Distribution and Temporal Regulation of the Immune Response in the Rat Brain to Intracerebroventricular Injection of Interferon-γ

EXPERIMENTAL NEUROLOGY ARTICLE NO.

154, 403–417 (1998)

EN986943

Distribution and Temporal Regulation of the Immune Response in the Rat Brain to Intracerebroventricular Injection of Interferon-g Ze-Chun Peng,*,1 Krister Kristensson,† and Marina Bentivoglio* *Institute of Anatomy and Histology, Medical Faculty, University of Verona, Italy; and †Department of Neuroscience, Karolinska Institute, Stockholm, Sweden Received October 30, 1997; accepted September 4, 1998

INTRODUCTION The response to intracerebroventricular administration of interferon (IFN)-g was examined in the adult Wistar rat brain: major histocompatibility complex (MHC) antigens class I and II, CD8 and CD4 antigens, and the macrophage/microglia antigen OX42 were analyzed in respect to saline-injected cases over 1 week. The glial cell type expressing MHC antigens was characterized with double labeling. IFN-g was thus found to induce MHC class I and II expression in microglia, identified by tomato lectin histochemistry, and not in GFAP-immunostained astrocytes. MHC antigen-expressing microglia was detected in the periventricular parenchyma, several fields of the cerebral cortex, cerebellum, major fiber tracts, and brainstem superficial parenchyma. Different gradients of density and staining intensity of the MHC-immunopositive elements were observed in these regions, in which MHC class I antigens persisted up to 1 week, when MHC class II induction had declined. Quantitative analysis pointed out the proliferation of OX42-immunoreactive cells in periventricular and basal brain regions. CD81 T cells were observed in periventricular regions, basal forebrain, and fiber tracts 3 days after IFN-g injection and their density markedly increased by 7 days. CD41 T cells were also seen and they were fewer than CD81 ones. However, numerous CD41 microglial cells were observed in periventricular and subpial regions, especially 1 week after IFN-g injection. Our data indicate that this proinflammatory cytokine mediates in vivo microglia activation and T cell infiltration in the brain and that the cells involved in this immune response display a regional selectivity and a different temporal regulation of antigen expression. r 1998 Academic Press Key Words: microglia; major histocompatibility complex; CD41 T cells; CD81 T cells; cytokines; periventricular brain regions; white matter.

1 On leave from the Department of Anatomy, Hunan Medical University, Changsha, People’s Republic of China.

Expression of human leukocyte antigens encoded by the major histocompatibility complex (MHC) is low or absent in the normal adult human and rat brain (see 15, 45, 46, for review), but increases in inflammatory demyelinating diseases (20, 24, 26, 45) and other pathological brain conditions (6, 8, 14, 31, 37, 38). Experimental studies demonstrated that MHC antigens can be induced in the nervous tissue under diverse conditions (see 1, 39 for review), including the exposure to the cytokine interferon (IFN)-g, which is a key molecule in the regulation of immune responses. Administration of IFN-g to cultured animal or human brain cells was found to induce in glial cells the expression of MHC class I antigens (10, 44), which present antigens to CD81 T cells (cytotoxic/suppressor lymphocytes) and therefore play a critical role in tissue graft rejection and in immune responses to viruses and to neoplastically transformed cells (48). Most of the data indicating that IFN-g induces MHC class II expression in cells of the nervous tissue derive from in vitro studies, in which such induction was found mainly in astrocytes among cultured fetal or neonatal brain cells (5, 44). In contrast, experiments based on cell cultures from the adult brain (10), and studies in vivo in adult rats (33, 35, 41), indicated that in the central nervous system (CNS) IFN-g induces MHC class II antigens mostly in microglia, whereas such induction is rare or absent in astrocytes. The cellular correlates of this type of immune response in the brain are, therefore, still unclear. Since the first demonstration that T cells can recognize antigens only in the context of self MHC antigens on antigen-presenting cells (47), an increasing body of evidence indicated that aberrant expression of MHC class I and class II molecules may be associated with the pathogenesis of autoimmune demyelinating disorders (1, 20, 24). In humans, systemic administration of IFN-g worsened the clinical course of multiple sclerosis (MS) (28), and the number of IFN-g-secreting cells and the IFN-g level were found to be increased in the cerebrospinal fluid (CSF) of MS patients (18, 27). MHC

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0014-4886/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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class II antigen expression was found to be very high in the MS demyelinating plaques, which are most frequently observed in the periventricular white matter (19). However, the relationship between IFN-g circulating in the CSF and the immune response in periventricular regions of the adult brain remains to be documented. On this basis, we here investigated in vivo by means of immunohistochemistry the effect of IFN-g on microglia/macrophages, on the expression of MHC antigens, and on T cells homing in the adult rat brain after intracerebroventricular (icv) administration of IFN-g. Our study was aimed at analyzing in detail the topographical features of this response, as well as its time course over a period of 1 week, and the involved cell types. For this latter step of the study, the immunofluorescent revelation of MHC antigens was combined with immunofluorescent labeling of astrocytes or histochemical labeling of microglial cells (2). MATERIALS AND METHODS

Twenty-four 2- to 3-month-old male outbred Wistar rats (200–350 g body weight; Harlan Nossan, Italy) were used. The animals were kept in basal conditions (12 h/12 h light–dark cycle with lights on at 7 a.m.; food and water freely available). Surgery and Tissue Preparation The experiments were conducted following a protocol that received institutional approval and authorization of the Italian Ministry of Health. Under deep barbiturate anesthesia (pentobarbital, 50 mg/kg i.p.), the skin of the head of the rats was sagittally incised and a small window was opened in the skull with a dental drill. Fifteen rats were injected stereotaxically with 10 µl of recombinant IFN-g (Genzyme Customer Service, Code 80-3575-01; $2 3 105 units/ml) into the right lateral cerebral ventricle through a Hamilton microsyringe in 2 min. The other nine rats were injected with equivalent volumes of saline as control. One day (n 5 8 IFN-g-treated, n 5 3 saline-injected controls), 3 days (n 5 3 IFN-g-treated, n 5 3 saline-injected), or 7 days (n 5 4 IFN-g-treated, n 5 3 saline-injected) after the icv injections, the animals were again deeply anesthesized and perfused transcardially with phosphatebuffered saline (PBS; 0.01 M, pH 7.4) to wash out the blood, followed by freshly prepared 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) for a very short period of time (no longer than 5 min). Since MHC antigens are sensitive to paraformaldehyde fixation (as indicated by the supplier of the antibodies), the fixative was immediately washed out continuing the perfusion with 100 ml of PBS. The brains were then removed and soaked in 30% sucrose in PBS at room temperature until they sank. Forty-micrometer-thick coronal sections were cut on a freezing microtome. Ten series of

sections were collected in PBS and processed with different regimens of immunohistochemistry, immunofluorescence, histofluorescence, or double labeling, respectively, as detailed below. One series of sections was stained with cresyl violet for cytoarchitectonic study. Immunohistochemistry Series of sections from either control or IFN-gtreated rats were processed for immunohistochemistry in the same solutions. These sections were repeatedly washed in PBS and preincubated in a solution containing 5% normal horse serum and 0.3% Triton X-100 in PBS for 1 h. Consecutive sections were then incubated overnight in different antibodies diluted in PBS containing 1% normal horse serum, 0.2% Triton X-100, and 0.1% NaN3. The following monoclonal antibodies (purified from ascites; supplied by Serotec) were used: OX6 (diluted 1:1000), which recognizes MHC class II antigens; OX18 (1:500), which recognizes MHC class I antigens; OX42 (1:500), which labels the complement type 3 receptor of the cells of the macrophage/microglia lineage; W3/25 (1:500), which recognizes the CD4 antigen of T-helper cells; and OX8 (1:300), which recognizes the rat CD8 antigen of suppressor/cytotoxic T cells. After the incubation in primary antibodies, the sections were washed and then incubated in biotinylated horse anti-mouse IgGs (Vector; 1:200). They were then processed with the avidin-biotin peroxidase protocol (ABC kit, Vectastain; Vector) using 3,38-diaminobenzidine (Sigma) as chromogen. An additional series of sections was incubated in 5% normal goat serum for 1 h and then incubated overnight at 4°C in anti-glial fibrillary acidic protein (GFAP) polyclonal antibodies raised in rabbit (Sigma), diluted 1:100 in PBS containing 0.2% Triton X-100, 0.1% NaN3, and 1% normal goat serum. These sections were further processed for immunohistochemistry as mentioned above, using goat anti-rabbit IgGs as secondary antibodies (Vector; 1:200). After the final reaction in 3,38-diaminobenzidine and H2O2, all the sections were washed and mounted on gelatinized slides, air dried, dehydrated, cleared, coverslipped, and studied at the microscope with bright-field illumination. In some experiments, series of sections were processed as mentioned above but omitting the primary antibodies; no immunoreactivity was observed in these sections. Data Analysis The material was studied without knowledge of the animals’ group assignment. The cytological features and distribution of the immunostained elements were analyzed and all the sections were screened for the evaluation of the immunoreactivity using a semiquantitative criterion. A quantitative study was also performed in the tissue processed for OX42 immunohisto-

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FIG. 1. Microphotographs showing MHC class I-immunopositive cells in the cingulate cortex (A) and hypothalamus (B) 1 day after IFN-g icv injection. Note that the MHC class I-positive cells in the brain parenchyma display thin and ramified lightly stained processes. Note in B that the ependymal lining of the 3rd ventricle (on the right) and the endothelial cells in the blood vessels near the ependyma are darkly stained. Scale bars: 25 µm in A, 50 µm in B.

chemistry. Under the microscope, four equivalent levels through the anteroposterior extent of the thalamic paraventricular nucleus (PVT) and three sections through the nucleus of the vertical limb of the diagonal band (NDB), selected on the basis of the adjacent

Nissl-stained sections, were sampled for OX42-immunoreactive (ir) cell counts. This analysis was performed in 12 rats, including 3 animals of each of the experimental groups that were allowed to survive 1 day or 7 days after IFN-g or saline injections. The PVT and NDB

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TABLE 1 Double Labeling Procedures Series of sections

First primary antibody

First conjugated fluorescent dye

1 2 3 4

OX6 OX6 OX18 OX18

DAM-IgG-TR a DAM-IgG-TR a DAM-IgG-TR a DAM-IgG-TR a

Second primary antibody RA-GFAP b No RA-GFAP No

Second fluorescent dye b-GARb IgG c b-tomato lectin d b-GARb IgG b-tomato lectin

Fluorescein DSC Fluorescein DSC Fluorescein DSC Fluorescein DSC

a

Donkey anti-mouse-IgG conjugated Texas Red. Rabbit anti-GFAP. c Biotinylated goat anti-rabbit IgG. d Biotin-labeled tomato lectin. b

were selected among the structures containing OX42immunostained elements since they were easily identifiable on the basis of topographic criteria and delimitation from neighboring structures. All the OX42immunopositive cell bodies (i.e., somata displaying a round or oval shape, giving off several stained processes) were counted, using a grid in the eyepiece, in a 10,000-µm2 area extending from the ventral wall of the dorsal 3rd ventricle in PVT, or from the ventral border of the NDB. The difference between the mean numbers of immunostained cells in all sections from the control and IFN-g-treated rats was analyzed with the Student t test. Double Immunofluorescence and Immunofluorescence Combined with Histofluorescence In order to identify the cell types in which MHC antigen expression was induced by IFN-g icv administration, series of sections from 7 rats (n 5 1 salineinjected, 1 day survival; IFN-g treated: n 5 3, 1 day survival; n 5 1, 3 day survival; n 5 2, 7 day survival) were also processed for double labeling, combining immunofluorescence to MHC class I or II antigens with either GFAP immunofluorescence or tomato lectin histofluorescence (2). The parameters adopted in the double labeling procedures are listed in Table 1. Briefly, these sections were washed thoroughly in PBS before and after each incubation. Nonspecific binding sites were blocked by incubation in the normal serum from the species in which the secondary antibody had been raised. MHC class I or class II antigen immunofluorescence was performed first, with the same antibodies

and dilutions indicated above. Donkey anti-mouse Texas red-conjugated IgGs (Jackson Immunoresearch Laboratories; diluted 1:100) were used for the visualization of MHC antigens, and the sections were then processed for tomato lectin histochemistry or GFAP immunofluorescence. For tomato lectin histofluorescence, the sections were incubated overnight at 4°C in biotinylated tomato lectin (Sigma, No. L9389, 6 µg/ml) in PBS containing 0.5% Triton X-100. Avidin-conjugated fluorescein DSC (Vector) was used for the visualization of histofluorescence in the second step of this procedure. For GFAP immunofluorescence, the sections were incubated overnight at 4°C in primary polyclonal antibodies raised in rabbit against GFAP (Sigma; diluted 1:100) and then incubated for 2 h in biotinylated goat antirabbit IgGs. Finally, the sections were reacted with avidin-conjugated fluorescein DSC, washed in PBS, mounted on gelatinized slides, air dried, coverslipped with Entellan, and studied under a Leitz microscope equipped with Ploempack fluorescence illumination. Each field was observed with two different filtersystems, eliciting the red MHC antigen immunofluorescence and the yellow–green GFAP immunofluorescence or lectin positivity, respectively. RESULTS

MHC Antigen Expression The IFN-g-induced expression of MHC antigens in the meninges, well documented in previous investigations (40, 41), was not analyzed in the present study. Only the findings observed in the brain parenchyma

FIG. 2. Microphotographs showing MHC class II immunoreactivity in the brain of animals sampled 1 day after icv injections. (A) The section at the level of the septum and basal forebrain shows that no MHC class II-positive cells are evident in the brain parenchyma after saline icv injection. (B–D) In the animals treated with IFN-g numerous microglia-like cells are darkly stained in the periventricular areas and at the brain basal surface (B,C); the ependymal lining also displays MHC class II positivity. In addition, MHC class II-stained endothelia are seen in blood vessels in periventricular and basal regions (B,C). Note that the density of MHC class II-positive cells gradually decreases toward the deeply located brain parenchyma. At higher magnification, the immunostained cells display the morphological features of activated microglial cells (D). Abbreviations: ac, anterior commissure; cc, corpus callosum; LV, lateral ventricle; 3V, third ventricle. Scale bars, 350 µm in A, 400 µm in B and C, 40 µm in D.

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will be here described. Since, as mentioned above, MHC antigens are sensitive to aldehyde fixation, paraformaldehyde-fixed tissue may succeed in rendering false negative immunostaining, although the perfusion protocol we adopted resulted in a very light fixation of the tissue. However, as described in detail below, very specific positive immunostaining was detected in the brain parenchyma. In the saline-injected animals, MHC antigen-immunostained elements displayed the features of microglial cells. In the control cases, a few MHC class II (OX6)-ir cells were confined to the areas surrounding the injection needle track and within some fiber tracts, such as the corpus callosum, the internal capsule bilaterally and the ipsilateral fornix, as well as in the central white matter of the cerebellum. MHC class I (OX18)positive cells exhibited instead a more widely spread distribution in the control animals: they were more numerous in all structures in which MHC class II-ir cells were also observed; in addition, MHC class I-ir cells were seen in the external capsule, olfactory tract, optic tract and chiasm, pyramidal tracts, and rarely also in the cerebral and cerebellar cortices and subcortical gray matter. After IFN-g injections, immunoreactivity to MHC class I and class II antigens was prominently elevated in the brain in respect to the saline-injected animals. In the IFN-g-treated brains, the vast majority of the MHC class I or II-immunostained elements displayed the morphological features characteristic of microglia: MHC class I-positive glial cells were usually very lightly stained with relatively thin stained ramified processes (Fig. 1), whereas the MHC class II-positive glial cells displayed darkly stained cell bodies with relatively thick ramified stained processes (Fig. 2D). In addition, some scattered round lymphocyte-like MHC class Ipositive cells were observed (Figs. 1B and 3G). Induction of MHC class I immunoreactivity was very evident in the brain 1 day after IFN-g icv administration (Table 2). At this time point, the density of MHC class I-positive glial cells increased dramatically in respect to the control cases in the periventricular parenchyma (Fig. 1B), including the periacqueductal gray, in the corpus callosum, and in the major brain fiber bundles. In addition, glial cells exhibiting MHC class I positivity were evident in the cerebral cortex (Fig. 1A), in the cerebellar cortex and white matter, as well as throughout the brainstem parenchyma adjacent to the subarachnoid space. A band of periventricular tissue extending for about 0.2–0.3 mm from the ependyma was characterized by a high background and intense staining of endothelial cells in the blood vessels (Fig. 1B). In this periventricular band of tissue, MHC class I-positive glial cells were less dense than in the neighboring tissue more remote from the ventricles. On the other hand, the MHC class I-immunostained cell

population was most dense in the immediately adjacent tissue extending for about 2 mm in depth, and these glial cells became gradually more dispersed toward the deep core of the brain, e.g., in the caudate-putamen and thalamus. The density of MHC class I-ir glia decreased with increasing survival time, but these cells were still numerous 3 days after the IFN-g icv injection and persisted by 7 days (Table 2). As for the immunoreactivity detected in the cerebral cortex, 1 day after IFN-g administration MHC class I-positive glia could be detected in all layers of the ipsilateral cortex, whereas these cells were mainly confined to the superficial layers (I and II) of the contralateral cortex, except for the contralateral temporal fields, in which MHC class I-ir glial cells were detected through layers I–IV. In the animals sampled 3 and 7 days after the IFN-g icv injection, these immunopositive cells were mainly restricted bilaterally to layers I and II of the cingulate, frontal, parietal, and temporal cortices, and they were not detected in the most lateral portion of the frontal and parietal cortices. MHC class II-immunopositive glial cells were very numerous in the periventricular gray and white matter (Figs. 2B and 2C) in all the rats sampled 1 day after IFN-g icv injection (Table 2). In these cases, MHC class II-immunostained glial cells were also seen throughout most of the cerebral and cerebellar cortices, as well as in the brainstem superficial parenchyma beneath the pia mater. In comparison with the MHC class I-ir glial cells, those exhibiting MHC class II immunopositivity were denser (28 vs 42 positive cells per 0.01 mm2 in the sampled periventricular areas) and more intensely stained, but they displayed a more restricted distribution. Thus, MHC class II-ir glial cells were detected in a narrow band of periventricular tissue, and they were clustered in the superficial layers of the frontal and medial occipital cortices and in layers I–IV of the temporal cortex. No MHC class II-positive cells were observed in the lateral frontoparietal and occipital cortices and in the deep layers of most of the cerebral cortex, where, as mentioned above, MHC class I induction had instead been observed 1 day after IFN-g icv injections. On the other hand, MHC class II-ir glial cells were widely distributed throughout the brainstem, being absent only in the deep parenchyma remote from the ventricular walls. The density of MHC class II-immunostained elements decreased markedly with longer survival. In three of the four animals sampled 1 week after IFN-g icv injections, a few clusters of these immunopositive glial cells were seen in the periventricular areas; in addition, MHC class II-positive glial cells were scattered in the major fiber tracts, but they were no longer detectable in the cerebral cortex by 7 days (Table 2). However, in the other rat sampled after 7 days, MHC class II-ir cells were still evident in the periventricular

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FIG. 3. Microphotographs showing OX42- (A,E), MHC class II- (B,F), MHC class I- (C,G), and CD8- (D,H) immunoreactivities in consecutive sections through the floor of the fourth ventricle sampled 7 days after IFN-g icv injection. (E–H) Represent at higher magnification the insets in A–D, respectively. Note that some of the OX42- (E) and MHC class II- (F) immunostained cells display the morphological features of ameboid microglial cells. Some lymphocyte-like MHC class I-stained cells are located close to the blood vessels (arrows in F and G). CD81 cells display the features of T cells (H). Scale bars: 140 µm in D; 35 µm in H; the magnification of A–C is the same as in A, and that of E–G the same as in H.

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TABLE 2 Relative Density of IFN-g-Induced Immunoreactivity in the Brain MHC class I (OX18) a 1 day

MHC class II (OX6)

3 days 7 days

Periventricular parenchyma 111 11 Cerebral cortex: Cingulate cortex 111 11 Frontal cortex 111/1 11/1 Parietal cortex 11/1 1 Temporal cortex 111 11 Occipital cortex 11/1 1 Basal forebrain 11 1 Corpus callosum 11 11 Internal capsule 11 1 External capsule 1 1 Other fiber 11 1 bundles b Cerebellar cortex 111 11 Cerebellar white matter 11 11 Superficial brainstem parenchyma 111 11

1 day

CD81 (OX8) T cells

CD41 (W3/25) T cells

CD41 (W3/25) microglia

3 days 7 days 1 day 3 days 7 days 1 day 3 days 7 days 1 day 3 days 7 days

1

1111

11

(1)

2

1

111

2

6

6

2

2

111

1

1111

11

2

2

2

2

2

2

2

2

2

1

1

111*

11*

2

2

2

2

2

2

2

2

2

1

1

(11)

(1)

2

2

2

2

2

2

2

2

2

1

2

2

2

1

2

2

6

2

2

1

11

1111** 11**

1 1

111* 1111

1* 11

2 2

2 2

2 11

2 111

2 2

2 6

2 6

2 2

2 2

1 1

1

111

11

1

2

1

1

2

2

6

1

1

1

1

11

1

2

2

2

1

2

2

6

1

1

1

1

11

1

2

2

2

2

2

2

2

1

1

1

1

111

11

1

2

1

11

2

2

6

1

1

11

1

111

1

2

2

2

2

2

2

2

2

2

2

1

11

1

2

2

2

2

2

2

2

1

1

1

1

111

1

2

2

1

11

2

2

6

2

2

2

Note. * Restricted to certain layers, as specified in the text; ** Restricted to layer I–IV; ( ) Immunostained cells in clusters with a patchy distribution. 2, no immunoreactive cells; 6, immunoreactive cells observed occasionally in some sections; 1, Low density of immunoreactive cells; 11, moderate density; 111, high density; 1111, very high density. a Antigen (antibody). b Including olfactory tract, anterior commissure, optic tract, fornix, stria medullaris, mammillothalamic tracts.

areas, the NDB, the temporal cortex, and throughout the brainstem superficial parenchyma. In the brain of this latter animal, most of the MHC class II-positive elements displayed the morphological features of activated microglial cells, as seen in the rats sampled 1 day or 3 days after the IFN-g icv injections; in addition, numerous stained elements displayed the features of ameboid reactive microglia, especially in the areas bordering the floor of the 4th ventricle (Figs. 3B and 3F). MHC class II immunoreactivity was also detected in blood vessel endothelia of the periventricular regions and basal brain surface (Figs. 2B and 2C). OX42 Immunoreactivity Numerous OX42-ir cells were detected throughout the brain in both the control and IFN-g-treated animals

and they were hypertrophic around the needle track. In the saline-injected control animals, OX42-ir elements, which displayed the features of microglial cells, were also hypertrophic in the periventricular areas when compared to those in the areas remote from the ventricular system, but their stained processes were long and thin (Figs. 4A and 4B). After exposure to IFN-g, the processes of the hypertrophic OX42-ir cells were instead contracted (Figs. 4C–4F). The quantitative analysis showed that the density of the OX42-ir cell population increased significantly after IFN-g treatment at all the examined time points (Fig. 5). Such increase was most evident in the brain of the animals sampled 7 days after IFN-g icv administration (Figs. 4E and 5), in which many of the OX42-ir cell bodies were clustered in pairs. In these animals, many

FIG. 4. Microphotographs of OX42 immunoreactivity in the midline thalamus 1 day after saline (A,B) or IFN-g (C,D) icv injections, and 7 days after IFN-g icv injection (E,F). (B,D, and F) Represent at higher magnification the insets in A, C, and E, respectively. Note the hypertrophy of the immunostained cells 1 day after IFN-g injections (C,D) in respect to the control case (A,B), and that all the OX42-ir cells in C through F display contracted processes (compare D and F with B), suggesting that these cells are activated in response to the IFN-g icv administration. Note also in E an increase in the density of the immunopositive cells. Scale bars: 75 µm in A; 25 µm in B; the magnification of C and E is the same as in A, and that of D and F is the same as in B.

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CD8 Immunopositivity

FIG. 5. Histogram showing the density (mean number 6 SD) of OX42-ir cells in the vertical limb of the nucleus of the diagonal band (NDB) and paraventricular thalamic nucleus (PVT) after saline and IFN-g icv injections. **P , 0.01; ***P , 0.001.

CD81 (OX8-ir) cells were not seen in the brain of rats sampled 1 and 3 days after saline injections. Only a few CD81 lymphocyte-like cells were seen around the needle track in the control rats sampled after 7 days. As listed in Table 2, no CD81 elements were observed 1 day after IFN-g injections, whereas by 3 days a few lightly stained CD81 lymphocyte-like cells were seen close to the blood vessels in the periventricular regions, in the corpus callosum and other fiber tracts, in the basal forebrain, and throughout the brainstem superficial parenchyma (Table 2). The density of CD81 cells, which were darkly stained and displayed a round cell body with lymphocyte-like features (Figs. 3D, 3H, and 6), increased in the animals sampled 7 days after IFN-g injection (Table 2). Such CD81 T cells were located in the vicinity of blood vessels, especially in the dentate gyrus of the hippocampal formation, in the PVT and hypothalamus (Fig. 6A), and in the floor of the 4th ventricle (Figs. 3D and 3H), i.e., in regions in which numerous OX42-ir and MHC class I-positive ameboid microglial cells had also been observed. In addition, CD81 T cells were detected in the superficial portion of the ventral forebrain, including the NDB (Fig. 6B). At the same time point (i.e., 1 week after IFN-g injection), CD81 T cells were also seen in fiber tracts and at the brainstem surface (Table 2). CD4 Immunopositivity

OX42-ir glial cells were hypertrophic, with relatively short and rarely branched immunostained processes (Figs. 3E and 4F). OX42-ir cells displaying the morphological features of ameboid microglial cells were also observed in the periventricular regions, especially in the PVT (Fig. 4E) and in the brainstem dorsal tegmental nuclei (Fig. 3E), i.e., along the ventral walls of the dorsal 3rd and 4th ventricles, respectively.

CD41 (W3/25-ir) cells, which displayed the morphological features of lymphocytes, were rarely seen in the brain parenchyma of the control cases, scattered through the major fiber tracts, such as the corpus callosum, internal capsule, and optic chiasm. The walls of some blood vessels also exhibited CD4 immunostaining, with no evident difference between the control and the IFN-g-treated animals.

FIG. 6. Microphotographs of CD8 immunopositivity in the suprachiasmatic nucleus of the hypothalamus (A) and in the vertical limb of the nucleus of the diagonal band (B) 7 days after IFN-g icv injection. Note that the CD81 cells display the morphology of T lymphocytes and are in close relationship to the blood vessels. Scale bars, 40 µm in A, 85 µm in B.

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After IFN-g icv injections, in the animals sampled at 1 day and 3 days CD4 positivity did not display major differences in respect to the control cases (Table 2). However, an increase of CD41 T cells was evident in one of the rats sampled 3 days after the IFN-g icv injection and in one of the animals sampled by 7 days. Even in these cases, the density of CD41 T cells in the brain was much lower than that of CD81 T cells (Table 2). At variance with the saline-injected control animals, CD41 cells exhibiting the morphological features of microglia were also detected after IFN-g icv injections (Fig. 7). The density of the CD41 microglial cells increased in all the rats sampled in the 7th day in respect to those sampled after 1 and 3 days (Table 2). Such CD41 microglial cells were most commonly observed in the regions bordering the lateral and 3rd ventricles (Fig. 7), and they also frequently occurred in the brain fiber tracts 7 days after the IFN-g icv injections (Table 2). Double Labeling of Glial Cells and MHC Antigens In agreement with the findings described above, in the tissue from IFN-g-injected animals that was processed for double immunohistochemistry numerous MHC class II-positive glial cells were found in the areas surrounding the ventricular system, including the periacqueductal gray, and in the superficial portion of the entire brain beneath the pia mater, i.e., in areas close to the CSF in the subarachnoid space. MHC class Ipositive glial cells were also numerous in these regions, but their immunostaining was weaker than that of the MHC class II-positive elements. In the same sections, GFAP-immunofluorescent cells, which displayed the features of astrocytes, were seen throughout the brain, with a prevalence in the cerebel-

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lar cortex and in the superficial layers of the cerebral cortex. No substantial increase of the density of GFAP-ir cells was evident in the forebrain after the IFN-g icv injections when compared with standard lab preparations from untreated rats. No double GFAP-immunostained cells (MHC class I-GFAP-ir or MHC class II-GFAP-ir elements) were seen after IFN-g icv injections, although, as mentioned above, brilliant single-labeled cells (MHC class I or II-immunopositive glial cells and GFAP-immunofluorescent astrocytes) were seen throughout the brain in the sections processed for double immunofluorescence. In the tissue from IFN-g-injected animals processed for double MHC antigen immunofluorescence and tomato lectin histofluorescence, single tomato lectinstained cells displaying the features of microglia were located both in the deep brain areas remote from the ventricular system and at the brain surface. In these sections, all the MHC class II-positive cells were also stained by tomato lectin positivity, thus indicating that they were microglial cells (Fig. 8). Such double-labeled microglial cells were hypertrophic and displayed a topographical prevalence around the ventricular system and in the brain parenchyma beneath the pia mater in respect to the brain central core. All the MHC class I-positive glial cells were also tomato lectinstained. Thus, the double-labeling experiments showed that after IFN-g icv injections MHC antigen expression was induced in microglia and not in astrocytes in the brain. DISCUSSION

The present study focused on the brain response to IFN-g circulating in the CSF, a condition that may occur during pathological production of this proinflam-

FIG. 7. Microphotographs of CD4 immunostaining in the anterodorsal thalamic nucleus 7 days after IFN-g icv injection. Note that the CD41 cells display the morphological features of microglial cells. Scale bars, 90 µm in A, 32 µm in B.

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FIG. 8. The microphotographs illustrate the double labeling with MHC class II antigens (red) immunofluorescence (A) and tomato lectin (yellow-green) histofluorescence labeling of microglia (B). The same field was photographed with two different excitation wavelengths. Note that all the cells exhibiting MHC class II immunofluorescence in A are also stained in B by tomato lectin positivity. Scale bar, 35 µm.

matory cytokine by T cells in the CSF, such as that described in humans affected by MS (18, 27). An initial study on the effect of IFN-g on the brain reported an induction of MHC class II expression in different cell types in vitro (44). In the same study (44), similar results were observed in cell suspensions from brains of neonatal mice in which IFN-g had been injected in the brain parenchyma. In subsequent investigations, IFN-g was injected directly in the brain parenchyma in adult rats (33), or administered intrathecally to albino rats, in which the cellular and immune response was investigated in the spinal cord (41). Intravenous (35) and icv (40, 43) IFN-g injections have also been performed in Lewis rats, a strain susceptible to experimental allergic encephalomyelitis. The present data, obtained in adult Wistar rats, support the findings obtained in such previous investigations, showing that IFN-g induces a high expression of MHC class I and II antigens in the brain in vivo. In addition, our findings point out that the icv administration of a single dose of IFN-g can induce in vivo activation of microglial cells in the brain and the expression of CD4 antigen in microglia. We also provide evidence that IFN-g induces CD81 and CD41 T cell infiltration in the brain. The cells expressing MHC antigens and the recruited CD81 and CD41 T cells showed a close relationship with the CSF, i.e., they were mostly observed in the periventricular and subpial regions of the brain. In addition, microglial cells expressing MHC class I and II antigens were also

evident in several fields of the cerebral cortex and in the brain white matter after IFN-g injections. The administration of different doses of IFN-g, ranging from 25 Units in mice (44) to 50,000 Units in rats (33), have been used in previous investigations on the CNS response to this cytokine in vivo, making it difficult to compare the findings obtained in different studies in relation to the administered dose. It should be noted in this respect that a dose-dependent effect of IFN-g on MHC antigen induction was observed after intrathecal (41) and intracerebral (33) administration of the cytokine. Our study was based on the icv injection of a dose 25 times lower than the maximal dose of IFN-g administered intracerebrally in a previous investigation (33). The present findings point out that this was sufficient for MHC antigen induction in vivo from the CSF, in agreement with previous evidence that also relatively low doses of intrathecal IFN-g consistently resulted in MHC antigen induction (33). IFN-g Induces MHC Expression in Microglial Cells in the Brain in Vivo In response to inflammatory stimulation, microglial cells become hypertrophic with contracted processes; these ameboid or reactive microglial cells are morphologically and functionally similar to the inflammatory macrophages (9). Reactive microglial cells have been proposed to exert a cytotoxic effect via a direct cell-tocell contact or by releasing several potentially cytotoxic

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substances (3, 7). A wealth of data has reported that in vivo microglial cells are the major cell class that may constitutively express MHC class I (17, 41) and class II (11, 30, 32, 41) molecules and CD4 antigens (21, 29, 30) in the CNS, and in which the expression of immune molecules may be rapidly upregulated upon activation (4, 22, 29, 36). Human and experimental animal studies have also indicated that MHC class II antigen expression is absent (4, 40, 42) or only occasionally detectable (16, 41, 45) in astrocytes in the adult CNS. The cell type in which MHC antigen expression was induced in vivo by IFN-g in the brain was previously described on the basis of morphological features, or using GFAP as a marker of astrocytes (35, 40, 41, 43, 44), or the OX42 antigen in consecutive sections (33). In support to the indications provided by these studies, the present investigation unequivocally demonstrated that IFN-g circulating in the CSF induces MHC class I and II antigens in microglial cells in the brain and not in astrocytes. This conclusion was based not only upon the negative evidence provided by double GFAP immunocytochemistry, but also on the positive evidence provided by double staining with tomato lectin binding histochemistry, which labels both ameboid and ramified microglia in the adult rat brain (2). Differential Timing and Spatial Gradient of the IFN-g-Induced MHC Class I and II Antigens in the Brain in Vivo It is interesting to note that the expression of MHC class I and II antigens induced in microglia by IFN-g was here found to display a different temporal regulation: a high expression of MHC class I antigens persisted up to 7 days, when the induction of MHC class II antigens had markedly declined. This would tally with the kinetics of MHC class II gene induction by IFN-g, with mRNA levels peaking at 24–48 h and decreasing after 72 h (see 1 for review). After intraparenchymal injection of IFN-g, the induction of MHC class II staining in glial cells was found to peak after 4 days and to persist up to 1 month but no MHC class I staining was detected in microglial cells (33). The brain response to IFN-g seems to be based on secondary effects rather than on the direct action of this cytokine (33). Therefore, our data indicate that the timing of such response after IFN-g icv administration is different from that detected previously after intraparenchymal injection of the cytokine. The present findings also pointed out a different spatial gradient of distribution of the IFN-g-induced MHC class I or II antigen expression: MHC class II immunostaining was highest in the brain tissue bordering the ventricles, whereas MHC class I-ir microglial cells were less dense in the immediate vicinity of the ventricles than in the deeper neighboring band of tissue. This finding may reflect a different immune

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modulation exerted by IFN-g circulating in the CSF on the adjacent tissue. On the other hand, in spite of their overall lower density compared to MHC class II-ir microglial cells, the MHC class I-ir ones were found to be much more widely distributed throughout the brain after the icv IFN-g injections, especially in the cerebral cortex. These data not only indicate a differential response in the brain of MHC class I and II antigens to the exposure to IFN-g in the CSF, but also suggest a different modulation of brain cell subsets by this cytokine. IFN-g Induces Recruitment of T Cells in the Brain Experimental findings obtained both in vitro and in vivo demonstrated that IFN-g can recruit T cells into the CNS (see 33, 34 for review), but a previous intrathecal administration of a high dose of IFN-g could not confirm this phenomenon when examined up to 48 h in the spinal cord (41). In contrast to this latter finding, but in agreement with data obtained after IFN-g intraparenchymal injections (33), we could consistently observe in the brain some CD81 cytotoxic/suppressor T cells, and a few CD41 helper T cells, 3 days after IFN-g icv administration. CD81 T cells were relatively numerous in the parenchyma by 7 days after the injection, but CD41 T cells were not equally recruited into the brain at this time point. Thus, at variance with the data observed after direct intraparenchymal injection of IFN-g, which resulted in a maximal recruitment of inflammatory cells after 24 h (33), in the present study the administration of IFN-g in the CSF resulted in a delayed recruitment of T cells. This finding further supports the assumption that the occurrence of IFN-g in the CSF triggers a long-lasting series of events in the brain. Although the mechanism that initiates the extravasation and infiltration of T lymphocytes into CNS is unclear, it has been shown that only activated T cells that recognize CNS antigens persist (12) and that this is a multistep process involving cytokine-stimulated expression of MHC antigens and complementary adhesion receptor–ligand pairs by leukocytes, brain endothelial cells, and glial cells (see 23 for review). The present finding of an IFN-g-induced long-lasting persistence in microglia of MHC class I antigens, which are associated with the function of CD81 T cells, in contrast to a time-related decline of MHC class II antigens, which are associated with the function of CD41 T-helper lymphocytes, suggests that recruitment of T cells into the CNS is a slow process that may require continuous expression of MHC antigens in the CNS. IFN-g-Induced CD4 Expression in Microglia The expression of CD4 antigen in the mature microglial cells in the young adult brain is very low (21, 29)

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but can be upregulated by microglial cell differentiation during development and reactivation under pathological conditions (29), as well as with aging (30). In our experiments, CD4 upregulation in microglial cells was not observed 1 or 3 days after IFN-g icv injection, whereas CD41 microglial cells were seen by 7 days. This finding may indicate that the microglial cells were activated by IFN-g to differentiate, as supported by the present quantitative analysis of OX-42-immunostained cells, which confirmed the proliferation of microglia. In addition, the present data support the assumption that a cascade of inflammatory events is elicited by the exposure to IFN-g in the CSF. Implications for Brain Pathology In relation to the increased levels of IFN-g in the CSF of MS patients (18), it is interesting to note that the topographical distribution of IFN-g-induced MHC class II expression in the present study was similar to that of the predilection sites of demyelination plaques in MS (19). Although a causal relationship between demyelination and IFN-g was not investigated in the present study, our data support the hypothesis that IFN-g circulating in the CSF may play an important role in the pathogenesis of the periventricular lesions in the course of MS. It should also be considered that IFN-g is produced continuously in the CSF by T cells in the course of MS, in which, therefore, this cytokine can exert a long-lasting effect on the periventricular regions. Disruption of the blood–brain barrier and recruitment of leukocytes from the blood into the CNS have been recognized as the crucial steps in the development of inflammatory demyelinating diseases, such as MS and the animal model provided by experimental allergic encephalomyelitis (see 23 for review). Increasing evidence indicated that during MS the lesions develop through a local activation of lymphocytes and especially CD81 T cells. The CD8 glycoprotein is both an adhesion protein and a cosignalling receptor of cytotoxic/ suppressor T lymphocytes, and its function has been associated with MHC class I antigen (25). During MS, microglial cells appeared to be the predominant cell type synthesizing MHC antigens beyond the edge of MS lesion, followed by MHC antigen-expressing T lymphocytes, macrophages, and microglial cells within the hypercellular plaques and border regions (45). Interestingly, we have observed a recruitment of CD81 T lymphocytes in the brain 3 and 7 days after icv IFN-g administration, when the level of MHC class I antigens was still high. Microglial cells, which may act in the brain as scavenger and antigen-presenting cells, may also become the target of CD81 T cells (see 9 for review), and this may explain why IFN-g treatment just before the experimental allergic encephalomyelitis onset effectively inhibited clinical disease (43). In transgenic mice that expressed IFN-g construct to direct expression to oligodendrocytes, and thus represent a

model of constitutive IFN-g expression in the adult brain, the findings of increased expression of MHC class I and II, activation of macrophages/microglia, and infiltration of lymphocytes, predominantly of the CD8 phenotype (13), supported an active role of IFN-g in immune-mediated demyelination. Conclusions Altogether our data demonstrate that microglial cells are the major subpopulation of cells in the brain which can be rapidly activated to differentiate and express MHC antigens in response to IFN-g. In addition, CD81 and CD41 T cells can be recruited in the brain by IFN-g circulating in the CSF, and CD4 antigen expression can be induced by IFN-g in microglia. The present study pointed out a topographic predilection of different antigens and the involved cell types in the brain response to IFN-g in the CSF and a temporal sequence of these events. Thus, the cascade of events triggered by immune response in the brain may be regionally and temporally regulated. ACKNOWLEDGMENT This study was supported by the First Project on Multiple Sclerosis of the Italian Institute of Health (ISS) and by a grant of the Osterman Foundation (to K.K.).

REFERENCES 1. 2.

3. 4.

5.

6.

7.

8.

9.

10.

Abdulkadir, S. A., and S. J. Ono. 1995. How are class II MHC genes turned on and off. FASEB J. 9: 1429–1435. Acarin, L., J. M. Vela, B. Gonza´lez, and B. Castellano. 1994. Demonstration of poly-N-acetyl lactosamine residues in ameboid and ramified microglial cells in rat brain by tomato lectin binding. J. Histochem. Cytochem. 42: 1033–1041. Banati, R. B., J. Gehrmann, P. Schubert, and G. W. Kreutzberg. 1993. Cytotoxicity of microglia. Glia 7: 111–118. Bo¨, L., S. Mo¨rk, P. A. Kong, H. Nyland, C. A. Pardo, and B. D. Trapp. 1994. Detection of MHC class II-antigens on macrophages and microglia, but not on astrocytes and endothelia in active multiple sclerosis lesions. J. Neuroimmunol. 51: 135– 146. Cannella, B. and C. S. Raine. 1989. Cytokines up-regulate Ia expression in organotypic cultures of central nervous system tissue. J. Neuroimmunol. 24: 239–248. Finsen, B. R., M. B. Jørgensen, N. H. Diemer, and J. Zimmer. 1993. Microglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus. Glia 7: 41–49. Frei, K., C. Siepl, P. Groscurth, S. Bodmer, C. Schwerdel, and A. Fontana. 1987. Antigen presentation and tumor cytotoxicity by interferon-g treated microglial cells. Eur. J. Immunol. 17: 1271–1278. Gehrmann, J., R. Gold, C. Linington, J. Lannes-Vieira, H. Wekerle, and G. W. Kreutzberg. 1993. Microglial involvement in experimental autoimmune inflammation of the central and peripheral nervous system. Glia 7: 50–59. Gehrmann, J., and G. W. Kreutzberg. 1995. Microglia in experimental neuropathology. In Neuroglia (H. Kettenmann and B. R. Ransom, Eds.) pp. 883–904. Oxford Univ. Press, New York. Grenier, Y., T. C. G. Ruijs, Y. Robitaille, A. Olivier, and J. P. Antel. 1989. Immunohistochemical studies of adult human glial cells. J. Neuroimmunol. 21: 103–115.

IMMUNE RESPONSE TO INTERFERON-g IN THE BRAIN 11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25. 26.

27.

28.

29.

30.

31.

Hayes, G. M., M. N. Woodroofe, and M. L. Cuzner. 1987. Microglia are the major cell type expressing MHC class II in human white matter. J. Neurol. Sci. 80: 25–37. Hickey, W. F., B. L. Hsu, and H. Kimura. 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28: 254–260. Horwitz, M. S., C. F. Evans, D. B. McGavern, M. Rodriguez, and M. B. A. Oldstone. 1997. Primary demyelination in transgenic mice expressing interferon-g. Nature Med. 3: 1037–1041. Kono, H., T. Yamamoto, Y. Iwasaki, H. Suzuku, T. Saito, and H. Terunuma. 1989. Wallerian degeneration induces Ia-antigen expression in the rat brain. J. Neuroimmunol. 25: 151–159. Lampson, L. A. and W. F. Hickey. 1986. Monoclonal antibody analysis of MHC expression in human brain biopsies: Tissue ranging from ‘‘histologically normal’’ to that showing different levels of glial tumor involvement. J. Immunol. 136: 4054–4062. Lee, S. C. and C. S. Raine. 1989. Multiple sclerosis: Oligodendrocytes in active lesions do not express class II major histocompatibility complex molecules. J. Neuroimmunol. 25: 261–266. Ling, E. A., C. Kaur, and W. C. Wong. 1991. Expression of major histocompatibility complex and leukocyte common antigens in amoeboid microglia in postnatal rats. J. Anat. 177: 117–126. Link, J., M. So¨derstro¨m, T. Olsson, B. Ho¨jeberg, Å. Ljungdahl, and H. Link. 1994. Increased transforming growth factor-b, interleukin-4, and interferon-g in multiple sclerosis. Ann. Neurol. 36: 379–386. Martin, R., and H. F. McFarland. 1995. Immunological aspects of experimental allergic encephalomyelitis and multiple sclerosis. Crit. Rev. Clin. Lab. Sci. 32: 121–182. Martin, R., A. B. Vogt, H. Kropshofer, and R. Lichtenfels. 1994. Association of HLA and multiple sclerosis. Behring Inst. Mitt. 94: 158–170. Matsumoto, Y., N. Hara, R. Tanaka, and M. Fujiwara. 1986. Immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to Ia-positive cells with dendritic morphology. J. Immunol. 136: 3668–3676. McGeer, P. L., T. Kawamata, D. G. Walker, H. Akiyama, I. Tooyama, and E. G. McGeer. 1993. Microglia in degenerative neurological disease. Glia 7: 84–92. Merrill, J. E. and E. N. Benveniste. 1996. Cytokines in inflammatory brain lesions: Helpful and harmful. Trends Neurosci. 19: 331–338. Merrill, J. E., M. C. Graves, and D. G. Mulder. 1992. Autoimmune disease and the nervous system. Biochemical, molecular, and clinical update. West J. Med. 156: 639–646. O’Rourke, A. M., and M. F. Mescher. 1993. The roles of CD8 in cytotoxic T lymphocyte function. Immunol. Today 14: 183–188. Oksenberg, J. R., A. B. Begovich, H. A. Erlich, and L. Steinman. 1993. Genetic factors in multiple sclerosis. J. Am. Med. Assoc. 270: 2362–2369. Olsson, T. 1992. Cytokines in neuroinflammatory disease: role of myelin autoreactive T cell production of interferon-gamma. J. Neuroimmunol. 40: 211–218. Panitch, H. S., R. L. Hirsch, J. Schindler, and K. P. Johnson. 1987. Treatment of multiple sclerosis with gamma interferon: Exacerbations associated with activation of the immune system. Neurology 37: 1097–1102. Perry, V. H. and S. Gordon. 1987. Modulation of CD4 antigen on macrophages and microglia in rat brain. J. Exp. Med. 166: 1138–1143. Perry, V. H., M. K. Matyszak, and S. Fearn. 1993. Altered antigen expression of microglia in the aged rodent CNS. Glia 7: 60–67. Rao, K. and R. D. Lund. 1989. Degeneration of optic axons

32.

33.

34.

35.

36. 37.

38.

39. 40.

41.

42.

43.

44.

45.

46.

47.

48.

417

induces the expression of major histocompatibility antigens. Brain Res. 488: 332–335. Sedgwick, J. D., S. Schwender, R. Gregersen, R. Do¨rries, and V. ter Meulen. 1993. Resident macrophages (ramified microglia) of the adult brown Norway rat central nervous system are constitutively major histocompatibility complex class II positive. J. Exp. Med. 177: 1145–1152. Sethna, M. P. and L. A. Lampson. 1991. Immune modulation within the brain: Recruitment of inflammatory cells and increased major histocompatibility antigen expression following intracerebral injection of interferon-g. J. Neuroimmunol. 34: 121–132. Simmons, R. D. and D. O. Willenborg. 1990. Direct injection of cytokines into the spinal cord causes autoimmune encephalomyelitis-like inflammation. J. Neurol. Sci. 100: 37–42. Steiniger, B. and P. H. van der Meide. 1988. Rat ependyma and microglia cells express class II MHC antigens after intravenous infusion of recombinant gamma interferon. J. Neuroimmunol. 19: 111–118. Streit, W. J., M. B. Graeber, and G. W. Kreutzberg. 1988. Functional plasticity of microglia: A review. Glia 1: 301–307. Streit, W. J., M. B. Graeber, and G. W. Kreutzberg. 1989. Peripheral nerve lesion produces increased levels of major histocompatibility complex antigens in the central nervous system. J. Neuroimmunol. 21: 117–123. Tafti, M., S. Nishino, M. S. Aldrich, W. Liao, W. C. Dement, and E. Mignot. 1996. Major histocompatibility class II molecules in the CNS: increased microglial expression at the onset of narcolepsy in a canine model. J. Neurosci. 16: 4588–4595. Ting, J. P.-Y., and A. S. Baldwin. 1993. Regulation of MHC gene expression. Curr. Opin. Immunol. 5: 8–16. Uitdehaag, B. M. J., C. J. A. De Groot, A. Kreike, P. H. van der Meide, C. H. Polman, and C. D. Dijkstra. 1993. The significance of in-situ Ia antigen expression in the pathogenesis of autoimmune central nervous system disease. J. Autoimmun. 6: 323–335. Vass, K., and H. Lassmann. 1990. Intrathecal application of interferon gamma: progressive application of MHC antigens within the rat nervous system. Am. J. Pathol. 137: 789–800. Vass, K., H. Lassmann, H. Wekerle, and H. M. Wisniewski. 1986. The distribution of Ia antigen in the lesions of rat acute experimental allergic encephalomyelitis. Acta Neuropathol. 70: 149–160. Voorthuis, J. A. C., B. M. J. Uitdehaag, C. J. A. De Groot, P. H. Goede, P. H. Van der Meide, and C. D. Dijkstra. 1990. Suppression of experimental allergic encephalomyelitis by intraventricular administration of interferon-g in Lewis rats. Clin. Exp. Immunol. 81: 183–188. Wong, G. H. W., P. F. Bartlett, I. Clark-Lewis, F. Battye, and J. W. Schrader. 1984. Inducible expression of H-2 and Ia antigens on brain cells. Nature 310: 688–691. Woodroofe, M. N., A. S. Bellamy, M. Feldmann, A. N. Davison, and M. L. Cuzner. 1986. Immunocytochemical characterisation of the immune reaction in the central nervous system in multiple sclerosis. Possible role for microglia in lesion growth. J. Neurol. Sci. 74: 135–152. Yee, K. T., A. M. Smetanka, R. D. Lund, and K. Rao. 1990. Differential expression of class I and class II major histocompatibility complex antigen in early postnatal rats. Brain Res. 530: 121–125. Zinkernagel, R. M., and P. C. Doherty. 1974. Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 248: 701–702. Zinkernagel, R. M., and P. C. Doherty. 1997. The discovery of MHC restriction. Immunol. Today 18: 14–17.