Journal of Neuroimmunology, 30 (1990) 81-93 Elsevier
81
JNI 01008
Brain microglia constitutively express fl-2 integrins H. Aldyama and P.L. McGeer Department of Psychiatry, Kinsmen Laboratory of Neurological Research, Faculty of Medicine, University of British Columbia, Vancouver, BC V6T 1 W5, Canada (Received 1 March 1990) (Revised, received 1 June 1990) (Accepted 4 June 1990)
Key words: Microglia; 13-2 Integrin; Alzheimer's disease; Complement receptor; Leukocyte function-associated antigen
Summary Localization of fl-2 integrins in normal and Alzheimer disease temporal cortex was studied immunohistochemically. Resting microglia were found to express constitutively C D l l a (LFA-1), C D l l b (Mac-l, CR3), C D l l c (P150, 95; CR4), and CD18 (/3-2). They were also found to express constitutively leukocyte common antigen and the immunoglobulin receptor Fc~,RI. The intensity of expression of each of these antigens was enhanced on reactive microglia in Alzheimer disease tissue. HLA-DR was detected on only a few microglia in control tissue, but was intensely expressed on large numbers of reactive microglia in Alzheimer tissue. These data are consistent with a leukocyte origin and a phagocytic role for microglia. They provide further evidence of an inflammatory response of brain tissue in Alzheimer disease. The microglia were found to make up 9-12% of the total glial population in gray matter and 7.5-9% in white matter.
Introduction The integrins are a superfamily of cell adhesion molecules which promote cell-cell and cell-matrix interactions (Hynes, 1987). They are recognized, along with the immunoglobulin superfamily, as proteins of high evolutionary significance. Both families play key roles in developmental and immune function by providing chemotactic guidance of cells to their correct location and promoting their adherence to target struc-
Address for correspondence: Dr. Patrick L. McGeer, University of British Columbia, Kinsmen Laboratory of Neurological Research, Department of Psychiatry, 2255 Wesbrook Mall, Vancouver, BC V6T 1W5, Canada.
tures. Integrins are membrane spanning glycoproteins in which the intracellular domain is customarily linked to the cytoskeletal apparatus, while the extracellular domain attaches to receptors on membranes or matrix proteins. Each integrin is a heterodimer with a unique a subunit and one of three/3 subunits. The common /3 subunits divide the integrins into distinct families (/3-1, /3-2, /3-3). The fl-1 family, also called very late antigens (VLAs), is characterized by the M r 110,000 /3-1 subunit (Hemler et al., 1985). Their primary role in brain appears to be in association with capillaries, where some members promote adhesion of endothelial cells to basement membrane matrix proteins (McGeer et al., 1990). The/3-2 family is characterized by the M r 95,000 /3-2 suhunit, designated as CD18. Three a sub-
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82 units of M r 180,000, 170,000 and 150,000 are known. They have been designated as C D l l a (LFA-1), C D l l b (Mac-l, CR3) and C D l l c (P150,95; CR4) (Kishimoto et al., 1987; Arnaout et al., 1988; Corbi et al., 1988a, b; I_arson et al., 1988). The principal ligand of C D l l a (LFA-1) is thought to be intercellular adhesion molecule 1 (ICAM-1) (Martin and Springer, 1987). The principal ligands for C D l l b and C D l l c are thought to be complement components related to C3bi. They have been designated CR3 (Micklem and Sire, 1985) and CR4 (Myones et al., 1988) respectively. The function of B-2 integrins appears to be restricted to the immune system. They have been found only on leukocytes (Kishimoto et al., 1989), where they promote migration, adhesion and phagocytosis (Sanchez-Madrid et al., 1983; Anderson et al., 1987; Keizer and Te Velde, 1987; Kishimoto et al., 1987, 1989; Arnaout et al., 1988). They have therefore been referred to as leukocyte integrins, leukocyte adhesion proteins, and LeuCAMs (Kishimoto et al., 1989). A congenital abnormality in their production, leukocyte adhesion deficiency disease (LAD), is associated with vulnerability to repeated infections (Anderson et al., 1987). The role of /~-2 integrins in brain function is not yet clear, They have been reported to occur on microglial cells in the corona of Alzheimer disease senile plaques (Rozemuller et al., 1989). CD11b has been found on mouse (Robinson et al., 1986) and rat (Graeber et al., 1988) microglia, while C D l l c has been found on human microglia (Hogg et al., 1986). In view of the central role that fl-2 integrins are thought to play in immune function, an understanding of their expression in normal and pathological brain tissue should provide important information on how the immune system in brain operates. We describe here immunohistochemical staining of normal and Alzheimer brain tissue with a panel of antibodies that includes all members of the /~-2 integrin family. We compare the staining with that obtained using antibodies to three different leukocyte markers: leukocyte cornmon antigen (LCA), the immunoglobulin receptor Fc~,RI, and the major histocompatibility complex (MHC) class II glycoprotein HLA-DR.
LCA represents a family of major membrane glycoproteins found on all hematopoietic cells except erythrocytes and their progenitors (Thomas, 1989). F c y R I recognizes the immunoglobulin Fc chain. The receptor is a glycoprotein expressed on the surface of mononuclear phagocytes. It is highly localized to cells of this lineage (Fanger et al., 1989). H L A - D R is a group II MHC glycoprotein particularly associated with antigen presentation to T - h e l p e r / i n d u c e r lymphocytes (Stites et al., 1987),
Materials and methods Fifteen autopsied human brains obtained within 2-12 h of death were employed in the study: six without neurological disorder (average age 69, range 54-82) and nine with Alzheimer disease (average age 77, range 69-85). The control cases all died from acute cardiovascular disease; five from myocardial infarcts and one from a ruptured aneurysm. Small blocks of brain tissue were dissected from the temporal lobe. Brain blocks were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 2 days and transferred to a maintenance solution of 15% sucrose in 0.01 M phosphatebuffered saline (PBS) pH 7.4. Sections were cut on a freezing microtome at 30 t~m thickness, collected in the maintenance solution, and stored until stained. Some unfixed brain blocks were immediately frozen with dry ice and sectioned in a cryostat. Unfixed cryocut sections were mounted on glass slides, air-dried and fixed in acetone for 10 rain. The primary monoclonal antibodies employed in this study, as well as the source, type and dilution are shown in Table 1. MHM24 and SPVL7 were used as the source for antibodies against C D l l a , 2LPM19C and Bear-1 for C D l l b , Leu-M5 for C D l l c , 60.3 and MHM23 for the common t3-2 chain, HB104 for HLA-DR, 2 B l l + P D 7 / 2 6 for leukocyte common antigen (LCA, CD45), and 32.2 for the immunoglobulin G receptor Fc'fRI (CD64). The antibody to glial fibrillary acidic protein (GFAP, Dako), was a polyclonal rabbit antiserum.
83 TABLE 1 MONOCLONAL ANTIBODIES USED IN THIS STUDY Antigen
Antibody (Reference)
CD11a (LFA-1)
MHM24 Dakopatts (Hildreth et al., 1983) SPV-L7 a Sanbio (Spits et al., 1983) 2LPM19Ca Dakopatts Bear-1 Sanbio (Todd et al., 1982) Leu-M5 Becton-Dickinson (Schwarting et al., 1985) 60.3 a Oncogene (Beatty et al., 1983) MHM23 a Dakopatts (Hfldreth et al., 1983) HB104 ATCC (Charron and McDevitt, 1979) 2Bll + PD7/26 Dakopatts (Wamke et al., 1983) 32.2 Medarex (Anderson et al., 1986)
CDllb (Mac-l, C R 3 ) CDllc (p150,95, CR4) CD18 (fl-2 subunit)
HLA-DR CD45 (LCA) CD64 (Fc~,RI)
Source
Form
Dilution
Supernatant
1 : 100
Supernatant
1 : 10
Supcrnatant Supernatant
1 : 30 1 : 100
purified IgG (10 #g/ml)
1 : 10
Ascites
1 : 300
Supematant
1 : 30
Supernatant
1 : 1000
Supernatant
1 : 100
Purified IgG (1 mg/rrd)
1 : 1000
a Worked only in fresh-frozen, acetone-fixed tissue.
All sections were pretreated with 0.2% H 2 0 2 solution for 30 min to eliminate endogenous peroxidase. For immunostaining in the free-floating procedure, sections were incubated with primary antibody for 72 h in the cold. The primary antibody was diluted in PBS containing 0.3% Triton X-100 (PBS-Tx) and 10% normal horse serum to the concentration shown in Table 1. G F A P antiserum was used at a dilution of 1 : 1 0 , 0 0 0 i n PBSTx containing 10% normal goat serum. After incubation with primary antibody, sections were next treated with biotinylated secondary antibody (Vector Lab, diluted 1:1000) for 2 h at room temperature followed by incubation in the avidinbiotinylated horseradish peroxidase (HRP) cornplex (ABC Elite, Vector Lab., Burlingame, CA, U.S.A.) for 1 h at room temperature. Peroxidase labeling was detected by incubation with a solution containing 0.01% 3,3'-diaminobenzidine (DAB, Sigma), 0.6% nickel a m m o n i u m sulfate, 0.05 M imidazole and 0.00015% H202. A darkpurple reaction product appeared after about 1545 min, at which time the reaction was terminated by transferring the section to PBS. Sections were mounted on glass slides, dehydrated with graded
alcohols, and mounted with Entellan (Merck) with or without prior staining with neutral red. For double immunostaining, sections were treated for 30 min with 0.5% H 2 0 2 solution in PBS after the DAB reaction of the first cycle. The second immunohistochemical cycle was carried out similarly to the first cycle except that nickel ammonium sulfate was eliminated from the DAB solution, yielding a brown precipitate in the second cycle. The following combinations were investigated: leukocyte c o m m o n antigen (LCA) with M H M 2 4 ( C D l l a ) , Bear-1 ( C D l l b ) , Leu-M5 ( C D l l c ) , HB104 ( H L A - D R ) and 32.2 (FcyRI); H L A - D R with Leu-M5; and G F A P with MHM24, Bear-l, Leu-M5, LCA, and HB104. For staining of fresh-frozen acetone-fixed tissue, primary antibodies were diluted in PBS without Triton X-100. Following the pretreatment with 0.2% H202, primary antibody incubation was done overnight in a moist chamber at room ternperature. The rest of the procedure was the same as for free-floating sections except that they were carried out on the surface of glass slides. Three types of controls were used: elimination of the primary antiserum; substitution of rabbit
84
serum hyperimmunized with tobacco mosaic virus; and use of a mouse monoclonal antibody indifferent to brain antigens. N o positive staining of either normal or Alzheimer brain was obtained in control. To quantitate the ratio of microglia to total glial cells in the temporal cortex of control subjects, serial sections were cut from the middle temporal gyrus of the six control cases. Every fifth section was stained with Leu-M5 and an adjacent section stained with cresyl violet. Sections stained with Leu-M5 were further counterstained with neutral red. The numbers of nuclei of either LeuM5-positive cells (in Leu-M5-stained tissue) or total glial cells (in cresyl violet-stained tissue) were counted in a column 0.5 m m in width covering the whole cortical thickness as well as in an 0.5 m m square of subjacent white matter (at least 1 m m distant from the boundary of the gray and white), Approximately the same area was chosen from each section. The distinction between astroglia and small neurons was uncertain for a few of the cells (less than 5%) but these numbers were too small to have a significant influence on the final result. The ratio of Leu-M5-positive cells to total glial cells was calculated for each case by averaging the values of three sections. Results All members of the fl-2 integrin family were detected on microglial cells in both gray and white matter in normal and Alzheimer tissue. Positive staining was obtained only in fresh-frozen acetone-fixed tissue with monoclonal antibodies SPV-L7 ( C D l l a ) , 2LPM19C ( C D l l b ) , 60.3 and M H M 2 3 (CD18). On the other hand, strong positive staining with good morphological detail was obtained on paraformaldehyde-fixed tissue with
monoclonal antibodies M H M 2 4 ( C D l l a ) , Bear-1 ( C D l l b ) , and Leu-M5 ( C D l l c ) . These antibodies also stained leukocytes in the matrix of Alzheimer brain tissue which were present in small numbers. They did not stain neurons, astrocytes, oligodendroglia, or capillary endothelial cells. Similar staining was obtained with the antibody to LCA. The antibody to F c y R I strongly stained all microglia, while the antibody to H L A - D R stained only reactive microglia. Typical results of double immunostaining are shown in the color photomicrographs of Fig. 1. The full thickness of normal temporal cortex is illustrated in Fig. 1A, which is immunostained for C D l l c (brown) and H L A - D R (purple). The large complement of C D l l c - p o s i t i v e resting microglia is easily seen, with rare HLA-DR-positive microglia also appearing. This is in considerable contrast to the temporal cortex of Alzheimer disease, which is shown in Fig. lB. Many purple-colored HLADR-positive reactive microglia appear in both gray and white matter. These are often assembled in agglomerates. The reactive microglia are also strongly positive for C D l l c but the double staining of cells is not easily discerned under low power. Fig. 1C and D illustrates double immunostaining at higher power for C D l l c (purple) and G F A P (brown) in gray ( C ) and white ( D ) matter of Alzheimer temporal cortex. Here the C D l l c - p o s i tive microglia can be distinguished from the GFAP-positive astrocytes both by morphology and color. The microglia themselves appear in both resting and reactive forms. Resting microglia show small, round to somewhat elongated cell bodies and thin ramified processes, while reactive microglia show an enlarged cyctoplasm, as well as enlarged but retracted processes. These morphologies correspond to those originally described by
Fig. 1. Double immunostaining of normal (A) and Alzheimer (B, C and D) temporal cortex for CDllc (Leu-M5) and either HLA-DR (A and B) or GFAP (C and D). A and B show the full width of cortex with the pial surface at the top and subjacent white matter at the bottom. CDllc is stained brown and HLA-DR purple. Note that the control cortex has only rare HLA-DR-positire microglia while the Alzheimer cortex has very many appearing singly or as agglomerates. Higher power views of Alzheimer gray (C) and white (D) matter are shown in which GFAP is stained brown and CDllc purple. The GFAP-positive astrocytes can easily be distinguished from the CDllc-positive rnicroglia. Resting microglia stain weakly compared with the strongly staining reactive forms, some of which are aggregated. A and B are at the same magnification, bar = 250 #m. C and D are at the same magnification, bar = 100 #m.
85
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86 del Rio Hortega (1919) and Penfield (1925) using silver carbonate stains, Fig. 2 illustrates the staining of gray and white matter in control brain tissue for the/3-2 integrins C D l l a (MHM24, Fig. 2A and B), C D l l b (Bear-l, Fig. 2C and D), C D l l c (Leu-M5, Fig. 2 E and F ) and CD18 (60.3, Fig. 2G and H). In gray matter, microglia were only weakly positive for C D l l a (Fig. 2A), while in white matter they were moderately positive (Fig. 2B). They were also moderately positive for C D l l b (Fig. 2C and D) and C D l l c (Fig. 2 E and F ) in both gray and white matter, but the two monoclonal antibodies against the common/3-2 subunit stained only fresh-frozen acetone-fixed tissue. Microglial cells in white matter (Fig. 2 H ) were labeled more intensely than those in gray matter (Fig. 2G) by the/3-2 antibodies. Fig. 3 shows staining of sections nearby to those in Fig. 2 using different leucocyte markers, Fig. 3A and B shows staining of control gray and white matter for the FcTRI receptor. Fig. 3C and D shows staining for leukocyte common antigen (LCA) and Fig. 3E and F for HLA-DR. The results of control gray and white matter staining for LCA and FcTRI were identical to those for/3-2 integrin staining. Dense labeling of microglia was observed (Fig. 3A-D). The results of staining for H L A - D R were very different. Only rare positive cells were observed in gray matter (Figs. 1A and 3E) and a few in white matter (Figs. 1A and 3F). Fig. 4 shows staining of Alzheimer tissue for /3-2 integrins. Microglia in Alzheimer tissue were stained more intensely than in control tissue by each of the/3-2 integrin antibodies. A significant proportion of the positively stained cells had morphologies comparable to classical reactive microglia (Rio Hortega, 1919; Penfield, 1925). The staining was particularly intense in microglial aggregates which were closely associated with senile plaques (Itagaki et al., 1989; McGeer et al., 1989a).
Fig. 4A and B shows such dramatically enhanced expression of C D l l a by reactive microglia in Alzheimer gray and white matter. Heavily stained microglial aggregateg (arrow) were revealed to be localized in the center of a senile plaque when the slide was observed by fluorescence microscopy following thioflavin S counterstaining (data not shown). The expression of C D l l b and C D l l c was also enhanced in Alzheimer tissue compared with control but less prominently than that of C D l l a (Fig. 4C-F). Enhancement of CD18 expression seemed to be intermediate between C D l l a and C D l l b / C D l l c (Fig. 4G and H). Reactive microglia in Alzheimer gray and white matter also stained more intensely than resting microglia for FcTRI and LCA (Fig. 5A-D). The most dramatic change between control and Alzheimer tissue was seen with staining for HLA-DR. Instead of the occasional positive cell seen in control tissue, large numbers of microglia, mostly of reactive morphology, were seen in both gray and white matter (Figs. 1B and 5E and F). In order to determine more exactly the cell population expressing various /3-2 integrins, a complete series of double-immunostaining experiments was carried out for LCA in combination with the a chain of each /3-2 integrin and the FcTRI receptor. When LCA was stained purple in the first cycle, and C D l l a (MHM24), C D l l b (Bear-l), C D l l c ( L e u - M 5 ) o r FcTRI (32.2)brown in the second cycle, all microglia in control and Alzheimer tissue were stained either purple or purple-brown. Since the stronger purple color of the first cycle can obscure the weaker brown color of the second cycle, the experiments were repeated with the order of staining reversed. When the/3-2 integrins or F C T R I were stained purple in the first cycle and LCA was stained brown in the second cycle, all microglia similarly stained purple or purple-brown. Thus, no microglia were detected in Alzheimer or control tissue that stained singly for LCA, FcTRI or any of the/3-2 integrins.
Fig. 2. Staining of normal temporal cortical gray (A, C, E and G) and white (B, D, F and H) matter for/3-2 integrins. A and B, staining for CDlla (MHM24). C and D, staining for CDllb (Bear-l). E and F, staining for CDllc (Leu-M5). G and H, staining for CD18 (60.3). See Materials and Methods for experimental details. Each antibody stains similar numbers of resting microglia. Note, however,the weaker staining for CDlla in gray matter, and the relativelypoor morphologyobtained for CD18 staining in the fresh-frozenacetone-fixedmaterial. Bar in H = 50 ~tm.All photomicrographsin Figs. 2-5 are at the same magnification.
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Fig. 3. Staining of normal temporal cortical gray (A, C and E) and white (B, D and F ) matter with various leukocyte markers. A and B, staining of FcyR1 receptors with antibody 32.2. C and D, staining for LCA with the 2B11 + PD7/26 antibody. Note that a similar number of microglia with resting morphology are stained in these photomicrographs compared with those in Fig, 1. E and F, staining for HLA-DR. Notice that very few microglia are stained positively.
Very different results were obtained with double immunostaining for LCA and H L A - D R . When H L A - D R was stained purple in the first cycle and LCA brown in the second, many microglia were observed that were only stained brown. Such H L A - D R - / L C A + microglia had resting mor-
phology. They were particularly numerous in control tissue, but in Alzheimer tissue many microglia were reactive and H L A - D R positive. A complete set of double-immunostaining experiments was also conducted for G F A P and the c~ chain of each /~-2 integrin as well as LCA and
89 FcTRI to determine if astrocytes express integrins or other leukocyte markers. Whether GFAP was used in the first or second cycle, no GFAP-positive astrocytes were found to stain for C D l l c , LCA or HLA-DR. The results of microglial cell counts in the six control cases using C D l l c as the microglial marker showed the microglial population to be 10.6 ___0.9% (range 8.9-12) of the total in gray matter and 8.2 + 0.5% (range 7.6-8.9) in white matter. These data are in keeping with previous reports (Peters et al., 1976). The ratio in gray matter was fairly constant through the six layers of the cerebral cortex,
Discussion These data demonstrate the constitutive expression of/3-2 integrins on microglia in the resting as well as the reactive state. They confirm and extend previous reports on the occurrence of/3-2 integrins on these cells (Hogg et al., 1986; Robinson et al., 1986; Graeber et al., 1988; Rozemuller et al., 1989). They also confirm previous data on the constitutive expression of LCA and FcTRI on microglia (Itagaki et al., 1988; McGeer et al., 1989a). In the periphery, the FcTRI is almost exclusively restricted to mononuclear phagocytes (Fanger et al., 1989). Normal peripheral blood monocytes have a range of 15,000-40,000 sites per cell but, upon activation by inducers such as 7-interferon, these receptor numbers can be upregulated 5- to 10-fold (Fanger et al., 1989). Comparable data are not available for the numbers of/3-2 integrin molecules on various leukocytes cell lines, but they are known to be highly expressed on monocytes and granulocytes. The existence of these molecules on resting microglia, and their upregulation when microglia become reactive and undergo morphological change are powerful evidence supporting long-held theories that microglia are the brain representatives of the monocyte phagocytic system (Van Furth, 1982). Immunoglobulins and complement proteins are the key molecules which mark foreign or unwanted materials for disposal by phagocytes, Prominent staining of receptors for these proteins, even on resting microglia, is confirmation of much
previous evidence that phagocytosis is the primary function of these cells. The integrin molecules CD11b and C D l l c are receptors for C3bi (Micklem and Sim, 1985) and C3dg (Myones et al., 1988). These are products of complement proteins that have been chemically bound to tissue (McGeer et al., 1989). F c y R I is the receptor molecule for the immunoglobulin Fc chain. The function of C D l l a on microglia is less certain, but there may be important interactions with its ligand ICAM-1 acting as a guiding agent (Martin and Springer, 1987). Interaction between LFA-1 and ICAM-1 is considered fundamental to many immunologic reactions (Makgoba et al., 1989). In Alzheimer disease, it is known that plaques, tangles and dystrophic neurites are strongly stained by antibodies to complement proteins (Eikeleboom et al., 1989; McGeer et al., 1989a, b). In addition, it has been reported that /3-2 integrins are expressed by microglial cells in the corona of Alzheimer senile plaques (Rozernuller et al., 1989). Thus, the complement receptors represented by /3-2 integrins presumably assist microglial cells to adhere to these complement proteins in Alzheimer disease. The only known condition in which there is over-expression of /3-2 integrins is Down's syndrome (Taylor et al., 1988). The/3-2 subunit occurs on chromosome 21 in the obligatory region for this condition. Down's cases exhibit over-expression of LFA-1 and higher adhesiveness of lymphocytes due to the/3 chain gene dosage effect. It is not known whether this bears any relation to the increased susceptibility to infection of Down's syndrome patients or of their tendency to develop Alzheimer-type brain amyloid deposits (Glenner and Wong, 1984). Despite the long-standing idea of immunological privilege of brain, recent investigations have demonstrated presence of the relevant cellular elements of an immune response in brain tissue in several neurological diseases (Itagaki et al., 1988; McGeer et al., 1988, 1989a, b; Rogers et al., 1988; Eikelenboom et al., 1989; Rozemuller et al., 1989). Constitutive expression of /3-2 integrins appears further to characterize the resident microglial cells as the key component of the immune system in brain.
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Fig. 5. Staining of Alzheimer temporal cortical gray (A, C and E) and white (B, D and F ) matter for various leukocyte markers. A and B, staining for Fc~,RI (32.2). C and D, staining for LCA. G and H, staining for HLA-DR. Again, notice the similarity in microglial staining to the fl-2 integrin staining shown in Fig. 3. In A, C and E microglial aggregates (arrows) are visible which stain intensely. Similarly, in B, D and F most microglia stain intensely and are of reactive morphology. Also, notice that in Alzheimer disease most microglia are HLA-DR positive while very few are POsitive in control tissue.
Fig. 4. Immunohistochemical staining of Alzheimer temporal cortical gray (A, C, E and G) and white (B, D, F and H)mat.ter for fl-2 integrins. A and B, staining for C D l l a (MHM24). C and D, staining 'for C D l l b (Bear-l). E and F, staining for C D l l c (Leu-M5). G and H, staining for CD18 (60.3). Each fl-2 integrin stains comparable cells. Notice the intense staining of microglial aggregates in the gray matter photomicrographs (A, C, E and G, arrows). Some more lightly stained microglia, not in aggregates, are visible in the bacl~ground. In white matter, large microglial aggregates are not visible, but intensely staining microglia with reactive morphology form the majority of positively staining cells.
92
Acknowledgements The authors wish to thank Ms. Joane Sunahara
for technical assistance. This research was supported by grants from the Medical Research Council of Canada, the American Health Assistance Foundation, the Alzheimer Society of B.C., and private donors from British Columbia. References Anderson, D.C. and Springer, T.A. (1987) Leukocyte adhesion deficiency: an inherited defect in the Mac-l, LFA-1, and p150,95 glycoproteins. Annu. Rev. Med. 38, 175-194. Anderson, C.L., Guyre, P.M., Whitin, J.C., Ryan, D.H., Looney, R.J. and Fanger, M.L. (1986) Monoclonal antibodies to' the Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line. J. Biol. Chem. 261, 12856-12864. Arnaout, M.A., Gupta, S.K., Pierce, M.W, and Tenen, D.G. (1988) Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mol (complement receptor type 3). J. Cell Biol. 106, 2153-2158. Beatty, P.G., Ledbetter, J.A., Martin, P.J., Price, T.H. and Hansen, J.A. (1983) Definition of a common leukocyte cell-surface antigen (LP95-150) associated with diverse cell-mediated immune functions. J. Immunol. 131, 29132918. Charro n, D.J. and McDevitt, H.O. (1979) Analysis of HLA-D region-associated molecules with monoclonal antibody, Proc. Natl. Acad. Sci. U.S.A. 76, 6567-6571. del Rio Hortega, P. (1919) E1 'tercer elemento' de los centros nerviosos. Poder fagocitario y movilidad de la microglia. Bol. Soc. Esp. Biol. Ano ix, 154-166. Eikelenboom, P., Hack, C,E., Rozemuller, J.M. and Stare, F.C. (1989) Complement activation in amyloid plaques in Alzheimer's dementia. Virchows Arch. Abt. B Cell Pathol. 56, 259-262. Fanger, M.W., Shen, L., Graziano, R.F. and Guyre, P.M. (1989) Cytotoxicity mediated by human Fc receptors for IgG. Immunol. Today 10, 92-99. Glenner, G.G. and Wong, C.W. (1984) Alzheimer's disease and Down's syndrome. Sharing of a unique cerebrovascular amyloid fibril protein. Biochem. Biophys. Res. Commun. 122, 1131-1135. Graeber, M.B., Streit, W.J. and Kreutzberg, G.W. (1988) Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells. J. Neurosci. Res. 21, 18-24. Hemler, M.E., Huang, C. and Schwarz, L. (1985) The VLA protein family. J. Biol. Chem. 262, 3300-3309. Hildreth, J.E.K., Gotch, F.M., Hildreth, P.D. and McMichael, A.J. (1983) A human lymphocyte-associated antigen involved in cell-mediated lympholysis. Eur. J. Immunol. 13, 202-208.
Hogg, N., Takacs, L., Palmer, D.G., Selvendran, Y. and Allen, C. (1986) The P150,95 molecule is a marker of human mononuclear phagocytes: comparison with expression of class II molecules. Eur. J. Immunol. 16, 240-248. Hynes, R.O. (1987) Integrins: a family of cell surface receptors. Cell 48, 549-554. Itagaki, S., McGeer, P.L. and Akiyama, H. (1988) Presence of T-cytotoxic suppressor and leukocyte common antigen positive cells in Alzheimer disease brain tissue. Neurosci. Lett. 91, 259-264. Itagaki, S., McGeer, P.L,, Akiyama, H., Zhu, S. and Selkoe, D. (1989) Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease. J. Neuroimmunol. 24, 173182. Keizer, G.D., Te Velde, A.J., Schwarting, R., Figdor, C.G. and De Vries, J.E. (1987) Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of human monocytes. Eur. J. Immunol. 17, 1317-1322. Kishimoto, T.K., O'Connor, K., Lee, A., Roberts, T.M. and Springer, T.A. (1987) Cloning of the beta subunit of the leukocyte adhesion proteins: homology to an extracellular matrix receptor defines a novel supergene family. Cell 48, 681-690. Larson, R.S., Corbi, A.L., Berman, L. and Springer, T. (1988) Primary structure of the leukocyte function-association molecule-1 a-subunit: an integrin with an embedded domain defining a protein superfamily. J. Cell Biol. 108, 703-712. Makgoba, M.W., Sanders, M.E. and Shaw, S. (1989) The CD2-LFA-3 and LFA-I-ICAM pathways: relevance to Tcell recognition. Immunol. Today 10, 417 422. McGeer, P.L., Itagaki, S. and McGeer, E.G. (1988) Expression of the histocompatibility glycoprotein HLA-DR in neurological disease. Acta Neuropathol. 76, 550-557. McGeer, P.L., Akiyama, H., Itagaki, S. and McGeer, E.G. (1989a) Immune system response in Alzheimer disease. Can. J. Neurol. Sci. 16, 516-527. McGeer, P.L., Akiyama, H., ltagaki, S. and McGeer, E.G. (1989b) Activation of the classical complement pathway in brain tissue of Alzheimer patients. Neurosci. Lett. 107, 341-346. McGeer, P.L., Zhu, S.G. and Dedhar, S. (1990) lmmunostaining of human brain capillaries by antibodies to very late antigens. J. Neuroimmunol. 26, 213-218. Marlin, S.D, and Springer, T.A. (1987) Purified intercellular adhesion molecule-1 (ICAM-1)is a ligand for lymphocyte function-associated antigen-1 (LFA-1). Cell 51, 813-819. Matsumoto, Y., Watabe, K. and Ikuta, F. (1985) Immunohistochemical study on neuroglia identified by the monoclonal antibody against a macrophage differentiation antigen (Mac-l). J. Neuroimmunol. 9, 379-389. Micklem, K.J. and Sim, R.B. (1985) Isolation of complementfragment-iC3b-binding proteins by affinity chromatography. Biochem. J. 231,233-236. Myones, B.L., Dalzell, J.G., Hogg, N, and Ross, G.D. (1988) Neutrophil and monocyte cell surface p150,95 has iC3b-receptor (CR4) activity resembling CR 3. J. Clin. Invest. 82, 640-651.
93 Penfield, W. (1925) Microglia and the process of phagocytosis in gliomas. Am. J. Pathol. 1, 77-89. Peters, A., Palay, S.L. and Webster, H.deF. (1976) The Fine Structure of the Nervous System: The Neurons and Supporting Cells, W.B. Saunders Co., Philadelphia, PA, pp. 254-263. Robinson, A.P., White, T.M. and Mason, D.W. (1986) Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3. Immunology 57, 239-247. Rogers, J., Luber-Narod, J. and Styren, S.D. (1988) Expression of immune system-associated antigen by cells of the human central nervous system. Relationship to the pathology of Alzheimer's disease. Neurobiol. Aging 9, 330-349. Rozemuller, J.M., Eikelenboom, P., Pals, S.T. and Stare, F.C. (1989) Microglial cells around amyloid plaques in Alzheimer's disease express leukocyte adhesion molecules of the LFA-1 family. Neurosci. Lett. 101,288-292. Sanchez-Madrid, F., Nagy, J.A., Robbins, E., Simon, P. and Springer, T.A. (1983) A human leukocyte differentiation antigen family with distinct a-subunits and a common fl-subunit. The lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J. Exp. Med. 158, 1785-1803. Schwarting, R., Stein, H. and Wang, C.Y. (1985) The monoclonal antibodies aS-HCL 1 (aLeu-14) and aS-HCL 3 (aLeu M5) allow the diagnosis of hairy cell leukemia, Blood 65, 974-983.
Spits, H., Keizer, G., Borst, J., Terhorst, C., Hekman, A. and DeVries, J.E. (1983) Characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned CTL lines. Hybridoma 2, 423-437. Stites, D.P., Stobo, J.D. and Wells, J.V. (1987) Basic and Clinical Immunology, 6th edn., Appleton & Lange, Norwalk. Suomalainen, H.A., Gahmberg, C.G., Patarroyo, M., Beatty, P.G. and Schr~Sder, J. (1986) Genetic assignment of GP90, leukocyte adhesion glycoprotein to human chromosome 21. Somatic Cell Mol. Genet. 12, 297-302. Taylor, G.M., Haigh, H., Williams, A., D'Souza, S.W. and Harris, R. (1988) Down's syndrome lymphoid cell lines exhibit increased adhesion due to the over-expressi0n of lymphocyte function-associated antigen (LFA-1). Immunology 64, 451-456. Thomas, M.L. (1989) The leukocyte common antigen family. Annu. Rev. Immunol. 7, 339-369, 1989. Todd, R.F., Van Agthoven, A., Schlossman, S.F. and Terhorst, C. (1982) Structural analysis of differentiation antigens Mol and Mo2 on human monocytes. Hybridoma 1, 329337. Van Furth, R. (1982) Current view on the mononuclear phagocyte system. Immunobiology 161, 178-185. Warnke, R.A., Gatter, K.C., Falini, B. et al. (1983) Diagnosis of human lympboma with monoclonal antileukocyte antibodies. New Engl. J. Med. 309, 1275-1281.
81
JNI 01008
Brain microglia constitutively express fl-2 integrins H. Aldyama and P.L. McGeer Department of Psychiatry, Kinsmen Laboratory of Neurological Research, Faculty of Medicine, University of British Columbia, Vancouver, BC V6T 1 W5, Canada (Received 1 March 1990) (Revised, received 1 June 1990) (Accepted 4 June 1990)
Key words: Microglia; 13-2 Integrin; Alzheimer's disease; Complement receptor; Leukocyte function-associated antigen
Summary Localization of fl-2 integrins in normal and Alzheimer disease temporal cortex was studied immunohistochemically. Resting microglia were found to express constitutively C D l l a (LFA-1), C D l l b (Mac-l, CR3), C D l l c (P150, 95; CR4), and CD18 (/3-2). They were also found to express constitutively leukocyte common antigen and the immunoglobulin receptor Fc~,RI. The intensity of expression of each of these antigens was enhanced on reactive microglia in Alzheimer disease tissue. HLA-DR was detected on only a few microglia in control tissue, but was intensely expressed on large numbers of reactive microglia in Alzheimer tissue. These data are consistent with a leukocyte origin and a phagocytic role for microglia. They provide further evidence of an inflammatory response of brain tissue in Alzheimer disease. The microglia were found to make up 9-12% of the total glial population in gray matter and 7.5-9% in white matter.
Introduction The integrins are a superfamily of cell adhesion molecules which promote cell-cell and cell-matrix interactions (Hynes, 1987). They are recognized, along with the immunoglobulin superfamily, as proteins of high evolutionary significance. Both families play key roles in developmental and immune function by providing chemotactic guidance of cells to their correct location and promoting their adherence to target struc-
Address for correspondence: Dr. Patrick L. McGeer, University of British Columbia, Kinsmen Laboratory of Neurological Research, Department of Psychiatry, 2255 Wesbrook Mall, Vancouver, BC V6T 1W5, Canada.
tures. Integrins are membrane spanning glycoproteins in which the intracellular domain is customarily linked to the cytoskeletal apparatus, while the extracellular domain attaches to receptors on membranes or matrix proteins. Each integrin is a heterodimer with a unique a subunit and one of three/3 subunits. The common /3 subunits divide the integrins into distinct families (/3-1, /3-2, /3-3). The fl-1 family, also called very late antigens (VLAs), is characterized by the M r 110,000 /3-1 subunit (Hemler et al., 1985). Their primary role in brain appears to be in association with capillaries, where some members promote adhesion of endothelial cells to basement membrane matrix proteins (McGeer et al., 1990). The/3-2 family is characterized by the M r 95,000 /3-2 suhunit, designated as CD18. Three a sub-
0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
82 units of M r 180,000, 170,000 and 150,000 are known. They have been designated as C D l l a (LFA-1), C D l l b (Mac-l, CR3) and C D l l c (P150,95; CR4) (Kishimoto et al., 1987; Arnaout et al., 1988; Corbi et al., 1988a, b; I_arson et al., 1988). The principal ligand of C D l l a (LFA-1) is thought to be intercellular adhesion molecule 1 (ICAM-1) (Martin and Springer, 1987). The principal ligands for C D l l b and C D l l c are thought to be complement components related to C3bi. They have been designated CR3 (Micklem and Sire, 1985) and CR4 (Myones et al., 1988) respectively. The function of B-2 integrins appears to be restricted to the immune system. They have been found only on leukocytes (Kishimoto et al., 1989), where they promote migration, adhesion and phagocytosis (Sanchez-Madrid et al., 1983; Anderson et al., 1987; Keizer and Te Velde, 1987; Kishimoto et al., 1987, 1989; Arnaout et al., 1988). They have therefore been referred to as leukocyte integrins, leukocyte adhesion proteins, and LeuCAMs (Kishimoto et al., 1989). A congenital abnormality in their production, leukocyte adhesion deficiency disease (LAD), is associated with vulnerability to repeated infections (Anderson et al., 1987). The role of /~-2 integrins in brain function is not yet clear, They have been reported to occur on microglial cells in the corona of Alzheimer disease senile plaques (Rozemuller et al., 1989). CD11b has been found on mouse (Robinson et al., 1986) and rat (Graeber et al., 1988) microglia, while C D l l c has been found on human microglia (Hogg et al., 1986). In view of the central role that fl-2 integrins are thought to play in immune function, an understanding of their expression in normal and pathological brain tissue should provide important information on how the immune system in brain operates. We describe here immunohistochemical staining of normal and Alzheimer brain tissue with a panel of antibodies that includes all members of the /~-2 integrin family. We compare the staining with that obtained using antibodies to three different leukocyte markers: leukocyte cornmon antigen (LCA), the immunoglobulin receptor Fc~,RI, and the major histocompatibility complex (MHC) class II glycoprotein HLA-DR.
LCA represents a family of major membrane glycoproteins found on all hematopoietic cells except erythrocytes and their progenitors (Thomas, 1989). F c y R I recognizes the immunoglobulin Fc chain. The receptor is a glycoprotein expressed on the surface of mononuclear phagocytes. It is highly localized to cells of this lineage (Fanger et al., 1989). H L A - D R is a group II MHC glycoprotein particularly associated with antigen presentation to T - h e l p e r / i n d u c e r lymphocytes (Stites et al., 1987),
Materials and methods Fifteen autopsied human brains obtained within 2-12 h of death were employed in the study: six without neurological disorder (average age 69, range 54-82) and nine with Alzheimer disease (average age 77, range 69-85). The control cases all died from acute cardiovascular disease; five from myocardial infarcts and one from a ruptured aneurysm. Small blocks of brain tissue were dissected from the temporal lobe. Brain blocks were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 2 days and transferred to a maintenance solution of 15% sucrose in 0.01 M phosphatebuffered saline (PBS) pH 7.4. Sections were cut on a freezing microtome at 30 t~m thickness, collected in the maintenance solution, and stored until stained. Some unfixed brain blocks were immediately frozen with dry ice and sectioned in a cryostat. Unfixed cryocut sections were mounted on glass slides, air-dried and fixed in acetone for 10 rain. The primary monoclonal antibodies employed in this study, as well as the source, type and dilution are shown in Table 1. MHM24 and SPVL7 were used as the source for antibodies against C D l l a , 2LPM19C and Bear-1 for C D l l b , Leu-M5 for C D l l c , 60.3 and MHM23 for the common t3-2 chain, HB104 for HLA-DR, 2 B l l + P D 7 / 2 6 for leukocyte common antigen (LCA, CD45), and 32.2 for the immunoglobulin G receptor Fc'fRI (CD64). The antibody to glial fibrillary acidic protein (GFAP, Dako), was a polyclonal rabbit antiserum.
83 TABLE 1 MONOCLONAL ANTIBODIES USED IN THIS STUDY Antigen
Antibody (Reference)
CD11a (LFA-1)
MHM24 Dakopatts (Hildreth et al., 1983) SPV-L7 a Sanbio (Spits et al., 1983) 2LPM19Ca Dakopatts Bear-1 Sanbio (Todd et al., 1982) Leu-M5 Becton-Dickinson (Schwarting et al., 1985) 60.3 a Oncogene (Beatty et al., 1983) MHM23 a Dakopatts (Hfldreth et al., 1983) HB104 ATCC (Charron and McDevitt, 1979) 2Bll + PD7/26 Dakopatts (Wamke et al., 1983) 32.2 Medarex (Anderson et al., 1986)
CDllb (Mac-l, C R 3 ) CDllc (p150,95, CR4) CD18 (fl-2 subunit)
HLA-DR CD45 (LCA) CD64 (Fc~,RI)
Source
Form
Dilution
Supernatant
1 : 100
Supernatant
1 : 10
Supcrnatant Supernatant
1 : 30 1 : 100
purified IgG (10 #g/ml)
1 : 10
Ascites
1 : 300
Supematant
1 : 30
Supernatant
1 : 1000
Supernatant
1 : 100
Purified IgG (1 mg/rrd)
1 : 1000
a Worked only in fresh-frozen, acetone-fixed tissue.
All sections were pretreated with 0.2% H 2 0 2 solution for 30 min to eliminate endogenous peroxidase. For immunostaining in the free-floating procedure, sections were incubated with primary antibody for 72 h in the cold. The primary antibody was diluted in PBS containing 0.3% Triton X-100 (PBS-Tx) and 10% normal horse serum to the concentration shown in Table 1. G F A P antiserum was used at a dilution of 1 : 1 0 , 0 0 0 i n PBSTx containing 10% normal goat serum. After incubation with primary antibody, sections were next treated with biotinylated secondary antibody (Vector Lab, diluted 1:1000) for 2 h at room temperature followed by incubation in the avidinbiotinylated horseradish peroxidase (HRP) cornplex (ABC Elite, Vector Lab., Burlingame, CA, U.S.A.) for 1 h at room temperature. Peroxidase labeling was detected by incubation with a solution containing 0.01% 3,3'-diaminobenzidine (DAB, Sigma), 0.6% nickel a m m o n i u m sulfate, 0.05 M imidazole and 0.00015% H202. A darkpurple reaction product appeared after about 1545 min, at which time the reaction was terminated by transferring the section to PBS. Sections were mounted on glass slides, dehydrated with graded
alcohols, and mounted with Entellan (Merck) with or without prior staining with neutral red. For double immunostaining, sections were treated for 30 min with 0.5% H 2 0 2 solution in PBS after the DAB reaction of the first cycle. The second immunohistochemical cycle was carried out similarly to the first cycle except that nickel ammonium sulfate was eliminated from the DAB solution, yielding a brown precipitate in the second cycle. The following combinations were investigated: leukocyte c o m m o n antigen (LCA) with M H M 2 4 ( C D l l a ) , Bear-1 ( C D l l b ) , Leu-M5 ( C D l l c ) , HB104 ( H L A - D R ) and 32.2 (FcyRI); H L A - D R with Leu-M5; and G F A P with MHM24, Bear-l, Leu-M5, LCA, and HB104. For staining of fresh-frozen acetone-fixed tissue, primary antibodies were diluted in PBS without Triton X-100. Following the pretreatment with 0.2% H202, primary antibody incubation was done overnight in a moist chamber at room ternperature. The rest of the procedure was the same as for free-floating sections except that they were carried out on the surface of glass slides. Three types of controls were used: elimination of the primary antiserum; substitution of rabbit
84
serum hyperimmunized with tobacco mosaic virus; and use of a mouse monoclonal antibody indifferent to brain antigens. N o positive staining of either normal or Alzheimer brain was obtained in control. To quantitate the ratio of microglia to total glial cells in the temporal cortex of control subjects, serial sections were cut from the middle temporal gyrus of the six control cases. Every fifth section was stained with Leu-M5 and an adjacent section stained with cresyl violet. Sections stained with Leu-M5 were further counterstained with neutral red. The numbers of nuclei of either LeuM5-positive cells (in Leu-M5-stained tissue) or total glial cells (in cresyl violet-stained tissue) were counted in a column 0.5 m m in width covering the whole cortical thickness as well as in an 0.5 m m square of subjacent white matter (at least 1 m m distant from the boundary of the gray and white), Approximately the same area was chosen from each section. The distinction between astroglia and small neurons was uncertain for a few of the cells (less than 5%) but these numbers were too small to have a significant influence on the final result. The ratio of Leu-M5-positive cells to total glial cells was calculated for each case by averaging the values of three sections. Results All members of the fl-2 integrin family were detected on microglial cells in both gray and white matter in normal and Alzheimer tissue. Positive staining was obtained only in fresh-frozen acetone-fixed tissue with monoclonal antibodies SPV-L7 ( C D l l a ) , 2LPM19C ( C D l l b ) , 60.3 and M H M 2 3 (CD18). On the other hand, strong positive staining with good morphological detail was obtained on paraformaldehyde-fixed tissue with
monoclonal antibodies M H M 2 4 ( C D l l a ) , Bear-1 ( C D l l b ) , and Leu-M5 ( C D l l c ) . These antibodies also stained leukocytes in the matrix of Alzheimer brain tissue which were present in small numbers. They did not stain neurons, astrocytes, oligodendroglia, or capillary endothelial cells. Similar staining was obtained with the antibody to LCA. The antibody to F c y R I strongly stained all microglia, while the antibody to H L A - D R stained only reactive microglia. Typical results of double immunostaining are shown in the color photomicrographs of Fig. 1. The full thickness of normal temporal cortex is illustrated in Fig. 1A, which is immunostained for C D l l c (brown) and H L A - D R (purple). The large complement of C D l l c - p o s i t i v e resting microglia is easily seen, with rare HLA-DR-positive microglia also appearing. This is in considerable contrast to the temporal cortex of Alzheimer disease, which is shown in Fig. lB. Many purple-colored HLADR-positive reactive microglia appear in both gray and white matter. These are often assembled in agglomerates. The reactive microglia are also strongly positive for C D l l c but the double staining of cells is not easily discerned under low power. Fig. 1C and D illustrates double immunostaining at higher power for C D l l c (purple) and G F A P (brown) in gray ( C ) and white ( D ) matter of Alzheimer temporal cortex. Here the C D l l c - p o s i tive microglia can be distinguished from the GFAP-positive astrocytes both by morphology and color. The microglia themselves appear in both resting and reactive forms. Resting microglia show small, round to somewhat elongated cell bodies and thin ramified processes, while reactive microglia show an enlarged cyctoplasm, as well as enlarged but retracted processes. These morphologies correspond to those originally described by
Fig. 1. Double immunostaining of normal (A) and Alzheimer (B, C and D) temporal cortex for CDllc (Leu-M5) and either HLA-DR (A and B) or GFAP (C and D). A and B show the full width of cortex with the pial surface at the top and subjacent white matter at the bottom. CDllc is stained brown and HLA-DR purple. Note that the control cortex has only rare HLA-DR-positire microglia while the Alzheimer cortex has very many appearing singly or as agglomerates. Higher power views of Alzheimer gray (C) and white (D) matter are shown in which GFAP is stained brown and CDllc purple. The GFAP-positive astrocytes can easily be distinguished from the CDllc-positive rnicroglia. Resting microglia stain weakly compared with the strongly staining reactive forms, some of which are aggregated. A and B are at the same magnification, bar = 250 #m. C and D are at the same magnification, bar = 100 #m.
85
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86 del Rio Hortega (1919) and Penfield (1925) using silver carbonate stains, Fig. 2 illustrates the staining of gray and white matter in control brain tissue for the/3-2 integrins C D l l a (MHM24, Fig. 2A and B), C D l l b (Bear-l, Fig. 2C and D), C D l l c (Leu-M5, Fig. 2 E and F ) and CD18 (60.3, Fig. 2G and H). In gray matter, microglia were only weakly positive for C D l l a (Fig. 2A), while in white matter they were moderately positive (Fig. 2B). They were also moderately positive for C D l l b (Fig. 2C and D) and C D l l c (Fig. 2 E and F ) in both gray and white matter, but the two monoclonal antibodies against the common/3-2 subunit stained only fresh-frozen acetone-fixed tissue. Microglial cells in white matter (Fig. 2 H ) were labeled more intensely than those in gray matter (Fig. 2G) by the/3-2 antibodies. Fig. 3 shows staining of sections nearby to those in Fig. 2 using different leucocyte markers, Fig. 3A and B shows staining of control gray and white matter for the FcTRI receptor. Fig. 3C and D shows staining for leukocyte common antigen (LCA) and Fig. 3E and F for HLA-DR. The results of control gray and white matter staining for LCA and FcTRI were identical to those for/3-2 integrin staining. Dense labeling of microglia was observed (Fig. 3A-D). The results of staining for H L A - D R were very different. Only rare positive cells were observed in gray matter (Figs. 1A and 3E) and a few in white matter (Figs. 1A and 3F). Fig. 4 shows staining of Alzheimer tissue for /3-2 integrins. Microglia in Alzheimer tissue were stained more intensely than in control tissue by each of the/3-2 integrin antibodies. A significant proportion of the positively stained cells had morphologies comparable to classical reactive microglia (Rio Hortega, 1919; Penfield, 1925). The staining was particularly intense in microglial aggregates which were closely associated with senile plaques (Itagaki et al., 1989; McGeer et al., 1989a).
Fig. 4A and B shows such dramatically enhanced expression of C D l l a by reactive microglia in Alzheimer gray and white matter. Heavily stained microglial aggregateg (arrow) were revealed to be localized in the center of a senile plaque when the slide was observed by fluorescence microscopy following thioflavin S counterstaining (data not shown). The expression of C D l l b and C D l l c was also enhanced in Alzheimer tissue compared with control but less prominently than that of C D l l a (Fig. 4C-F). Enhancement of CD18 expression seemed to be intermediate between C D l l a and C D l l b / C D l l c (Fig. 4G and H). Reactive microglia in Alzheimer gray and white matter also stained more intensely than resting microglia for FcTRI and LCA (Fig. 5A-D). The most dramatic change between control and Alzheimer tissue was seen with staining for HLA-DR. Instead of the occasional positive cell seen in control tissue, large numbers of microglia, mostly of reactive morphology, were seen in both gray and white matter (Figs. 1B and 5E and F). In order to determine more exactly the cell population expressing various /3-2 integrins, a complete series of double-immunostaining experiments was carried out for LCA in combination with the a chain of each /3-2 integrin and the FcTRI receptor. When LCA was stained purple in the first cycle, and C D l l a (MHM24), C D l l b (Bear-l), C D l l c ( L e u - M 5 ) o r FcTRI (32.2)brown in the second cycle, all microglia in control and Alzheimer tissue were stained either purple or purple-brown. Since the stronger purple color of the first cycle can obscure the weaker brown color of the second cycle, the experiments were repeated with the order of staining reversed. When the/3-2 integrins or F C T R I were stained purple in the first cycle and LCA was stained brown in the second cycle, all microglia similarly stained purple or purple-brown. Thus, no microglia were detected in Alzheimer or control tissue that stained singly for LCA, FcTRI or any of the/3-2 integrins.
Fig. 2. Staining of normal temporal cortical gray (A, C, E and G) and white (B, D, F and H) matter for/3-2 integrins. A and B, staining for CDlla (MHM24). C and D, staining for CDllb (Bear-l). E and F, staining for CDllc (Leu-M5). G and H, staining for CD18 (60.3). See Materials and Methods for experimental details. Each antibody stains similar numbers of resting microglia. Note, however,the weaker staining for CDlla in gray matter, and the relativelypoor morphologyobtained for CD18 staining in the fresh-frozenacetone-fixedmaterial. Bar in H = 50 ~tm.All photomicrographsin Figs. 2-5 are at the same magnification.
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Fig. 3. Staining of normal temporal cortical gray (A, C and E) and white (B, D and F ) matter with various leukocyte markers. A and B, staining of FcyR1 receptors with antibody 32.2. C and D, staining for LCA with the 2B11 + PD7/26 antibody. Note that a similar number of microglia with resting morphology are stained in these photomicrographs compared with those in Fig, 1. E and F, staining for HLA-DR. Notice that very few microglia are stained positively.
Very different results were obtained with double immunostaining for LCA and H L A - D R . When H L A - D R was stained purple in the first cycle and LCA brown in the second, many microglia were observed that were only stained brown. Such H L A - D R - / L C A + microglia had resting mor-
phology. They were particularly numerous in control tissue, but in Alzheimer tissue many microglia were reactive and H L A - D R positive. A complete set of double-immunostaining experiments was also conducted for G F A P and the c~ chain of each /~-2 integrin as well as LCA and
89 FcTRI to determine if astrocytes express integrins or other leukocyte markers. Whether GFAP was used in the first or second cycle, no GFAP-positive astrocytes were found to stain for C D l l c , LCA or HLA-DR. The results of microglial cell counts in the six control cases using C D l l c as the microglial marker showed the microglial population to be 10.6 ___0.9% (range 8.9-12) of the total in gray matter and 8.2 + 0.5% (range 7.6-8.9) in white matter. These data are in keeping with previous reports (Peters et al., 1976). The ratio in gray matter was fairly constant through the six layers of the cerebral cortex,
Discussion These data demonstrate the constitutive expression of/3-2 integrins on microglia in the resting as well as the reactive state. They confirm and extend previous reports on the occurrence of/3-2 integrins on these cells (Hogg et al., 1986; Robinson et al., 1986; Graeber et al., 1988; Rozemuller et al., 1989). They also confirm previous data on the constitutive expression of LCA and FcTRI on microglia (Itagaki et al., 1988; McGeer et al., 1989a). In the periphery, the FcTRI is almost exclusively restricted to mononuclear phagocytes (Fanger et al., 1989). Normal peripheral blood monocytes have a range of 15,000-40,000 sites per cell but, upon activation by inducers such as 7-interferon, these receptor numbers can be upregulated 5- to 10-fold (Fanger et al., 1989). Comparable data are not available for the numbers of/3-2 integrin molecules on various leukocytes cell lines, but they are known to be highly expressed on monocytes and granulocytes. The existence of these molecules on resting microglia, and their upregulation when microglia become reactive and undergo morphological change are powerful evidence supporting long-held theories that microglia are the brain representatives of the monocyte phagocytic system (Van Furth, 1982). Immunoglobulins and complement proteins are the key molecules which mark foreign or unwanted materials for disposal by phagocytes, Prominent staining of receptors for these proteins, even on resting microglia, is confirmation of much
previous evidence that phagocytosis is the primary function of these cells. The integrin molecules CD11b and C D l l c are receptors for C3bi (Micklem and Sim, 1985) and C3dg (Myones et al., 1988). These are products of complement proteins that have been chemically bound to tissue (McGeer et al., 1989). F c y R I is the receptor molecule for the immunoglobulin Fc chain. The function of C D l l a on microglia is less certain, but there may be important interactions with its ligand ICAM-1 acting as a guiding agent (Martin and Springer, 1987). Interaction between LFA-1 and ICAM-1 is considered fundamental to many immunologic reactions (Makgoba et al., 1989). In Alzheimer disease, it is known that plaques, tangles and dystrophic neurites are strongly stained by antibodies to complement proteins (Eikeleboom et al., 1989; McGeer et al., 1989a, b). In addition, it has been reported that /3-2 integrins are expressed by microglial cells in the corona of Alzheimer senile plaques (Rozernuller et al., 1989). Thus, the complement receptors represented by /3-2 integrins presumably assist microglial cells to adhere to these complement proteins in Alzheimer disease. The only known condition in which there is over-expression of /3-2 integrins is Down's syndrome (Taylor et al., 1988). The/3-2 subunit occurs on chromosome 21 in the obligatory region for this condition. Down's cases exhibit over-expression of LFA-1 and higher adhesiveness of lymphocytes due to the/3 chain gene dosage effect. It is not known whether this bears any relation to the increased susceptibility to infection of Down's syndrome patients or of their tendency to develop Alzheimer-type brain amyloid deposits (Glenner and Wong, 1984). Despite the long-standing idea of immunological privilege of brain, recent investigations have demonstrated presence of the relevant cellular elements of an immune response in brain tissue in several neurological diseases (Itagaki et al., 1988; McGeer et al., 1988, 1989a, b; Rogers et al., 1988; Eikelenboom et al., 1989; Rozemuller et al., 1989). Constitutive expression of /3-2 integrins appears further to characterize the resident microglial cells as the key component of the immune system in brain.
90
91
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Fig. 5. Staining of Alzheimer temporal cortical gray (A, C and E) and white (B, D and F ) matter for various leukocyte markers. A and B, staining for Fc~,RI (32.2). C and D, staining for LCA. G and H, staining for HLA-DR. Again, notice the similarity in microglial staining to the fl-2 integrin staining shown in Fig. 3. In A, C and E microglial aggregates (arrows) are visible which stain intensely. Similarly, in B, D and F most microglia stain intensely and are of reactive morphology. Also, notice that in Alzheimer disease most microglia are HLA-DR positive while very few are POsitive in control tissue.
Fig. 4. Immunohistochemical staining of Alzheimer temporal cortical gray (A, C, E and G) and white (B, D, F and H)mat.ter for fl-2 integrins. A and B, staining for C D l l a (MHM24). C and D, staining 'for C D l l b (Bear-l). E and F, staining for C D l l c (Leu-M5). G and H, staining for CD18 (60.3). Each fl-2 integrin stains comparable cells. Notice the intense staining of microglial aggregates in the gray matter photomicrographs (A, C, E and G, arrows). Some more lightly stained microglia, not in aggregates, are visible in the bacl~ground. In white matter, large microglial aggregates are not visible, but intensely staining microglia with reactive morphology form the majority of positively staining cells.
92
Acknowledgements The authors wish to thank Ms. Joane Sunahara
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