate the presence of intact nuclei within GFP cells.
RESULTS
First set of experiments
In lesioned mice, GFP-expressing, activated T cells were found at the injection site and attached to the ventricular surface and the choroid plexus. Although no quantification was performed, their distance to the entorhinal area (injection site) appeared to increase with time, and their distribution suggested migration along the alveus (a myelinated fiber tract) to the ventral horn of the ventricle. At 6 hpi, first cells were found in the connective tissue around, but not within, the cervical lymph nodes. At 12 hpi, some GFP cells resided in one of the deep cervical lymph nodes of individual animals (Fig. 1
), and GFP cells were absent in superficial cervical and inguinal lymph nodes and the spleen. The number of cells subsequently increased, involving several deep cervical nodes leading to dense cellular accumulations at 48 hpi (Fig. 1)
, which were still present at 7 days. There was also an evident increase of GFP cells in superficial cervical and inguinal nodes from 36 to 48 hpi but not between 48 hpi and 7 days. The population in inguinal and superficial cervical nodes never reached the density observed in the deep cervical group. In the spleen, only single cells were observed throughout all experiments. It is important that corresponding observations were made in animals receiving injections into the ventricle.
Viability of GFP-expressing cells in the lymph nodes was tested using DAPI staining at 7 days after injection into the site of entorhinal lesion. Intact nuclei were apparent in many GFP cells, proving their viability (Fig. 2 ).
Second set of experiments
Once the time-frame of T cell arrival had been established, we sought to identify the trafficking route from brain to cervical lymph nodes. To this end, we performed whole head/neck sections to monitor the efflux directly. In fact, from 12 to 48 hpi, T cells were found in close proximity to the olfactory nerves penetrating the cribroid plate (Fig. 3
) and within the nasal mucosa, where the cells were located beneath the epithelial layer (Fig. 4
). In keeping with the finding that one deep cervical lymph node of individual animals was populated specifically by GFP cells at 24 hpi, the respective whole head/neck section revealed many cells in one lymph node, and fluorescence was virtually absent in the neighboring nodes (Fig. 5
). The distribution of cells did not evidently differ between animals receiving injection into the ventricle and those with entorhinal injection.
DISCUSSION
The concept that cells from the subarachnoid space can reach lymphoid organs is not new and was first demonstrated for erythrocytes, which primarily drained into cervical lymph nodes [23 , 24 ]. Carson et al. [25 ] extended this knowledge to intrathecally injected dendritic cells, and others [26 , 27 ] have recently reported similar results. However, none of these studies identified the anatomical exit for cells from the cranial subarachnoid space. To this end, we first established the time-frame of arrival of intrathecally injected GFP-expressing CD4+ T cells in lymphoid organs. We then developed a fixation protocol, which allowed us to directly observe the injected cells and monitor their way out of the brain. We found cells, first along the olfactory nerves, then beneath the nasal mucosa, and eventually, in specific, deep cervical lymph nodes. Our interpretation of this sequential distribution is that at least the T cells injected in this study use the same route, which is known for the drainage of soluble antigens [18 ].
One important question remains open at present: Is the passage from brain to cervical lymph nodes a result of passive transportation within draining cerebrospinal fluids and the lymph or rather a process of active migration? As erythrocytes, which clearly cannot migrate, also reached cervical lymph nodes upon intrathecal injection [23 , 24 ], it seems that migration is not a prerequisite. However, we do not want to exclude that site-specific chemotactic cues contribute to the accumulation of the T cells in specific lymph nodes, and the neighboring ones were almost devoid of cells. However, brain insult was not apparently necessary for an induction of putative homing signals, as the distribution did not differ between nodes from animals with cell injection into the lesioned entorhinal cortex and those with injection into the ventricle.
Recently, Wenkel et al. [28 ] demonstrated that OVA injected into the brain induced an immune deviation that could be adoptively transferred by cervical lymph node cells. This may be attributed to drainage of OVA along the known routes [18 ] and subsequent tolerogenic antigen presentation as a result of lack of proinflammatory stimuli. Based on the findings reported here, an alternate explanation would be that regulatory T cells, which are induced in the neuropil as a result of insufficient costimulation [29 30 31 32 ], may down-modulate adaptive immune responses in an antigen-specific manner.
Using entorhinal cortex lesion, we have demonstrated that not only T cells [31 ] but also monocytes infiltrate layers of axonal degeneration, where they transform into ramified microglia [13 ]. As the total number of microglia, upon initial massive increase, declines to prelesional levels at 30 days after lesion [33 ], it may be speculated that (a subpopulation of) microglia leave the neuropil and reach cervical lymph nodes. Addressing this issue, however, still awaits a conclusive technical approach. However, migration of DC from the brain into cervical lymph nodes has been demonstrated recently [25 26 27 ] and may underlie the presence of myelin fragments in the lymph nodes of humans and monkeys suffering from multiple sclerosis or its animal model autoimmune encephalomyelitis [34 , 35 ]. Although the old picture of the BBB keeping leukocytes out of the brain has been defeated, it remains an experimental challenge to study the extent to which intraparenchymal populations are capable of leaving the neuropil toward lymphoid organs. We may eventually learn that the brain is entered and left by immune cells, just as any other organ, and that "immune privilege" is a result of site-specific cues modulating the activation state of antigen-presenting cells and lymphocytes.
ACKNOWLEDGEMENTS
This study has been supported by DFG Grants Be 2272/1-2 and SFB 507 B16 (I. B.). J. G. was a scholar of the Studienstiftung des Deutschen Volkes and is a recipient of a stipend from Charité. The authors thank Sabine Winkler and Angela Hildebrandt for their excellent technical assistance. Kimberly Rosegger carefully revised this manuscript.
Received March 6, 2006; revised May 22, 2006; accepted June 1, 2006.
REFERENCES
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