THE JOURNAL OF COMPARATIVE NEUROLOGY 313:409-430 (1991)

Characterisation of Two New Monoclonal Antibodies Directed Against Rat Microglia JOCHEN GEHRMA" AND GEORG W. KREUTZBERG Department of Neuromorphology, Max-Planck-Institute for Psychiatry, D-8033 Martinsried, Federal Republic of Germany

ABSTRACT With the aid of cultured rat microglial cells as immunogen, we raised two monoclonal antibodies, designated murine clone (MUC) 101 and 102, which recognised subsets of resident microglial cells in the normal central nervous system and cells of the mononuclear phagocyte system in peripheral organs. These antibodies were charaderised by immunoperoxidase immunocytochemistry, immunoelectron microscopy, and immunoblotting. The immunostained cells were identified as microglial cells by double-immunofluorescence labelling with the B,-isolectin from Griffonia simplicifolia, an established microglial cell marker. Under normal conditions, both antibodies labeled resident microglia but with different distribution patterns. Under pathological conditions, e.g., after facial nerve transection, they labeled activated, perineuronal microglia in the operated facial nucleus. Immunoelectron microscopy demonstrated a membrane localisation of the antigen recognised by MUC 102. In peripheral organs, MUC 101 and 102 reacted with different cell populations of the mononuclear phagocyte system, particularly in thymus, spleen, and peripheral lymph node. Western blot experiments showed that MUC 101 recognised two proteins of 116 and 95 kD in fractions obtained from operated facial nucleus while MUC 102 reacted with two proteins of 62 and 70 kD molecular weight. These immunocytochemical results 1)confirm the antigenic similarity between microglia and cells of the monocyte-macrophage cell lineage, and 2) indicate that considerable antigen heterogeneity might exist among resident microglia. MUC 101 and 102 could thus become useful for studying the function of microglial cells both under normal and pathological conditions. Key words: macrophage, perivascular cell, antigen heterogeneity,axotomy, regeneration

Microglial cells are a morphologically distinct cell type of the central nervous system (CNS) which constitute between 5 and 20% of the total glial cell population (Peters et al., '76; Kreutzberg, '87; Lawson et al., '90). The morphological plasticity of microglial cells was first described in detail in silver-stained preparations by del Rio-Hortega ('19, '32). Two forms of microglial cells have been distinguished: the resting or ramified microglia in mature brain and the ameboid microglia that occur perinatally. At the light microscopical level, resting microglial cells are best identified by means of monoclonal antibodies and lectins (for review see Streit and Kreutzberg, '87; Streit et al., '88; Perry and Gordon, '88; Graeber and Streit, '90a). These stains show resting microglialcells to have a highly branched morphology with several crenellated processes quite distinct from that of astrocytes or oligodendrocytes. Resting microglial cells are swiftly activated in response to even subtle pathological stimuli (for review see Streit et al., '88; Kreutzberg, '87; Kreutzberg et al., '89a,b; Graeber and Streit, '90a). Transection of cranial nerves, e.g., the o 1991 WILEY-LISS, INC.

facial nerve, has proved to be especially appropriate for studying microglial activation. Under these conditions resting microglial cells rapidly proliferate and transform into an activated form without becoming truly phagocytic cells (Cammermeyer, '65; Sjostrand, '65; Watson, '65; Kreutzberg, '66, '68). If neuronal degeneration occurs, for instance after injection of a neurotoxic lectin into a cranial nerve, activated microglial cells further develop into phagocytic brain macrophages (Streit and Kreutzberg, '88). Thus at least three different types of microglia can be distinguished: resting and activated microglia, and microgliaderived brain macrophages. Although the precise function of microglial cells is not yet fully understood, they have been proposed to be involved in several processes, including phagocytosis, respiratory burst activity, and the secretion of cytokines (Streit and Kreutzberg, '88; Streit et al., '88; Graeber and Streit, '90a; Fagan and Gage, '90; Giulian et al., '91; Banati et al., '91; Accepted July 11, 1991.

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Gehrmann et al., '91a,b). Microglial activation is involved in penetrating brain injury (Giulian et al., '89), autoimmune diseases (Matsumoto et al., '80, '89; Lassmann et al., '86), neurodegenerative diseases (McGeer et al., '881, viral infections (Weinstein et al., 'go), and peripheral nerve lesion (Cammermeyer, '65; Sjostrand, '65; Kreutzberg, '66; Kreutzberg et al., '89a,b; Gehrmann et al., '91a). The activation of microglial cells in these models is apparent through changes in their morphology, immunophenotype, migration, and proliferation (Kreutzberg, '66, '68; Blinzinger and Kreutzberg, '68; Graeber et al., '88; Streit and Kreutzberg, '88; Streit et al., '89a,b). The identification of microglial cells in tissue sections is based on a number of histochemical, immunocytochemical, and ultrastructural criteria. These include the histochemical localisation of enzymes associated with purine metabolism, e.g., 5'-nucleotidase (Kreutzberg and Barron, '781, as well as the immunocytochemical detection of monocytei macrophage antigens, such as the CR3 complement receptor (Perry and Gordon, '88; Graeber et al., '88). Microglial cells can also be detected by the binding of the B,-isolectin of Griffonia simplicifolia (GSI-B,) which recognises a membrane-associated glycoconjugate (Streit and Kreutzberg, '87; Streit, '90). The hybridoma technique by Koehler and Milstein ('75) has made it possible to define and distinguish heterogenous cell populations by the detection of specific epitopes expressed on different types of cells. In an attempt to distinguish microglial cells from other cells of the monocytemacrophage cell lineage, we have raised two new monoclonal antibodies which recognise distinct epitopes on rat microglial cells. Rat microglial cells cultured according to the technique of Giulian and Baker ('86) were used as the immunogen. Growing hybridoma cells were screened immunocytochemically on cryostat sections from rat brainstem after unilateral transection of the facial nerve. Under these conditions both resting and activated microglial cells are present. We report here the immunocytochemical and biochemical characterisation of two new monoclonal antibodies, MUC 101 and 102, recognising different epitopes on microglial cells. These antibodies have proved to be useful not only for the detection of microglial cells in tissue sections, but also for the study of questions related to microglial cell heterogeneity and function in various neuropathologies.

MATERIALS AND METHODS Primary cell cultures Cultures of microglial cells were prepared from newborn rat brain according to the protocol of Giulian and Baker ('86). After removal of the meninges followed by mechanical dissociation of brain tissue, primary glial cultures were kept in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 15% fetal calf serum (BoehringerMannheim) at 3% CO, and 37°C for 4-6 weeks. Cells growing on top of a confluent astrocyte layer were removed by gentle shaking and separated from other more slowly adhering cells, e.g., astrocytes or oligodendrocytes, by a short adherence step of 30 minutes. About 95% of the adhering cells displayed a typical microglial morphology of either the ameboid or the ramified type and were positive for nonspecific esterase, CR3 complement receptor, and the low-density lipoprotein (LDL) receptor. They were negative for astrocyte markers (e.g., glial fibrillary acidic protein

(GFAP))and oligodendrocyte markers [e.g.,the anti-myelinoligodendrocyte monoclonal antibody (anti-MOG)] (Linington et al., '84). Following a short adherence microglial cells were mechanically removed from the culture flask, washed three times in phosphate-buffered saline (PBS) to remove serum components, and used for the immunisation of balbic mice. In order to test the cross-reactivities of our monoclonal antibodies, the following cell suspensions were prepared 1) blood monocytes from heparinised blood isolated by a Ficoll/sodium diatrizoate gradient (Boyum, '68); 2) peritoneal macrophages according to the method of Daems ('80); and 3) bone marrow cells of rat femur (Dijkstra et al., '85). All cell suspensions were plated onto glass coverslips, grown under conditions identical to those of microglia, and used for immunocytochemistry as previously described (Graeber et al., '89a).

Establishment of murine hybridoma clones Balb/c mice were immunised with cultured rat microglial cells. They were primed intraperitoneally (i.p.1with approximately 5 x lo6 microglial cells emulsified with an equal volume of Freund's complete adjuvant. Injections were repeated five times at monthly intervals with approximately the same number of cultured rat microglial cells emulsified with incomplete Freund's adjuvant. Booster injections were given on days 35 and 36 i.p. and on days 37 and 38 intravenously (i.v.1 via the tail vein after the last monthly injection. Fusion of mouse spleen cells with the feeder-independent PAI-mouse myeloma cell line was performed according to the procedure of Debus et al. ('83). The fused cells were cultured in 24-well tissue culture plates in HAT-selective RPMI-1640 medium (Gibco) supplemented with 15% fetal calf serum (Boehringer-Mannheim) and incubated at 5%CO,. Screening of the hybridoma supernatants was performed by a three-step avidin-biotin-peroxidase method as described below on 20-pm cryostat sections of rat brainstem containing the facial nuclei 5 days after axotomy of the right facial nerve. Clones reacting with cells of microglial morphology were selected and cloned three times by the method of limiting dilution. Hybridoma cells were cultured in roller bottles (Costar) for large-scale production of monoclonal antibodies. The antibodies were concentrated from the resulting tissue culture supernatants by precipitation with 50% ammonium sulphate. The pellet was redissolved in saline and then dialysed against 1.5 M glycine, 3 M NaC1, pH 8.9. The monoclonal antibodies were then purified by affinity chromatography on protein-A Sepharose according to the manufacturer's instructions (Pharmacia-LKl3). The class and subclass of the immunoglobulins secreted by the antibody producing clones were determined by using a mouse IgG subtyping kit (Amersham-Buchler). MUC 101 belonged to the IgG1, and MUC 102 to the IgG2a subclass (for comparison see Table 2).

Surgery and tissue collection Wistar rats (10 weeks old, weighing approximately 200 g) were operated under deep ether anaesthesia. The right facial nerve was cut near its exit from the stylomastoid foramen, while the intact contralateral side served as a control. After various time intervals, animals were anaesthetized, killed, and the brainstem removed and immediately snap frozen in high-pressure CO,. Cryostat sections (20 p,m) of rat brainstem containing the facial nuclei were

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA collected on gelatin-coated glass slides and stored at -70°C until immunocytochemical investigation. Crossreactivities were tested on 20-pm sections of the following peripheral tissues: lung, liver, thymus, spleen, cervical lymph nodes, kidney, and skin.

Light immunocytochemistry Cryostat sections were fixed in 3.7% formalin for 5 minutes followed by acetone (50% 2 minutes, 100% 2 minutes, and 50% 2 minutes) at room temperature. The sections were then rehydrated in 0.0 1M tris-bufferedsaline (TBS), pH 7.4, for 5 minutes and in TBS containing 0.1% bovine serum albumin (BSA) for another 5 minutes. After incubation with 2% normal horse serum (DAKO, Hamburg, FRG) for 30 minutes, mouse monoclonal antibodies (1 pg/ml for MUC 101 and 0.1 pg/ml for MUC 102) were added to the tissue sections overnight at 4°C. Subsequent antibody detection was carried out using a biotinylated horse anti-mouse secondary antibody (Serotec) and the Vectastain ABC-Elite kit (Vector Labs., Burlingame, CA, USA) with 3,3'-diaminobenzidine (DAB) as peroxidase substrate. Selected sections were counterstained with cresyl violet to visualise neighbouring structures, such as neurons and blood vessels. Prior to incubation with the ABC complex, endogenous peroxidase activity was suppressed by incubating the sections for 10 minutes in a 0.3% hydrogen peroxide solution. Acid phosphatase activity was demonstrated on selected cryostat sections from peripheral tissues according to Burnstone (Pearse, '68) following the immunoperoxidase procedure. The staining patterns of MUC 101 and 102 were further compared with those of established antimacrophage monoclonal antibodies. Adjacent sections were therefore incubated with one of the following antibodies (the specificity and references are shown in parenthesis): OX-42 (CR3 complement receptor; Robinson et al., '861, OX6 [major histocompatibility complex (MHC) class 11; McMaster and Williams, '791, OX18 (MHC class I; Fukumoto et al., '82), W3/25 (CD4; Williams et al., '771, and W3/13 (pan T-cell marker; Williams et al., '77). These monoclonal antibodies were purchased from Serotec, UK. Control sections were prepared by omission of primary antibody or, alternatively, replacement of primary antibody by two irrelevant antibodies of the same IgG subclass. Double-immunofluorescencemicroscopy studies were carried out on 20-pm tissue sections from rat brainstem after axotomy of the right facial nerve. After blocking the fixed sections with either 2% goat or rabbit normal serum, sections were first incubated with either MUC 101 or MUC 102, followed by goat anti-mouse IgG TRITC conjugate (Sigma). This was followed by rabbit anti-GFAP antibody (DAKO), with subsequent visualisation with goat anti rabbit IgG-FITC conjugate F(ab')2 fragment (Pel-Freez)or with the FITC-labeled microglia-binding lectin GSI-B, (Sigma) according to the procedure of Streit and Kreutzberg ('87). Likewise MUC 102-positive microglial cells in white matter were double labeled with MUC 102 and GSI-B,. Adjacent sections were labeled with a rabbit anti-GFAP polyclonal antibody followed by a goat anti-rabbit IgG FITC conjugate F(ab')2 fragment and then with the anti-MOG monoclonal antibody (Linnington et al., '84) followed by rabbit anti-mouse TRITC conjugate F(ab')2 fragment (Dako). This double immunofluorescence allowed us to compare the staining patterns of astrocytes (GFAP) and

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oligodendrocytes/myelinated axons (anti-MOG) with the MUC 102 immunoreactivity pattern. Appropriate controls were carried out by 1)replacing the primary antibody with TBS; 2) substituting the primary antibody with an irrelevant antibody of the same IgG subclass; and 3) the aid of a secondary antibody directed against immunoglobulins from species other than the one in which the primary antibody was raised.

Electron microscopy Animals were perfused 5 days after axotomy of the right facial nerve under chloral hydrate anaesthesia with 2% paraformaldehyde in 0.01 M PBS, pH 7.4. After immersion in the same fixative for 1-2 hours, vibratome sections of rat brainstem containing the facial nuclei were cut at 60 pm and immunostained free-floating. For electron microscopy, the incubation times with the primary antibody and the detection reagents were increased to approximately 12 hours for each step at 4°C to allow better antibody penetration into the relatively thick vibratome sections. Following immunostaining, selected sections were fixed in Dalton's osmium, dehydrated, and embedded in Epon according to standard procedures. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined in a Zeiss EM10 electron microscope.

Biochemistry Samples of cultured rat microglia and operated facial nuclei were used as the starting material for Western blot experiments. Cultured rat microglial cells were prepared as described. The facial nuclei were isolated by microdissection from snap-frozen rat brainstem 10 days after transection of the facial nerve and stored at -70°C. Two facial nuclei were pooled for each analysis. Fractions either from cultured rat microglial cells or operated facial nuclei were: 1) crude homogenate; 2) supernatant; and 3) membrane fraction obtained after centrifugation of crude homogenates at 100 OOOg for 30 minutes. Extracts were analysed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. In the case of MUC 101 epitope analysis, extracts were submitted to immunoprecipitation with the antibody coupled to Sepharose beads since otherwise no specific proteins recognised by MUC 101 could be detected in the samples. MUC 101 (1 mg) monoclonal antibody, affinity purified as previously described (for comparison see Establishment of murine hybridoma clones), was coupled to CNBr-activated Sepharose according to the manufacturer's instructions (Pharmacia-LKB). Coupled antibodySepharose (100 pl) was incubated overnight at 4°C with 100 pl of Triton X-100 solubilized tissue fractions. Antigenantibody complexes were purified three times by centrifugation through 10% sucrose and the final pellet resuspended in electrophoresis buffer containing 2% P-mercaptoethanol, briefly boiled, and subjected to SDS-PAGE and immunoblotting. SDS-PAGE was performed in 7.5% (MUC 101) or 12.5% (MUC 102) polyacrylamide minigels according to the method of Poehling and Neuhoff ('80). Gels were either stained for protein with Coomassie Blue or electroblotted onto Immobilon P transfer membrane (Millipore) according to the procedure of Towbin et al. ('79). Prior to staining with antibodies the blots were incubated with 5% dried milk powder containing 0.1% Triton X-100 in 0.01 M TBS, pH 7.4, for 30 minutes at room temperature in order to block nonspecific binding sites. After incubation

J. GEHRMA” AND G.W. KREUTZBERG

412 with the primary antibody overnight at 4°C (MUC 101 1 p,g/ml; MUC 102 0.1 Fgiml) the Immobilon membrane was incubated with a 1500 dilution of a goat anti-mouse peroxidase-labeled secondary antibody (Dionova) for 60 minutes at room temperature. 4-Chlor-1-naphthol was used as a substrate for peroxidase visualisation. Controls included immunoprecipitation with an irrelevant antibody of the same IgG class (mouse monoclonal antibody 100/1 recognising 100- and 115-kD epitopes of bovine clathrin assembly proteins; gift from Dr. Ungewickell, MPI for Biochemistry, Martinsried) coupled to the Sepharose beads. The immunoblotting was further controlled by using an irrelevant antibody of the same IgG subclass as well as omission of the secondary antibody.

RESULTS Light microscopy At the light microscopical level, resting microglial cells immunostained by MUC 101 and 102 were identified on the basis of their typical morphology (del Rio-Hortega, ’19, ’32; Peters et al., ’76; Perry and Gordon, ’88; Kreutzberg, ’87; Graeber and Streit, ’90a). Fitting the classical description, they appeared as ramified, radially branched cells having several crenellated processes arising from a round or slightly elongated cell body. In normal rat brain, MUC 101 stained resting microglial cells in white matter areas and, infrequently in grey matter (Fig. 1A). MUC 102 strongly stained resting microglial cells throughout different brain areas including white and grey matter (Fig. 1B). However, the staining was slightly more conspicuous in white matter compared to grey matter (for comparison see Table 1). In some grey matter areas, including brainstem for example, MUC 102 stained resident microglial cells uniformly. In general, MUC 102 staining was much more intensive and more widely distributed on resting microglial cells than MUC 101immunoreactivity (for comparison see Fig. lA,B). Both MUC 101 and MUC 102 immunostaining revealed resting microglial cells with two different morphologies, i.e., a stellate or a bipolar morphology (Fig. 2A,B). The geometry of microglial cell processes seemed to depend on their location. In grey matter microglial cells had a predominantly stellate morphology (Fig. 2A). In white matter cells of the bipolar type were more frequent and often had their processes oriented along the long axis of fibre tracts (Fig. 2B). Different types of cells associated with blood vessels appeared to be stained by the two antibodies. MUC 101 stained perivascular cells located in the vicinity of brain blood vessels (Fig. 2C). MUC 101-positiveperivascular cells did not exhibit ramified cell processes, in contrast to intraparenchymal resting microglial cells (cf. Figs. 1A,B and 2A,B,D). The morphology and distribution of MUC 101-positive perivascular cells were similar to the previously described ED2-positive,perivascular cells (Graeber et al., ’89b).At the ultrastructural level, ED2-positiveperivascular cells have been identified as a specialized type of pericyte (Graeber et al., ’89b; Graeber and Streit, ’90b). Accordingly, MUC 101-positive cells in the vicinity of blood vessels are termed “perivascular cells.” MUC 101-positive perivascular cells exhibited the following characteristics: 1) they were relatively large, elongated, or spindle-shaped cells closely aligned with the wall of an adjacent blood vessel; 2) they did not have any direct contact with neuronal or other cellular elements in the CNS parenchyma, and they partic-

TABLE 1. Reactivities of Monoclonal Antibodies With Different Types of Microglial and Perivascular Cells in Rat Brain Cell t w e recognised Resting microglia In white matter In grey matter Activated microglia Perivascular microglia Perivascular cell

MUC 101

MUC 102

+

++ + ++ ++

(t)

+ +

-

‘Immunocytochemicallabelling code: -, absent; (21, only a few cells stained; 2 , weak, +, well discernable; + t , strongly positive. The classification of microglial and perivascular cells is based on the terminology suggested by Streit et al., ’88 and by Graeber and Streit, ’90b.

ularly lacked cellular processes radiating into the neuropil; and 3) their localisation along blood vessels and distribution in the CNS were similar to that of the previously described ED2-positive perivascular cells. MUC 101, like the ED2 antibody, stained perivascular cells in the CNS but not in the peripheral organs (for comparison see Fig. 9). In contrast to MUC 101, MUC 102 stained ramified, juxtavascular microglial cells other than the MUC 101 or ED2-positive perivascular cells (cf. Fig. 2D). At the light microscopical level, processes of MUC 102-positive cells appeared to form contacts with the surface of adjoining blood vessels (Fig. 2D). However, it was difficult to distinguish between the type of MUC 101-positive perivascular cell and MUC 102-positive perivascular microglial cell on the basis of light microscopical evaluation alone. Immunoelectron microscopy further identified such perivascular processes of MUC 102-positivemicroglial cells. Such terminal processes of microglial cells were attached to the basal membrane of an adjoining blood vessel rather than being surrounded by a basal membrane as is the case with ED2-positiveperivascular cells (for comparison see Fig. 8C; Graeber et al., ’89b). These ultrastructural characteristics confirmed that MUC 102-positive perivascular microglial cells were true components of the CNS parenchyma. Based on these criteria, juxtavascular MUC 102-positive microglial cells are therefore termed “perivascular microglia.” They are distinct from the MUC 101- or ED2-positive perivascular cells as well as from other resident, intraparenchymal microglial cells. The staining patterns of MUC 101 and 102 on the different types of microglial and perivascular cells present in rat brain are summarised in Table 1. In the leptomeninges, MUC 101 stained round cells resembling meningeal macrophages (Fig. 2E). In contrast, MUC 102-positivecells were less numerous in the meninges. MUC 102 rather stained resting microglial cells that came into close contact with the meningeal surface (Fig. 2F). In the choroid plexus, MUC 101stained cells within the plexus which resembled macrophages with a round or ovoid nucleus. These cells had a few short processes and were scattered throughout the choroid plexus (Fig. 2G). In addition, MUC 101 labeled cells on the ventricular surface of the choroid plexus, the so-called epiplexus (Kolmer) cells (Fig. 2G). In comparison, MUC 102 seemed to label epiplexus cells preferentially rather than cells within the choroid plexus (Fig. 2H). Neither MUC 101 nor MUC 102 reacted with any other cell population in the CNS. In particular, they stained no other glial cells apart from microglial and perivascular cells, i.e., no staining of astrocytes or oligodendrocytes was observed. Both monoclonal antibodies were strictly speciesspecific, i.e., they did not crossreact with other species tested (mouse, rabbit, and guinea pig).

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA

Fig. 1. Staining of resident microglial cells in the normal rat CNS. A MUC 101 immunoreactivity, white matter of the cerebellum. ~ 2 4 0B: . MUC 102 immunoreactivity, cochlear nucleus. ~ 2 6 0 .

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Figure 2

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA Resting microglial cells become rapidly activated under various pathological conditions. Activated microglial cells can be distinguished from the resting form through differences in their immunophenotype and functional state. Transection of the rat facial nerve, for example, leads to the rapid proliferation of resting microglial cells as well as their translocation from the neuropil to a perineuronal position (Cammermeyer, '65; Sjostrand, '65; Kreutzberg, '66, '68; Blinzinger and Kreutzberg, '68).Perineuronal, activated microglial cells show an increased expression of cellular markers, such as the CR3 complement receptor (Graeber et al., '88) or newly express markers, such as MHC class I1 molecules (Streit et al., '89b). This well-characterised model proved to be valuable for testing the reactivities of the new monoclonal antibodies with activated microglial cells. Both MUC 101 and 102 recognised activated, perineuronal microglial cells occurring in the facial nucleus after transection of the facial nerve. MUC 101 strongly labeled activated, perineuronal microglial cells on the operated side with a maximum around day 10 after facial nerve transection (Fig. 3A). MUC 102 immunoreactivity was strongly increased in the operated facial nucleus with a maximum around day 5 after facial nerve transection (Fig. 3B). As observed for the staining of resting microglial cells under normal conditions (for comparison see Fig. 11, the staining intensity of MUC 102 in the operated facial nucleus surpassed that of MUC 101. In addition, MUC 102 immunoreactivity appeared on activated microglia much earlier and remained elevated for a longer period of time following axotomy than did MUC 101 immunoreactivity (data not shown). In the operated facial nucleus, both monoclonal antibodies stained activated microglial cells that surround the injured facial motoneurons with their hypertrophic processes (Fig. 4B,D). In the control facial nucleus, MUC 101 occasionally stained cells in a perivascular position similar to those previously demonstrated (for comparison see Fig. 2C) rather than ramified microglial cells (Fig. 4C). In contrast, MUC 102 intensively labeled ramified, resting microglia in the control facial nucleus (Fig. 4A). The immunostained cells were further identified by colocalisation with the microglia-binding lectin, the B,isolectin from Griffonia simplicifolia (GSI-B,). In brain parenchyma, GSI-B, binds selectively to resting and activated microglial cells (Streit and Kreutzberg, '87; Streit, '90). In the operated facial nucleus, MUC 101immunoreac-

Fig. 2. Staining of microglial and perivascular cells in different locations of the normal rat CNS. A MUC 102, trigeminal nucleus. Microglial cells have a stellate morphology and are located in the vicinity of neuronal cell bodies counterstained with cresyl violet. ~ 2 4 0 . B: MUC 102, corpus callosum. Microglial cells display a bipolar morphology, their processes being oriented along the long axis of fibre tracts. ~ 2 8 0 .C: MUC 101, brainstem. Elongated perivascular cells lacking ramified processes are located within the wall of a blood vessel. Phase contrast microscopy. ~ 5 8 0D . MUC 102, brainstem. A ramified perivascular microglial cell comes into contact with the tissue side of a blood vessel. Counterstained section. x 880. E-H Sections counterstained with cresyl violet. E: MUC 101, meninges. Round cells, likely to represent local macrophages, are stained in the meninges. X 170. F MUC 102, meninges. Compared to the MUC 101 immunoreactivity in E only a few cells within the meninges are stained. Occasionally, resident microglial cells seem to extend their processes along the meningeal surface. X170. G MUC 101, choroid plexus. Cells within the plexus of either round or elongated shape are stained. ~ 4 8 0 H . : MUC 102, choroid plexus. Mostly cells located on the ventricular surface, likely to represent epiplexus cells, are stained. x 480.

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tivity formed an almost continuous rim surrounding the soma of the chromatolytic facial motoneurons (Fig. 5A). As shown in Figure 5C,D, the staining patterns of MUC 101 and GSI-B, were identical, thus identifying the cells in the operated facial nucleus as microglial cells. On the other hand, comparison of MUC 101 staining with that for GFAP, an intracellular marker for astrocytes (Bignami et al., '72), gave a nonoverlapping staining pattern indicating that MUC 101 did not bind to astrocytes (Fig. 5A,B). Due to the staining of surface structures by both MUC 101 and 102 (for comparison see Figs. 7, 8, and 111, immunostained microglial cells tended to display a blurred appearance with typical, crenellated outlines. In contrast, immunostained astrocytes appeared as sharply delineated cellular profiles due to the staining of abundant stacks of GFAP, their major cytoskeletal component. MUC 102 immunoreactivity could also be colocalized on activated microglial cells in the operated facial nucleus with GSI-B, binding (data not shown). Furthermore, by applying GSI-B, to cerebellar white matter, it was demonstrated that MUC 102 also stained ramified microglia in white matter areas (Fig. 6A,B). Double immunofluorescence with the anti-MOG antibody, which recognises the oligodendrocytemyelin continuum (Linnington et al., '84), and with GFAP showed that both the network of oligodendrocyteslmyelinated axons as well as the astrocyte staining pattern were antigenically and morphologically distinct from the MUC 102-positive microglial cells in white matter (Fig. 6C,D).

Electron microscopy Further identification of immunostained cells was obtained using immunoelectron microscopy. For this reason, sections were prepared for ultrastructural immunocytochemistry from the operated facial nucleus and various other brain areas, including cerebellum and thalamus, which had been stained with MUC 101 or MUC 102. However, MUC 101 immunoreactivity was abolished even after mild perfusion with low concentrations of paraformaldehyde. Therefore it was impossible to use MUC 101 for ultrastructural immunocytochemistry. For this reason, it is not possible to define precisely the type of perivascular cell recognised by MUC 101. However, additional antibodies against CNS perivascular cells are currently being used in an attempt to clarify this matter. In contrast, MUC 102 immunoreactivity was well preserved after perfusion with 2% paraformaldehyde, thus allowing its localisation at the ultrastructural level. Microglia were usually identified in the electron microscopy preparations on the basis of cell size, prominent heterochromatin, dense cytoplasmic matrix, and prominent strands of wide rough endoplasmic reticulum (Vaughn and Peters, '68; Blakemore, '75; Peters et al., '76; Kreutzberg, '87). Activated microglia occurring in response to axotomy were found in a close perineuronal position 5 days after axotomy. MUC 102 reaction product was detected on the plasma membranes of activated microglial cells (Fig. 7A-C). These perineuronal microglial cells covered large areas of the neuronal soma, extending long cytoplasmic processes. Microglial processes formed multiple, convoluted folds and were often found in apposition to dendrites, sometimes engulfing them (Fig. 7A,B). The neuronal surface, however, was free of reaction product. Although the reaction product was mainly found on the microglial plasma membranes, some diffuse cytoplasmic staining was occasionally observed. In these cases, the DAB deposits were localized

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Fig. 3. Staining of activated microglial cells. Overviews of rat brainstem, containing the facial nuclei, 5 days after transection of the right facial nerve. x 42. A. MUC 101 immunoreadivity is increased on the right, operated facial nucleus as compared to the control side. Small immunostained cells, likely to represent perivascular cells, are scat-

J. GEHRMANN AND G.W. KREUTZBERG

tered throughout the brainstem section. B: Compared to MUC 101, MUC 102 immunoreactivity is even more strongly increased on the operated facial nucleus. Immunostained, resting microglial cells appear to be equally distributed throughout the section.

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA within the rough endoplasmic reticulum, probably indicating the sites of protein synthesis (Fig. 7D). Furthermore, reaction product was occasionally localized in intracellular vacuoles which indicates a possible association of the MUC 102 epitope with intracellular degradation processes (Fig. 7E). Apart from activated microglia, MUC 102immunoreactivity was ultrastructurally detected on resting microglia. Resting microglia were identified in different locations including grey matter areas, such as the control facial nucleus, and in white matter areas, e.g., white matter of the cerebellum and the capsula interna. Resting microglia in the control facial nucleus exhibited some cellular processes that displayed reaction product on their surface (Fig. 8A). These processes were found in close proximity to neighbouring structures, e.g., s o n s and dendrites. In contrast, white matter microglia also showed reaction product on their surface but displayed fewer cytoplasmic processes (Fig. 8B). There, microglial cells were surrounded by numerous myelinated axon bundles. Occasionally they were situated in direct apposition to an adjoining oligodendrocyte which was always free of reaction product. Resident microglial cells were frequently identified either in direct apposition to the basal membrane of a blood capillary or having contact with the basal membrane through a terminal process (Fig. 8C). These perivascular microglia were always situated outside the basal membrane. Occasionally their processes were associated with structures of the neighbouring endothelium reminiscent of tight junctions. Due to their localisation outside the basal membrane these perivascular microglia were distinguishable from perivascular cells, e.g., the ED2-positive perivascular cells which are enclosed within the basal membrane (Graeber et al., '89b; Graeber and Streit, '90b). Thus immunoelectron microscopy confirmed the existence of truly MUC 102-positive, perivascular microglial cells as suggested by the light microscopy (for comparison see Fig. 2D). Reaction product was always absent from structures other than microglia, i.e., astrocytes, oligodendrocytes, neurons, or endothelial cells.

Cross-reactivitieswith other macrophage populations Microglial cells share antigens with other cells of the monocyte-macrophage cell lineage (Perry and Gordon, '88; Streit et al., '88). Thus several monoclonal antibodies directed against peripheral macrophages cross-react with related cells in the brain: resting microglia (Perry and Gordon, '88; Robinson et al., '86; Streit et al., '88),activated microglia (Graeber et al., ,881, and perivascular cells (Graeber et al., '89b). We therefore tested the cross-reactivities of our two monoclonal antibodies in different peripheral rat tissues. The staining patterns and distributions are summarised in Table 2. MUC 101 and MUC 102 both reacted either with large, round cells or with cells with a dendritic appearance characterised by numerous slender processes. These labeled cells always showed acid phosphatase activity as demonstrated by a combination of the immunoperoxidase procedure with detection of acid phosphatase activity in the same cryostat section (data not shown). In thymus, MUC 101reacted with cells having a dendritic morphology and displaying slender processes. These cells were found both in the cortex and the medulla of rat thymus, being most conspicuous in the corticomedullary junction. They showed considerable ramification, with elon-

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gated processes (Fig. 9A,B). The staining pattern was suggestive of labelling of the plasma membranes. The morphology and distribution of cells stained with MUC 102 in thymus, however, was different (Fig. 10A,B). They were less ramified and their morphology resembled more closely that of ED1-3-positive thymic macrophages. They were preferentially localized in the corticomedullary junction, but also near the intralobular space. In lymph nodes, both monoclonal antibodies labeled cells with a dendritic morphology. MUC 102 immunoreactivity (Fig. 10C,D), however, was more widely distributed in lymph node than MUC 101 (Fig. 9C,D). Spleen macrophages were stained with MUC 101 mostly in the outer periarterial sheath (PALS), but also to a small extent in the inner PALS, (Fig. 9E). MUC 102 immunoreactivity was more clearly concentrated on spleen macrophages in the outer PALS and also diffusely in the red pulp (Fig. 10E). Follicular dendritic cells, detected by the ED5 monoclonal antibody (Jeurrissen et al., '881, were not stained in the spleen by MUC 101 or 102. In lung, the staining patterns of the two monoclonal antibodies were conspicuously different. While MUC 101 only stained a very few alveolar macrophages [either at the periphery of the bronchus-associated lymphoid tissue (BALT) or within the alveolar space], macrophages within the BALT were reactive with MUC 102 with some weaker staining in the intraalveolar space (Figs. 9F, 10F). In liver, neither of the monoclonal antibody cross-reacted with the Kupffer cells or any sinusoidal or parenchymal cell (Figs. 9G, 10G). Langerhans cells in the skin did not seem to be conspicuously recognised by either of the two monoclonal antibodies. Langerhans cells were identified on the basis of their strong reactivity with anti-Ia antibodies (Rowden et al., '77; McMaster and Williams, '79; Tamaki et al., '79). Dermal macrophages, not constitutively expressing the Ia antigen (Hsiao et al., '89), were weakly stained with MUC 101 and more intensively with MUC 102 (Figs. 9H, 10H). Thus, MUC 101 cross-reacts with cells having a dendritic morphology in thymus and lymph nodes. In addition, MUC 101 reacts with cells (presumably representing peripheral macrophages) in the spleen and weakly with dermal and alveolar macrophages. In contrast to the staining of perivascular cells in the CNS (for comparison see Fig. 2C), MUC 101 did not cross-react with cells in a perivascular position in the peripheral tissues. MUC 102 similarly detected cells of a dendritic morphology in lymph nodes, but also cells of classical macrophage morphology in the thymus. It also cross-reacted with spleen macrophages and strongly with macrophages in the BALT. Neither Kupffer nor Langerhans cells were detected by these monoclonal antibodies. The distributions of MUC 101-positive cells in thymus and lymph nodes and MUC 102-positive cells in lymph node partially matched the staining pattern obtained with the OX-6 antibody recognising a monomorphic determinant of the Ia antigen (McMaster and Williams, '79). In summary, these antibodies recognise cell populations of the mononuclear phagocyte system as assessed by morphological criteria, tissue distribution, and correlation with acid phosphatase activity and established monoclonal antibodies. They did not react with other cell types or tissue structures, i.e., granulocytes, lymphocytes, follicular dendritic cells, vascular endothelium, mucosal or bronchial epithelium, or liver parenchymal cells.

Figure 4

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA We also tested the monoclonal antibodies on the following cell preparations: blood monocytes and macrophages, peritoneal macrophages, and microglial cells. While MUC 101 stained neither cultured blood monocyte-macrophages nor peritoneal macrophages, it weakly reacted with cultured rat microglia. In contrast, MUC 102 strongly reacted with all three cultured cell populations (Table 2). The interactions of the two monoclonal antibodies with cultured rat microglial cells, the starting material for immunisation, are currently being characterised by fluorescence activated cell sorter analysis.

Western blot analysis

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fractions from homogenates of operated facial nuclei or cultured rat microglia. However, they appeared stronger on the immunoblots when homogenates of operated facial nuclei had been used as samples for SDS-PAGE. These bands could not be detected with samples of unoperated control facial nuclei (data not shown). Therefore there appears to be an increase in the amount of antigen detected by MUC 102 in response to axotomy in the operated facial nucleus. Control experiments were performed using an irrelevant antibody of the same IgG subclass (clone 2/40/15 directed against tyrosinhydroxylase, Boehringer-Mannheim). They consistently gave negative results (lane 7).

Western blot analysis of either microglial cells or operDISCUSSION ated facial nuclei was performed to define the target Since the introduction of the hybridoma technology by epitopes of the two monoclonal antibodies. For MUC 101 analysis, the immunoprecipitation step was included in Koehler and Milstein ('75), monoclonal antibodies have order to isolate the specific antigens from a large amount of become invaluable tools for studying macrophage heterogetissue extract. Figure 11A shows the results of immunopre- neity. Macrophages in different tissues and body cavities cipitation experiments of operated facial nuclei fractions indeed form a heterogenous population as demonstrated by immunoadsorbed with MUC 101 coupled to Sepharose differences in their morphology, distribution, enzyme activbeads. MUC 101 recognised two proteins of 116 kD and 95 ities, cell surface properties, and functional capacities (cf. kD in samples obtained from operated facial nuclei (Fig. Sorg, '82). In contrast to most T- and B-cell markers, 11A, lanes 1, 3). These proteins could be equally well antimacrophage antibodies generally detect epitopes on the precipitated from crude homogenates (lanes 1,7) or a crude cell surface which are expressed only during certain stages membrane fraction (lanes 3, 9) of operated facial nuclei. of their activation. Thus they only recognise macrophage They were not detectable in the supernatant fraction (lanes subpopulations (Hogg et al., '84; Zwadlo et al., '85; Esiri 2, 81, consistent with the immunocytochemical data that and McGee, '86). Several monoclonal antibodies against the antigen is more abundant on the plasma membranes human and mouse macrophages have been described. than in the cytosol. In contrast, immunoprecipitations Among these are the antibodies EBMll (Esiri and McGee, using homogenates obtained either from unoperated con- '86) or AMC30 (Cras et al., '90) which react with human trol facial nuclei or cultured rat microglia gave negative microglial cells. Other antibodies define epitopes on mouse results (data not shown). Control experiments both with an microglial cells such as the F4/80 antigen (Austyn and irrelevant antibody of the same IgG subclass coupled to the Gordon, '81; Perry et al., '85; Lawson et al., '90). In the rat, Sepharose beads (see Materials and Methods) and with a few antimacrophage antibodies have been reported (Dijkoperated facial nuclei homogenates were also performed stra et al., '85; Takeya et al., '89; Colic et al., '90). Here we and gave negative results (Fig. 1lA, lane 4). Only the heavy describe two new anti-rat monoclonal antibodies, MUC 101 and light chains of the mouse immunoglobulins released and 102, reacting with distinct populations of microglial from the Sepharose beads prior to SDS-PAGE were de- cells and other cells of the mononuclear phagocyte system tected as were control blots with the boiled Sepharose beads in peripheral organs. The specificity and reliability of the alone (lane 5). Omission of the secondary anti-mouse staining obtained with these antibodies were controlled by antibody (lane 6) completely abolished staining of the immunoperoxidase immunocytochemistry, immunofluorescence double-labelling studies, and immunoelectron micros116-kD and 95-kD bands. Western blot analysis using fractions of cultured rat COPY. microglial homogenates and similar fractions from operComparison of MUC 101 and 102 with ated facial nuclei showed that MUC 102 is directed against established antimacrophage antibodies two proteins of approximately 70 and 62 kD (Fig. l l B , lanes In rat brain, several macrophage antigens, such as Fc and 1, 3 , 4 , and 6) with a weaker staining polypeptide at 60 kD molecular weight. These bands could only be detected in the complement receptors, have been demonstrated on resident crude homogenates (lanes 1,4) and in the crude membrane microglial cells (Perry and Gordon, '88; Streit et al., '88). fractions (lanes 3, 6) but not in the supernatant fraction Other antigens expressed on peripheral macrophages, e.g., (lanes 2, 5). The same pattern was observed when using those recognised by the ED1-3 monoclonal antibodies (Dijkstra et al., '85) or by Ki-M2R (Wacker et al.,'85), however, are not detected on resident microglial cells (Graeber et al., '88). On the contrary, the ED2 monoclonal antibody has been used to distinguish perivascular cells from ramified Fig. 4. MUC 101 and 102 immunoreactivity in the control versus microglial cells (Graeber et al., '89b). Such myelomonocytic the operated facial nucleus 10 days after facial nerve transection. Sections counterstained with cresyl violet. A MUC 102, control antigens are only expressed by microglial cells under pathonucleus. Ramified microglial cells are numerous in the neuropil. x 380. logical conditions (Graeber et al., '90). The distribution of MUC 101immunoreactivity in the rat B: MUC 102, operated nucleus. Counterstained motoneurons are surrounded by immunopositive microglial cells similar to the staining CNS resembles to some extent the distribution of the CD4 shown in D. ~ 3 8 0C: . MUC 101, control nucleus. A perivascular cell is antigen found with the W3/25 monoclonal antibody (Perry stained with some diffuse background staining in the neuropil due to nonspecific staining of the facial nucleus neuropil. X540. D: MUC 101, and Gordon, '87). Both MUC 101 immunoreactivity and operated nucleus. Strongly immunostained perineuronal microglial CD4 antigen are expressed by a few resident microglial cells ensheath with their hypertrophic processes the slightly counter- cells, activated microglial cells, and cells in the leptostained facial motoneurons. x540. meninges and in the choroid plexus, as well as by perivascu~

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Fig. 5. MUC 101.Double immunofluorescence microscopy within the operated facial nucleus, 10 days after axotomy. Comparison of MUC 101 immunoreactivity (A) and GFAP immunoreactivity (B)demonstrates a nonoverlapping staining pattern. Comparison of MUC 101 immunoreactivity (C) with the GSI-B, isolectin-binding on microglial cells (D) shows a congruent staining pattern. Arrows in C and D

J. GEHRMA" AND G.W. KREUTZBERG

indicate the cell nuclei of double-labeled, perineuronal microglial cells. In D blood capillaries are additionally stained due to the reactivity of GSI-B, with endothelia. This also applies to Figure 6B. Due to the surface staining of MUC 101, the outlines of microglial cells shown in A and C appear slightly blurred as compared to e.g., the GFAP staining. This also applies to Figures 6A. A, B, ~ 2 8 0C, ; D, x480.

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA

Fig. 6. MUC 102. Double immunofluorescence microscopy within normal cerebellar white matter. Comparison of MUC 102 immunoreactivity (A) and GSI-B, binding of microglial cells (B) demonstrates a congruent staining pattern (arrows indicate the positions of double-

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stained microglial cells) which is distinct from the staining pattern obtained in an adjacent section with the anti-MOG antibody (C), which recognises oligodendrocytes and CNS myelin fibres, and with GFAP (D),which stains astrocytes. A-D, X400.

Figure I

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA lar cells. In the peripheral organs, however, they exhibit quite different distribution patterns. CD4 antigen, detected by the W3/25 antibody, is mainly expressed by T-helper lymphocytes and macrophages. In contrast, MUC 101 did not recognise T lymphocytes, but mainly macrophages and cells of a dendritic morphology in the peripheral organs. A further distinction emerges from the Western blot data. Whereas the W3/25 antibody recognises a 56-kD glycoprotein (likely to be the homologue of the human OKT4 antigen) (Williams et al., '77; Barclay, '811, MUC 101 recognises two proteins of 116-and 95-kD molecular weight. The molecular weights of the MUC 101 target epitopes, however, showed a conspicuous similarity to leucocyte adhesion molecules, particularly the p-2 integrins. p-2 integrins are a/p heterodimers with three unique subunits of 180 kD,170 kD,and 150 kD and a common p subunit of 95 kD (for review see Springer, '90). The molecular weight of one of the proteins recognised by MUC 101 in Western blots was identical with that of the common p chain of p-2 integrins. The molecular weights, however, did not correlate with those of any of the a-chains. In the human CNS p-2 integrin expression has been demonstrated on resting microglial cells as well as activated microglial cells, in particular around the amyloid plaques in Alzheimer's disease (Rozemuller et al., '89; Akiyama and McGeer, '90). Therefore, MUC 101 might recognise a member of the leucocyte adhesion molecule family. As appropriate monoclonal antibodies are not yet available against the rat homologues of p-2 integrins, the similarity in molecular weights of the MUC 101 determinant and the p-2 integrins has to be studied further. The staining pattern of MUC 102 is distinct from that of MUC 101 both in the CNS and in the peripheral tissues. MUC 102 stained resting microglial cells more intensively than MUC 101. A more conspicuous difference emerges from the staining characteristics of cells in the vicinity of blood vessels. While MUC 101 stained perivascular cells, like the ED2-positive perivascular cell (Graeber et al., '89b), MUC 102 intensively labeled perivascular microglial cells with direct contact with the basal membrane (for comparison see Figs. 2D, 8C). In general, the MUC 102 staining pattern resembled to some extent the distribution of the CR3 complement receptor on microglial cells recognised by the OX-42 antibody, Both antibodies intensively stained resident and activated microglial cells, but did not label perivascular cells. Compared to the OX-42 immunoreactivity, MUC 102 staining was slightly more conspicuous on resting microglial cells in white matter while OX-42-

Fig. 7. MUC 102 immunoelectron microscopy. Facial nucleus 5 days after facial nerve transection. A Microglial cell processes exhibiting reaction product (arrows) cover the surface of a chromatolyticmotoneuron (N). The bottom left shows the cell body and the characteristic nucleus with dense heterochromatin of the corresponding microglial cell (MI. x 12,000. B: These microglial cell processes are highly convoluted, sometimes engulfing dendrites (arrows). X 16,000. C: Further example of a perineuronal microglial cell (MI, in close apposition to a chromatolytic motoneuron (N). The reaction product is present on the plasma membrane which surrounds the microglial cell body. Arrows indicate the position of reaction product. x 12,000. D Microglial cell process with reaction product on its surface. Some reaction product is also found either diffusely distributed within the cytoplasm or along the endoplasmic reticulum (arrows). ~28,000.E: Microglial cell process displaying reaction product on its surface. Reaction product is also found on the surface of some cytoplasmic vacuoles, indicated by arrows. x 13,000.

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positive microglial cells were more evenly distributed between white and grey matter (data not shown). Nevertheless, our immunocytochemical data do not exclude the possibility that MUC 102 stained a subpopulation of resident OX-42-positive microglial cells. In Western blots, however, the molecular weights of the OX-42 determinants and MUC 101 determinants are distinct. Whereas OX-42 precipitates three polypeptides of 160-, 103-, and 95-kD molecular weight (Robinson et al.,'86), MUC 102 recognised two proteins of 70 and 62 kD and a weaker staining polypeptide of approximately 60 kD. In the peripheral tissues the distributions of both MUC 101 and 102 immunoreactivity also differed from those of established macrophage-specific antibodies. In spleen, for example, MUC 102 staining resembled the staining and distribution pattern of ED1 (Dijkstra et al., '85), being distinct from ED1 in the other tissues investigated. Unlike several other anti-rat macrophage antibodies, e.g., OX-42, MUC 101and 102 did not cross-react with Kupffer cells, the liver macrophage. Many of the cells recognised by MUC 101 in thymus and lymph node and some cells stained by MUC 102 in lymph node had a dendritic appearance. It is therefore necessary to compare the immunostained cells with other dendritic cells described in lymphoid organs, i.e., the follicular dendritic cell and the interdigitating dendritic cell. None of the monoclonal antibodies reacted with the follicular dendritic cells (Jeurissen and Dijkstra, '88). This is not surprising since the follicular dendritic cells are thought to arise from precursor cells, the reticulum cells (Dijkstra et al., '84), different from the precursors of the mononuclear phagocyte system. Apart from their high constitutive expression of MHC class I1 molecules, however, no specific marker for dendritic cells is available (for review see Klinkert, '90).Whether the cells of dendritic morphology stained by MUC 101 and partially by MUC 102 belong to the system of interdigitating dendritic cells present in T-cell-dependent areas of lymphoid organs (Veerman, '74; Dijkstra, '82) or are subtypes of peripheral macrophages therefore remains open.

Antigen heterogeneity of microglial cells In this study, two monoclonal antibodies have been described which recognise different antigenic determinants on rat microglial cells. In the peripheral organs these antibodies cross-react with subpopulations of cells of the mononuclear phagocyte system. This finding supports the theory of a mesodermal origin of microglial cells originally proposed by del Rio-Hortega ('19, '32). Given their antigenic similarity with other mononuclear phagocytes, microglial cells should be considered as the intrinsic brain equivalent of a specialized tissue macrophage. The functional and antigenic heterogeneity of peripheral macrophages has been well documented (Foerster and Landy, '81; Sorg, '82; Springer and Unkeless, '84). Evidence is now also emerging for both the functional and the antigenic heterogeneity of microglial cells. In a recent study, a defined function, i.e., the selective phagocytosis of terminal parts of neurosecretory neurons, has been ascribed to microglial cells in the neurohypophysis of the rat (Pow et al., '89). These microglial cells are immunophenotypically distinct from other resident microglial cells, since they constitutively express the ED1 determinant (Perry and Gordon, '90) which is otherwise absent from resting microglial cells (Sminia et al., '87; Graeber et al, '89b; Perry and Gordon, '90; Graeber and Streit, '90a).

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Fig. 8. MUC 102 immunoelectron microscopy in normal rat CNS. A: A resident microglial cell in the normal facial nucleus is characterized by reaction product on its surface (arrows). The microglial cell (M) is found close to two adjoining dendrites (D).~ 1 5 , 0 0 0B: . A resident microglial cell (M) in the capsula interna exhibits reaction product (arrows) on its surface and is surrounded by myelinated axons (A) of

J. GEHRMANN AND G.W. KREUTZBERG

different size. ~14,000. C: A microglial cell process (M) in the normal facial nucleus which exhibits reaction product on its surface is found in ultimate contact with the basal membrane (long arrow) of an adjoining capillary. Sites of reaction product of the microglial cell process along the basal membrane are indicated by the open arrows. x 23,000.

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA TABLE 2. Cross-Reactivitiesof Monoclonal Antibodies With Different Peripheral Macrophage Populations and Cultured Macrophages'

Monoclonal antibodies

MUC 101'

MUC 1023

Tissue Thymus Medulla Corticomedullary junction Cortex Lymph node Cortex Paracortex Follicles Medulla Spleen White pulp Inner PALS Outer PALS Red pulp

Lung BALT Alveolar Liver Kupffer cells Kidney Skin Langerhans cell Dermal macrophages Cultured cells Brain macrophages Blood monocytes Bone marrow macrophages Peritoneal macrmhaees

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Lassmann et al., '91). In vivo the threshold for MHC class I1 antigen expression appears to be low for perivascular microglial cells. Upon treatment with even low concentrations of interferon-gamma, perivascular microglial cells rapidly express MHC class I1 antigens prior to any other cell type in the CNS, including other parenchymal microglial cells mass and Lassmann, '90). In the present study, MUC 102 immunoelectron microscopy confirms the presence of perivascular microglial cells proximal to the glia limitans. Perivascular microglial cells could therefore represent a morphologically, immunophenotypically, and functionally distinct subtype of resident microglial cell. In summary, the precise biological basis of microglial heterogeneity remains unclear. On the one hand, it cannot be excluded that true subpopulations exist in terms of different cell lineages as is known for the astrocyte/ oligodendrocyte lineage (Raff et al., '83; Wilkin et al., '90). Our data, however, suggest rather that microglial cells display a considerable antigen heterogeneity that could reflect a possible functional diversity of resident microglial cells in the mature CNS.

Microglial cells during the axonal reaction

There are only few cell types in the CNS which can be as easily and rapidly activated as microglial cells. We have studied microglial reactions under pathological conditions using the facial nerve paradigm. The facial nerve is transected near its exit at the stylomastoid foramen, leaving the blood-brain barrier untouched (for review see Streit The tissue distribution, staining pattern, and biochemi- et al., '88; Kreutzberg et al., '89a,b). Due to this remote cal data of MUC 101 and 102 are not congruent with those lesion, the microglial reaction in the facial nucleus can be of any of the macrophage-specific antigens described so far studied in the absence of extrinsic haematogenous cells. on rat microglial cells. The distributions of both MUC 101 During peripheral nerve regeneration several functions and 102 immunoreactivity thus suggest the existence of have been ascribed to activated microglial cells. Perineuheterogenous microglial cell populations in the rat CNS. ronal microglial cells detach afferent synaptic terminals They suggest at least that resident microglial cells exhibit from the surface of injured motoneurons, a process now antigen heterogeneity. Although the precise distinction generally referred to as "synaptic stripping" (Blinzinger between MUC 101-positive perivascular cells and MUC and Kreutzberg, '68). Furthermore, activated microglial 102-positiveperivascular microglial cells is difficult, based cells could play a pivotal role during regeneration by virtue on the present light and electron microscopy they seem to of their capacity to secrete cytokines, such as interleukin-1 represent two morphologically distinct populations of cells or colony-stimulating factors in response to CNS injury of the monocytelmacrophage cell lineage in the vicinity of (David et al., '90; Fagan and Gage, '90; Giulian and the blood-brain barrier. Due to differences in their morphol- Lachmann, '85; Giulian et al., '89). Due to their crucial role ogy, immunophenotype, and localisation within or outside in CNS repair, the characterisation of activated microglial the basal membrane, two distinct cell types can be distin- cells or subtypes by means of the two monoclonal antibodies guished: 1)extraparenchymal perivascular cells which rep- MUC 101 and 102 could help to elucidate the involvement resent a specializedtype of pericyte and are surrounded by a of microglial cells in these pathologies. The different time basal membrane; they are recognised by the MUC 101 and courses of changes in both MUC 101 and 102 immunoreacthe ED2 antibodies (for comparison see Fig. 2C; Graeber et tivity in the operated facial nucleus indeed indicate that the al., '89b); and 2) intraparenchymal perivascular microglial expression of MUC 101 and 102 determinants is differencells which are not enclosed within the basal membrane. tially regulated on activated microglial cells. Further work Processes of perivascular microglia form contacts with the is required to establish the possible functional role of MUC basal membrane on the abluminal side. Both at the light 101 and MUC 102 epitopes during the transition of microand electron microscopical level they are stained by MUC glial cells from the resting to the activated state. Such studies are also important for elucidating the 102 (for comparison see Figs. 2D, 8C). These perivascular microglial cells are thought to repre- involvement of microglial cells in a variety of neurological sent a functionally distinct subpopulation of resident micro- disorders, including Alzheimer's disease, Parkinson's disglial cells (Streit et al., '88; Graeber and Streit, '90b; Vass ease, and retroviral infections, e.g., the aquired immune and Lassmann, '90). This extends the original description deficiency syndrome (AIDS) (Rozemuller et al., '86, '89; of juxtavascular microglial cells by del Rio-Hortega ('32). McGeer et al., '87; Michaelis et al., '88; Itagaki et al., '89). Several studies have demonstrated that perivascular micro- As revealed by a recent immunocytochemical study on glial cells form a predominant source of intrinsic MHC class peritumoral biopsy tissue and human gliomas (Morimura et I1 antigen-expressing cells (Hickey and Kimura, '88; Vass al., 'go), human microglial cells could also acquire, espeand Lassmann, '90; Graeber and Streit, '9Ob; Hickey, '91; cially under pathological conditions, a functional and anti-

'Immunocytochemical labelling code: -, absent; ?, weak; +, well discernable; ++, strongly positive. Ahreviations: PALS: periarterial lymphatic sheath; BALT: bronchusassociated lymphoid tissue. 'IgG1 isotype, membrane stainingpattern. 31gGZaisotype, membrane stainingpattern.

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Fig. 9. MUC 101 immunoreactivity in peripheral organs. A, B: Thymus. Immunostained cells are mainly localized in the corticomedullary junction (arrows) M, medulla; C, cortex. x 120. B: Cells display a dendritic morphology with slender processes. The staining appears to be membranous. ~ 8 0 0 C, . D Cervical lymph node. Immunostained cells (arrows in C) are present both in the cortex and paracortex. D: These cells also show considerable ramification with slender processes; C, ~ 1 2 0 D, ; ~ 7 2 0E . : Spleen. Cells are stained mainly in the outer

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periarterial lymphatic sheath (PALS) (arrows). x 120. F Lung. A few macrophages (arrow)either at the periphery of the bronchus-associated lymphoid tissue (BALT) or situated intraalveolarly are positive for MUC 101 B, bronchus. ~ 8 0 G . Liver. In the liver parenchyma no specific staining is observed. The relatively high staining background is due to the high endogenous biotin content of liver. This also applies to Figure 10G. X120. H: Skin. A few cells, presumably macrophages (arrow),are stained. ~ 9 0 .

MONOCLONAL ANTIBODIES AGAINST RAT MICROGLIA

Fig. 10. MUC 102 immunoreactivity in peripheral organs. A, B: Thymus. M, medulla; C, cortex. Immunostained cells are mostly localized in the corticomedullary junction but also in the cortex. A, x 120; B, ~ 8 8 0C, , D.Cervical lymph node. Labeled cells (arrows) are localised in the cortex and paracortex. C, x 120. D: These cells have a dendritic morphology with crenellated processes (arrows). x 740. E:

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Spleen. Most of the macrophages in the outer PALS (arrows) are intensively stained. ~ 1 2 0 F: . Lung. Mostly cells in close association with a small bronchus (B) are stained. ~ 8 0G: . Liver parenchyma is free of MUC 102 staining. x 120. H: Skin. Some cells, presumably macrophages (arrows), are stained. X 90.

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J. G E H R M A " AND G.W. KREUTZBERG could exist among resting microglia. Whether this heterogeneity could also reflect the existence of true subpopulations of microglia remains open. Microglial cells are thought to represent the intrinsic immune defense system of the CNS (for review see Streit et al., '88; Graeber and Streit, 'goal. They are rapidly activated in response even to subtle pathological stimuli. Their rapid involvement in different pathologies underlines the crucial role of this cell type in both immunological and nonimmunological processes of the CNS. The monoclonal antibodies, designated MUC 101 and MUC 102, which recognise defined antigens on subsets of microglial cells, therefore supplement the repertoire of tools now available for answering questions of microglial heterogeneity and activation particular under pathological conditions. These monoclonal antibodies should thus contribute to our understanding of the nature of microglial cells and their role in normal and pathological conditions in the central nervous system.

ACKNOWLEDGMENTS

Fig. 11. Results of Western blot analysis of MUC 101 (A) and MUC 102 (B) epitopes. A Samples of operated facial nucleus 10 days after axotomy were immunoprecipitated using MUC 101-coupled Sepharose beads and submitted to 7.5% SDS-PAGE under reducing conditions. Proteins were then transferred to an Immobilon membrane and reacted with MUC 101 (lanes 1-51 or stained with amido black (lanes 7-9). The samples were: crude homogenate fraction (lanes 1 and 71, supernatant fraction (lanes2 and 81, crude membrane fraction (lanes 3 and 9). Arrows indicate the positions of the immunoprecipitated 116 and 95 kD polypeptides. Lanes 4-6 were controls. Lane 4 Immunoprecipitation from a crude homogenate with an irrelevant IgGl antibody coupled to the Sepharose beads; the immunoblot was then treated with MUC 101. Lane 5 Immunoblot with the immunobeads alone showing detection of the heaw and light immunodobulin chains bv the secondary goat anti-mouse"antibod;r. Lane 6identical to lane"5 except for omission of the secondary antibody which abolished staining of the immunoglobulin chains released from the immunobeads. B: Samples obtained from either microglid cultures (lanes 1-3) or operated facial nucleus (lanes 4-71 were submitted to 12.5% SDS-PAGE under reducing conditions. Proteins were then transferred to an Immobilon membrane and processed with MUC 102. Arrows indicate the positions of the 62 and 70 kD polypeptides. Samples were: Lanes 1-3: Cultured rat microglia. Lane 1: Crude homogenate. Lane 2: Supernatant fraction. Lane 3 Crude membrane fraction. Lanes 4-7: Operated facial nuclei. Lane 4: Crude homogenate. Lane 5: Supernatant fraction. Lane 6 Crude membrane fraction. Lane 7: Control, i.e., immunoblot of crude facial nuclei homogenate processed with an irrelevant IgG2a antibody as mentioned in Materials and Methods.

genic status distinct from other cells of the monocytemacrophage cell lineage. In summary, these two new monoclonal antibodies, MUC 101 and MUC 102, detect resting and activated microglial cells in the rat CNS. Furthermore, distinct types of cells in the vicinity of blood vessels are labeled, i.e., perivascular cells as opposed to perivascular microglia. In the peripheral organs, both antibodies cross-react with different populations of the mononuclear phagocyte system, emphasizing the antigenic similarity between microglial cells and other cells of the monocyte-macrophage cell lineage. Our immunocytochemical data suggest that antigen heterogeneity

We appreciate the technical assistance of Bettina Liebstein, Petra Grammel, Dietmute Buringer, and Irmtraud Milojevic. We wish to thank Karin Briickner and Susanne Luh for photography as well as Prof. Renate RenkawitzPohl (MPI for Biochemistry, Martinsried) for help with phase contrast microscopy. We thank Dr. Ernst Ungewickell (MPI for Biochemistry, Martinsried) for the gift of PAI-mouse myeloma cells and Uta Eichelsbacher, Roland Gorlich, Karin Liins, Stephen Morris, Stephan Schroder, and Robert Lindner (MPI for Biochemistry, Martinsried) for their advice on hybridoma cultures and antibody purification. We thank Dr. Christopher Linington (Department of Neuroimmunology, MPI for Psychiatry) for the gift of anti-MOG antibody and Dr. Martin Reddington, Dr. Carola Haas, and Dr. Siegfried W. Schoen for reading the manuscript.

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