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Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To explore the second explanation, it is useful to examine another immune privileged site, the anterior chamber of the eye. Immune privilege and immune deviation in the eye have been extensively studied. Evidence derived from recent studies has revealed that immune privilege in the eye results from an active down-regulation of systemic and local immunity rather than passive "ignorance" (9, 10, 11, 12, 13, 14, 15). After injection of soluble Ag into the anterior chamber of the eye, local F4/80 APC leave the eye, enter the blood stream, and carry an immunological signal to the spleen (14). Within 1 wk, regulatory T cells emerge in the spleen and create Ag-specific suppression of delayed-type hypersensitivity (DTH) (9, 10). This deviant immune response (termed anterior chamber-associated immune deviation (ACAID)) can be adoptively transferred to naive animals with an i.v. injection of spleen cells from donors that received an anterior chamber injection of Ag 7 days previously (10). Additional features of ACAID are the presence of primed Ag-specific cytotoxic T cells that fail to become activated inside the eye (12) and B cells that secrete noncomplement-fixing rather than complement-fixing Abs (15).
Our overall goal is to reassess immune privilege in the CNS and to evaluate whether the CNS facilitates the induction of deviant immune responses after injection of soluble Ag into the striatum. Additionally, we wanted to determine whether the systemic immune response to CNS Ag resembles ACAID.
We have chosen to examine the striatum in large part because this region is the site in which neural transplants have been placed by others in an attempt to ameliorate symptoms of parkinsonism (16). Therefore, understanding the immunological status of this site under various conditions may help us better understand the variable survival of grafted tissue in these experiments.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult female BALB/c mice, aged 6–10 wk, were obtained from the animal facilities at the Schepens Eye Research Institute or from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in a common room of a vivarium. Injections and ear measurements were conducted under anesthesia induced by i.p. injection of 0.075 mg/kg body weight ketamine (Ketalar; Parke Davis, Ann Arbor, MI) and 0.006 mg/kg body weight xylazine (xyla-ject, Phoenix Pharmaceuticals, St. Joseph, MO). Five mice were used for each group and experiments were repeated at least twice. All experimental procedures conformed to the National Institutes of Health and Harvard Medical School regulations on the use of animals in research.
Injections into the striatum
OVA (a heterologous protein Ag; Sigma, St. Louis, MO) was used
as a soluble Ag and was injected stereotactically into the striatum of
adult BALB/c mice. A small craniotomy was performed over the region to
be injected. A total volume of 1 µl of OVA (100 µg in PBS) was
injected into the dorsal caudate nucleus (stereotaxic coordinates: 2.0
mm lateral, 3.5 mm deep, 0.5 mm caudal to Bregma) via a pulled (tip
diameter, 
10 µm) 6.66-µl Microcap pipette (Drummund Scientific,
Broomall, PA). The micropipette was attached to a 50-µl Hamilton
syringe with polyethylene tubing, and the syringe was placed into a
Microdrive apparatus (L. S. Starrett, Athol, MA) to allow slow,
controlled injections. In additional groups of mice, the striatum
received a lesion with an unpulled micropipette (outside diameter, 1
mm) 0–4 days before Ag injection. Control experiments demonstrated
that injections made into previously ablated animals were indeed placed
into ablated regions (data not shown).
Assessment of BBB integrity
Animals received an i.v. injection of HRP (5% in Tris buffer (pH 9.0), 1 mg/5 g body weight, grade I; Boehringer Mannheim, Minneapolis, MN) and were then sacrificed 15 min later. The brains were fixed and processed for histology. HRP was developed with Hanker-Yates reagent, and control sections were stained with hematoxylin-eosin. The sections were viewed under light microscopy and examined for the presence of extravasated reaction product, indicative of a leaky BBB.
Immunohistochemical study of CNS tissue
CNS tissue removed after insertion of pulled or unpulled micropipettes was fixed in 4% paraformaldehyde and cryopreserved in 30% sucrose/PBS. Frozen sections were then cut at 30 µm on a sledge microtome. Sections were stained with the following reagents: anti-CD45, to identify bone marrow-derived cells (PharMingen, San Diego, CA); Griffonia simplicifolia (GS)-isolectin-biotin conjugate, to identify vascular endothelial cells and microglia (Sigma); and anti-glial fibrillary acidic protein, to identify astrocytes (Zymed, South San Francisco, CA). Secondary Abs conjugated to Cy3 or FITC (Jackson ImmunoResearch, West Grove, PA,), or in the case of GS-isolectin, avidin-PE reagent, were used to visualize the location of primary Abs via fluorescence microscopy.
Assay for DTH
DTH was evaluated 14 days after OVA injection into the striatum. Seven days after injection of OVA into the CNS, animals were immunized s.c. with 100 µg OVA and CFA. Ear-swelling analysis was performed 7 days later. DTH was measured based on ear swelling, as described previously (13, 17). Briefly, 200 µg OVA in 10 µl was injected into the left ear pinnae of the mice. The right ear served as untreated control. Both ear pinnae were measured immediately before injection and 24 h later with an engineer’s micrometer (Mitutoyo, Tokyo, Japan). The measurements were performed in triplicate. Results were expressed as specific ear swelling = (24-h measurement - 0-h measurement) experimental ear - (24-h measurement - 0-h measurement) negative control ear x 10-3 mm. A two-tailed Student’s t test was used and significance assumed if p < 0.05.
Splenectomy
In anesthetized mice, the spleen was exteriorized through an incision in the left abdominal wall, as described elsewhere (11). In sham controls, the spleen was merely replaced in the abdominal cavity, but in experimental animals the splenic stalk was ligated with sutures and excised. The wound was closed with clips.
Adoptive transfer assay for suppression of DTH
To evaluate whether the deviant immune response to OVA could be adoptively transferred into naive mice, splenocytes or cervical lymph node cells were harvested 8 days after injection of OVA into the striatum. These cells were injected i.v. (6 x 107 spleen or lymph node cells/recipient) into naive BALB/c mice, as described previously (13, 18). For both negative and positive controls, the same amount of spleen or lymph node cells of naive BALB/c mice was infused i.v. Within 24 h all experimental mice and the positive control mice were immunized s.c. with 100 µg OVA and CFA. Negative controls received no immunization. Seven days later, ear-swelling responses to OVA challenge were assessed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our first goal was to determine whether an injection of Ag into
the striatum of normal mice would elicit a systemic DTH response. With
the aid of a stereotactically guided pulled glass micropipette, OVA
(100 µg/1 µl) or PBS was injected into the dorsal caudate nucleus
of brains of adult naive BALB/c mice. Seven days later, these mice, as
well as normal BALB/c mice serving as positive controls, received s.c.
immunization with OVA (100 µg) mixed with CFA. One week thereafter,
the ear pinnae of these mice were challenged with OVA (200 µg), and
ear-swelling responses were assessed 24 and 48 h later. The
results of a representative experiment are displayed in Fig. 1
. Positive control mice, and mice that
were immunized with OVA after an injection of PBS into the striatum,
mounted intense DTH responses. In contrast, mice pretreated with OVA
into the striatum mounted only feeble ear-swelling responses, almost as
low as those of negative controls. The inability of mice exposed via
the striatum to OVA before immunization with OVA to acquire DTH implies
that systemic immune deviation was induced. For convenience of further
discussion, we have used the term brain-associated immune deviation
(BRAID).
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Injections placed in the dorsal caudate nucleus with the utmost
care must necessarily be disruptive to the local microanatomy, raising
the likelihood that the BBB is breached. We examined this possibility
using immunohistochemistry with the HRP method and with Abs capable of
detecting the presence of bone marrow-derived cells, activated
microglia, and astrocytes. Panels of BALB/c mice received injections of
PBS (1 µl) into the dorsal caudate nucleus. In some mice, the
striatum was ablated with an unpulled micropipette to create an
extensive breach in the BBB. Immediately after the insertion of the
micropipette, as well as 2 and 4 days later, HRP (1 mg/5 g body weight)
was injected i.v. into recipient mice. Fifteen minutes later, the mice
were killed and their brains were removed and prepared for histologic
analysis. Sections of the ablated and adjacent site were stained
for HRP reactivity. A summary of the results of this experiment is
presented in Fig. 2. Brains removed 15
min after intrastriatal injection revealed heavy staining with HRP at
the lesion site (Fig. 2A), which also diffused into adjacent
regions of the brainstem (Fig. 2B). After 2 days, no leakage
of reaction product was seen (Fig. 2, C and D),
indicating that the BBB had repaired itself by this time point.
Similarly, no leakage of reaction product was seen in the brains of
animals examined 4 days after ablation (Fig. 2, E and
F).
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A) and
GS-isolectin+ cells (data not shown) were found
in control or 0 day postlesion striatum, the margins of the lesion site
contained detectable microglial cells by day 2 (Fig. 3B). By
day 4 ,the margins of the lesion site no longer showed these cells,
whereas microglial cells were now identified peripheral to
the lesion site in the adjacent areas of the striatum (Fig. 3C). These cells displayed positive staining with
GS-isolectin and CD45, suggesting that they are microglia. The results
of these experiments are summarized in Table I.
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Systemic immune response to OVA injected into previously ablated striatum
The tempo of alterations in the BBB and the tempo of changes in
activated bone marrow-derived cells in and around the ablated site were
disparate. We next examined the extent to which injection of OVA into
ablated sites immediately or in a delayed fashion after lesion
placement would result in systemic immune deviation. Accordingly,
lesions were created in the dorsal caudate nucleus of normal BALB/c
mice with a blunt micropipette, as for the above described BBB
assessment. In one panel of mice, a pulled micropipette was used to
place a lesion in the dorsal caudate nucleus (minimal lesion). OVA was
then injected into the ablated site immediately thereafter or at 2 or 4
days later. As before, the mice received an immunizing dose of OVA s.c.
1 wk later and were then ear challenged with OVA 7 days thereafter. As
the results displayed in Fig. 4 indicate,
reduced ear-swelling responses, compared with positive controls, were
observed among mice that received intrastriatal OVA immediately after
lesion placement, as well as 4 days after lesion placement. In
contrast, mice that received intrastriatal injection of OVA 2 days
after lesion placement developed vigorous ear-swelling responses. These
results indicate that the presence of activated microglial cells at the
OVA injection site, rather than integrity of the BBB at that site,
correlates positively with the loss of the ablated site’s ability to
promote immune deviation.
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Injection of Ag into the anterior chamber produces a deviant
systemic immune response similar to our findings with Ag injected into
the striatum. The immune deviation associated with anterior chamber
injections has been found to depend on the presence of an intact
spleen. Anterior chamber injection of Ag into mice without spleens
fails to induce immune deviation (11). To determine
whether a similar situation applies to the brain, panels of BALB/c mice
were splenectomized or sham operated 1 wk before OVA was injected into
the dorsal caudate nucleus. The mice were immunized subsequently with
OVA plus CFA and ear challenged, as described above. Both mice with
intact spleens and mice whose spleens had been extirpated 1 wk before
intrastriatal injection of OVA mounted feeble ear-swelling responses.
This result indicates that, unlike ACAID, the immune deviation that
follows intrastriatal injection of Ag has no requirement for an
intact spleen (Fig. 5).
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In ACAID, failed DTH is mediated in part by regulatory T cells of
splenic origin. Cells of this type can adoptively transfer impaired DTH
when injected into naive recipients. We wished to determine whether
intrastriatal injection of Ag also generated regulatory T cells. The
finding that splenectomy failed to promote DTH in mice that received an
intrastriatal injection of OVA lessened the likelihood that regulatory
T cells would be present in spleens of recipients of intrastriatal OVA.
It has been previously demonstrated that Ags injected into the brain
and CSF drain into lymphatic channels that lead to the cervical lymph
nodes (6). This raised in our minds the possibility that
the cervical lymph nodes might be a source of regulatory T cells.
Accordingly, panels of BALB/c mice received injections of OVA into the
dorsal caudate nucleus. Eight days later, the mice were killed, and
their spleens and cervical lymph nodes were removed separately.
Single-cell suspensions (6 x 107 per
recipient) from spleen or from lymph nodes were injected i.v. into
naive BALB/c mice. Twenty-four h later, these mice were immunized s.c.
with OVA plus CFA, and their ear pinnae were challenged with OVA 1 wk
later. The results of representative experiments are presented in Figs. 6 and 7.
Mice that received spleen cells from donors into whose striatum OVA had
been injected displayed intense ear swelling similar to that of
positive controls (Fig. 6). In contrast, mice that received cervical
lymph nodes from similar donors displayed only feeble ear-swelling
responses (Fig. 7). These results indicate that OVA injected into the
striatum induced a population of regulatory lymphoid cells that emerged
in the draining cervical lymph nodes. Cells of this type suppressed the
development of DTH to OVA in recipients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An obvious methodological concern in these experiments involves the
leakage of soluble Ag from the site of the injection. It is clear that
regardless of the experimental procedure, Ag injected into the brain
will rapidly drain to the cervical lymph nodes (5).
Similarly, injections made into the CNS of animals with "intact
BBB" will cause a temporary breakdown of the BBB and subsequent
leakage of Ag into the blood, which has previously been shown to cause
an altered DTH response (19). However, if this was the
sole cause of the diminished DTH response in these experiments, one
would expect that injection of Ag into the brain would induce the
suppression of DTH. In fact, at 2 days postablation, no suppression of
DTH was observed. Moreover, the strongest suppression of DTH
occurred not when widespread BBB leakage was present (day 0), but at
day 4, when the BBB was reformed, and without the use of prior BBB
disrupting lesion (minimal trauma; see Fig. 4
). These results suggest
that the prime factor that determines the immunological response to Ag
(in the CNS, as well as the eye) is not the presence or absence of a
blood-tissue barrier or the amount of Ag "leakage", but rather the
environment in which initial Ag presentation takes place. This is
supported by previous work in the eye, in which ACAID can be induced in
spite of leakage of injected Ag or cells along the injection tract
(10). Our results support the hypothesis that the local
microenvironment is the strongest determinant of the immunological
state of the CNS.
Induction of a deviant immune response to CNS Ag injection was not dependent on an intact BBB since BRAID was elicited when OVA was injected into a newly created lesion site with extensive BBB leakage. Surprisingly, BRAID was not induced when OVA was injected into a site ablated 2 days previously. Our observation that 2-day-old striatal lesions contain large numbers of activated microglia suggests that these cells may interfere with immune deviation. In support of this hypothesis is the finding that BRAID was induced if Ag was injected into a striatal site ablated 4 days previously; at this time, activated microglia were no longer prominent at the lesion, although significant numbers of such cells were observed in brain tissue surrounding the lesion. We suspect that breakdown of the BBB after initial trauma results in activation of resident microglia, perhaps under the influence of serum proteins, and that activated microglia prevent the induction of BRAID. Recent reports associate the presence of activated microglia with a higher incidence of graft rejection in the CNS (20). In our model, it is not yet clear whether the presence of microglia alone abolishes the induction of BRAID or whether the activation of microglia under the altered conditions of a breached BBB is responsible for the loss of BRAID. Further investigations are under way to evaluate the role of microglia in Ag presentation in the CNS under physiological and diseased conditions.
Our results highlight an important quandary regarding the route that Ags take to leave the CNS and the part microglia play in the presentation of those Ags to the systemic immune system. In the eye, another immune privileged site, there is circumstantial evidence that resident F4/80 APC pick up Ag under the influence of the unique ocular microenvironment. These cells acquire the ability to induce systemic immune deviation, which they accomplish by migrating across the trabecular meshwork directly into the blood (14).
Many authors have described the brain as an immune privileged site, where transplanted tissues show a prolonged survival (1, 2, 3, 4). High Ab synthesis by both the cervical lymph nodes and the spleen have also been described as systemic characteristics of a deviant immune response elicited in the brain (6, 7, 8). Our results indicate that injection of soluble Ag into the striatum leads to a suppression of Ag-specific DTH similar to that described for ACAID (9). In ACAID, regulatory T cells have been found to be responsible for the suppression of Ag-specific DTH (10). These T cells emerge from the spleen, the primary draining lymphoid organ of the eye. In contrast to the immune deviation elicited in the eye (10, 13), an intact spleen was not required for BRAID, since BRAID could be induced in splenectomized mice. Additionally, we were not able to adoptively transfer BRAID into naive mice by spleen cells harvested after injection into the striatum. This indicates that suppression of DTH in BRAID does not follow the same splenic pathway as described for ACAID.
One of the immunological differences between the eye and the brain is the amount of lymphatic drainage out of each organ. Aqueous humor drains mainly via the trabecular meshwork into the venous system and only a very small portion leaves the eye via the uveoscleral route. Therefore, not more than 10% of protein injected into the eye ends up in the draining lymph nodes. The situation in the brain is probably different, because 14–47% of injected protein drains into the lymphatic system (5). The remaining protein leaves the brain with the cerebrospinal fluid through the arachnoid villi into the blood.
One report suggests that draining cervical lymph nodes are involved in an immune response elicited in the brain, stating that there is B cell expansion in the cervical lymph nodes after Ag injection into the brain (6). The Ab production is significantly increased compared with injections into other body sites (7).
We tested the influence of cervical lymph nodes on the suppression of DTH after OVA injection into the striatum. We harvested these nodes and reinjected the cells into naive animals. In contrast to similar experiments performed with spleen cells, cervical lymph node cells of mice following an OVA injection into the striatum were able to transfer the deviant immune response to naive mice. This result demonstrates that cells within the draining cervical lymph nodes possess the ability to suppress Ag-specific DTH. In ACAID, the regulatory cells are derived from the spleen and have been found to be to be CD8+ and Thy1.2+ T cells. The cells responsible for the deviant immune response elicited in the CNS remain to be identified and characterized.
Our data indicate that immune privilege in the brain is actively maintained. It exists even when the BBB is compromised, but is abolished at a lesion site with high local levels of microglial activation. BRAID is mediated by an immune deviation mechanism that differs from ACAID because an intact spleen is not required and can be adoptively transferred by cells isolated from cervical lymph nodes. Furthermore, these results suggest that studies in which tissue is transplanted into the CNS should take account of the importance of resident APC at the transplantation site in the pattern of the immune response generated to foreign Ags injected into the CNS.
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Acknowledgments |
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Footnotes |
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2 Current address: Augenklinik der Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany.
3 Address correspondence and reprint requests to Dr. Michael J. Young, Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114.
4 Abbreviations used in this paper: BBB, blood-brain barrier; ACAID, anterior chamber-associated immune deviation; CSF, cerebrospinal fluid; BRAID, brain-associated immune deviation; DTH, delayed-type hypersensitivity; GS, Griffonia simplicifolia.
Received for publication December 9, 1999. Accepted for publication March 2, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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