Acta Neuropathol (1992) 84:225 -233
Neuropathologga ~) Springer-Verlag1992 Regular papers
Ultrastructure of the microglia that phagocytose amyloid and the microglia that produce [5-amyloid fibrils* J. Frackowiak**, H. M.Wisniewski, J.Wegiel, G. S. Merz, K. Iqbal, and K. C.Wang Department of PathologicalNeurobiology,New York State Institute for Basic Research in DevelopmentalDisabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA Received October 11, 1991/Revised, accepted March 21, 1992
Summary. The function of microglia associated with ~-amyloid deposits still remains a controversial issue. On the basis of recent ultrastructural data, microglia were postulated to be cells that form amyloid fibrils, not phagocytes that remove amyloid deposits. In this electron microscopic study, we examined the ability of microglia to ingest and digest exogenous amyloid fibrils in vitro.We demonstrate that amyloid fibrils are ingested by cultured microglial cells and collected and stored in phagosomes. The ingested, nondegraded amyloid remains within phagosomes for up to 20 days, suggesting a very limited effectiveness of microglia in degrading ~-amyloid fibrils. On the other hand, we showed that in microglial cells of classical plaques in brain cortex of patients with Alzheimer's disease, amyloid fibrils appear first in altered endoplasmic reticulum and deep infoldings of cell membranes. These differences in intracellular distribution of amyloid fibrils in microglial cells support our observations that microglial cells associated with amyloid plaques are engaged in production of amyloid, but not in phagocytosis.
Key words: Alzheimer's disease - Tissue culture Microglia - Amyloid - Ultrastructure
Cells described as microglia, macrophages, or perivascular cells have been observed to surround amyloid deposits in the brains of Alzheimer's disease (AD) patients [8, 12, 17, 24, 34]. Because of this apposition, it has been suggested that cells associated with amyloid cores may be involved in either the phagocytosis [12] or * Supported by funds from the the New York State Office of Mental Retardation and DevelopmentalDisabilities and a grant from the National Institutes of Health, National Institute on Aging, Grant No. PO1-AGO-4220, AGO-5892 ** Visiting Scientist from the Department of Pathophysiology, Medical School, Gdansk, Poland Correspondence to: H. M. Wisniewski (address see above)
the formation of amyloid fibrils [9, 20-22, 26-29]. On the basis of what is known from other amyloidoses [11, 18, 32] and the morphology of amyloid and microglial cells [17, 24, 30, 31], it has been hypothesized that microglial cells in AD brains are engaged in production of amyloid fibrils. Ultrastructural studies and threedimensional reconstructions of amyloid plaques in brain biopsies from AD patients show amyloid fibrils in the altered cisternae of the smooth endoplasmic reticulum (SER) and in the channels confluent with the infoldings of the plasma membranes of microglial cells [24, 30]. On the other hand, typical images of phagocytosis of amyloid by macrophages were demonstrated recently in a study of removal of necrotic tissue in brains containing numerous amyloid plaques in stroke [33]. To study the ultrastructure of microglial cells engaged in phagocytosis of amyloid further, we exposed microglia in vitro to amyloid cores isolated from the brains of AD patients. Microglial cells were isolated from dogs animals known to develop ~-amyloid plaques during aging [23, 26, 32]. The aims of this study were (1) to characterize intracellular localization of amyloid ingested by microglial cells cultured in the presence of exogenous amyloid, (2) to examine the fate of ingested amyloid, and (3) to compare intracellular localization, morphology, and immunocytochemistry of amyloid ingested in vitro by microglial cells and of amyloid in microglial cells in AD brains.
Materials and methods Isolation of microglial cells Autopsy dog brains were obtained from SUNY Medical Center, Brooklyn,New York and were used for isolationof microglialcells within 2 h post mortem.To isolate microglia,we used the modified method described by Merz et al. [15].To eliminatemonocytesand meningeal macrophages, the brains were perfused with saline within 30 min post mortem, and onlycortex, stripped of meninges, was used for microgliaisolation. Finelymincedtissue was digested in 0.15% trypsin and 0.004% DNase, sieved through nylon
226 meshes, and centrifuged through Percoll (density 1.038). Pellets containing microglia and only occasional blood vessels were collected. Isolated cells were cultured in 75-cm2 flasks (Beckton Dickinson, Lincoln Park, N. J.) in RPMI 1640 (Whittaker Bioprod. Inc., Walkersville, Md.) supplemented with 15% FCS (containing less than 0.1 ng/ml of endotoxins; Whittaker Bioprod.) and 20 % of L-929 cell-conditioned medium. After 7-9 days of culture, microglia were harvested by orbital shaking at 250 rpm, 37 ~ for 1 h.
Ultrastructural studies Cultured microglial cells and biopsies from the brains of six AD patients were fixed with glutaraldehyde and osmic acid and embedded in Epon. For immunostaining ultrathin sections were collected on nickel grids without formvar membrane, deosmifcated with 5 % hydrogen peroxide (1 min), and stained for the presence of amyloid with ~-protein-speciflc 4G8 mAb [13], followed by protein A immunogold. Sections were incubated overnight with mAb diluted 1:10 in PBS/BSA with Tween 20. Protein A conjugated with 10-nm gold particles was used.
Identification of microglia Microglia were identifed as cells possessing surface MHC class II antigens, C3-receptor (Mac-1 antigen) and ferritin; exhibiting weak cytochemical staining for nonspecific esterases; and showing no reaction for GFAP and von Willebrand factor. Class II MHCpositive cells were detected with the mouse monoclonal antibodies (mAbs) DH59B and TH14B (VMRD Inc., Pullman,Wash.), which recognize species-nonspecific epitopes on class II MHC molecules in human, dog, and other species [4]. The anti-Mac-1 mAb (Boehringer Mannheim Biochem., Indianapolis, Ind.), a speciesnonspecific [2] marker of macrophages/microglia,was also found to immunostain canine phagocytes, peritoneal macrophages, peripheral blood monocytes, and granulocytes, but not fibroblasts or endothelial cells (J. Frackowiak, unpublished results). Ferritin was detected with rabbit serum anti-human ferritin (DAKO Corp., Santa Barbara, Calif.); antiferritin antibodies are known to show interspecies cross-reactivity [1]. An indirect immunofluorescence method was performed to immunostain living cells with DH59B and TH14B, Mac-1 mAbs, and anti-ferritin serum. Cells fixed with acetone (-20~ 15 rain) were immunostained with anti-GFAP mAb (Accurate Chem. Sci. Corp., Westburg, N.Y.) and rabbit serum against human von Willebrand factor (DAKO corp.). Esterase cytochemistry was performed according to Mueller et al. [16], at 37 ~ for 30 rain.
Isolation of amyloid cores Amyloid cores were isolated by the procedure of Iqbal et al. [10], except that the detergent treatment was substituted by digestion with collagenase and dispase [7]. The amyloid core-enriched fraction admixed with neurofibrillary tangles obtained from 1.8 M to 2.2 M sucrose interphase was diluted sixfold with water, homogenized in a ground-glass homogenizer, and centrifuged at 50 g for 6 min.The supernatant was rehomogenized twice again and centrifuged as above. All three 50 g pellets were highly enriched in amyloid cores and contained only small amounts of neurofibrillary tangles. The pellets resuspended in RPMI were sonicated before adding to cultured cells.
Experimental procedure The secondary cultures, containing 92 %-98 % of microglia, were established for light microscopy on eight-chamber Permanox slides (Nunc, Inc. Naperville, Ill.), and for light and electron microscopy (EM) studies, on Millicell-CM culture Plate Inserts (Millipore Corp., Bedford, Mass.) previously precoated with collagen, flbronectin, and laminin. Amyloid was added to cultured microglia and after 24 h, the amyloid not ingested by cells was washed out. Samples for light and electron microscopy studies were taken 3, 6, and 24 h after amyloid was added, and 6, 12, 19, and 20 days after washing out the extracellular amyloid. Cultures not treated with amyloid were used as control. Cultures on Permanox slides and Millicell-CM Inserts were immunostained for light microscopy for the cell markers listed above, as well as for amyloid using 4G8 and 6E10 mAbs [13], by immunofluorescence and immunoperoxidase methods.
Results
Dog microglial cells cultured with isolated amyloid cores T h e s e c o n d a r y c u l t u r e s u s e d in t h e e x p e r i m e n t s cont a i n e d 92 % - 9 8 % o f cells p o s i t i v e for m i c r o g l i a / m a c r o phage markers. Low content of strongly esterasep o s i t i v e cells c o n f i r m e d a n e g l i g i b l e c o n t a m i n a t i o n o f cultures with monocytes derived from the bloodstream o r w i t h m e n i n g e a l m a c r o p h a g e s (Table 1). M i c r o g l i a l Table 1. Characterization of cultures of microglial cells isolated from dog brains Marker
% of positive cells
Class II MHC antigens Mac 1 Ferritin Nonspecific esterase a) weak reaction b) strong reaction GFAP Von Willebrand factor
95 +_ 3 92 +_ 4 93 _+ 3 82 + 5
Neuropathologga ~) Springer-Verlag1992 Regular papers
Ultrastructure of the microglia that phagocytose amyloid and the microglia that produce [5-amyloid fibrils* J. Frackowiak**, H. M.Wisniewski, J.Wegiel, G. S. Merz, K. Iqbal, and K. C.Wang Department of PathologicalNeurobiology,New York State Institute for Basic Research in DevelopmentalDisabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA Received October 11, 1991/Revised, accepted March 21, 1992
Summary. The function of microglia associated with ~-amyloid deposits still remains a controversial issue. On the basis of recent ultrastructural data, microglia were postulated to be cells that form amyloid fibrils, not phagocytes that remove amyloid deposits. In this electron microscopic study, we examined the ability of microglia to ingest and digest exogenous amyloid fibrils in vitro.We demonstrate that amyloid fibrils are ingested by cultured microglial cells and collected and stored in phagosomes. The ingested, nondegraded amyloid remains within phagosomes for up to 20 days, suggesting a very limited effectiveness of microglia in degrading ~-amyloid fibrils. On the other hand, we showed that in microglial cells of classical plaques in brain cortex of patients with Alzheimer's disease, amyloid fibrils appear first in altered endoplasmic reticulum and deep infoldings of cell membranes. These differences in intracellular distribution of amyloid fibrils in microglial cells support our observations that microglial cells associated with amyloid plaques are engaged in production of amyloid, but not in phagocytosis.
Key words: Alzheimer's disease - Tissue culture Microglia - Amyloid - Ultrastructure
Cells described as microglia, macrophages, or perivascular cells have been observed to surround amyloid deposits in the brains of Alzheimer's disease (AD) patients [8, 12, 17, 24, 34]. Because of this apposition, it has been suggested that cells associated with amyloid cores may be involved in either the phagocytosis [12] or * Supported by funds from the the New York State Office of Mental Retardation and DevelopmentalDisabilities and a grant from the National Institutes of Health, National Institute on Aging, Grant No. PO1-AGO-4220, AGO-5892 ** Visiting Scientist from the Department of Pathophysiology, Medical School, Gdansk, Poland Correspondence to: H. M. Wisniewski (address see above)
the formation of amyloid fibrils [9, 20-22, 26-29]. On the basis of what is known from other amyloidoses [11, 18, 32] and the morphology of amyloid and microglial cells [17, 24, 30, 31], it has been hypothesized that microglial cells in AD brains are engaged in production of amyloid fibrils. Ultrastructural studies and threedimensional reconstructions of amyloid plaques in brain biopsies from AD patients show amyloid fibrils in the altered cisternae of the smooth endoplasmic reticulum (SER) and in the channels confluent with the infoldings of the plasma membranes of microglial cells [24, 30]. On the other hand, typical images of phagocytosis of amyloid by macrophages were demonstrated recently in a study of removal of necrotic tissue in brains containing numerous amyloid plaques in stroke [33]. To study the ultrastructure of microglial cells engaged in phagocytosis of amyloid further, we exposed microglia in vitro to amyloid cores isolated from the brains of AD patients. Microglial cells were isolated from dogs animals known to develop ~-amyloid plaques during aging [23, 26, 32]. The aims of this study were (1) to characterize intracellular localization of amyloid ingested by microglial cells cultured in the presence of exogenous amyloid, (2) to examine the fate of ingested amyloid, and (3) to compare intracellular localization, morphology, and immunocytochemistry of amyloid ingested in vitro by microglial cells and of amyloid in microglial cells in AD brains.
Materials and methods Isolation of microglial cells Autopsy dog brains were obtained from SUNY Medical Center, Brooklyn,New York and were used for isolationof microglialcells within 2 h post mortem.To isolate microglia,we used the modified method described by Merz et al. [15].To eliminatemonocytesand meningeal macrophages, the brains were perfused with saline within 30 min post mortem, and onlycortex, stripped of meninges, was used for microgliaisolation. Finelymincedtissue was digested in 0.15% trypsin and 0.004% DNase, sieved through nylon
226 meshes, and centrifuged through Percoll (density 1.038). Pellets containing microglia and only occasional blood vessels were collected. Isolated cells were cultured in 75-cm2 flasks (Beckton Dickinson, Lincoln Park, N. J.) in RPMI 1640 (Whittaker Bioprod. Inc., Walkersville, Md.) supplemented with 15% FCS (containing less than 0.1 ng/ml of endotoxins; Whittaker Bioprod.) and 20 % of L-929 cell-conditioned medium. After 7-9 days of culture, microglia were harvested by orbital shaking at 250 rpm, 37 ~ for 1 h.
Ultrastructural studies Cultured microglial cells and biopsies from the brains of six AD patients were fixed with glutaraldehyde and osmic acid and embedded in Epon. For immunostaining ultrathin sections were collected on nickel grids without formvar membrane, deosmifcated with 5 % hydrogen peroxide (1 min), and stained for the presence of amyloid with ~-protein-speciflc 4G8 mAb [13], followed by protein A immunogold. Sections were incubated overnight with mAb diluted 1:10 in PBS/BSA with Tween 20. Protein A conjugated with 10-nm gold particles was used.
Identification of microglia Microglia were identifed as cells possessing surface MHC class II antigens, C3-receptor (Mac-1 antigen) and ferritin; exhibiting weak cytochemical staining for nonspecific esterases; and showing no reaction for GFAP and von Willebrand factor. Class II MHCpositive cells were detected with the mouse monoclonal antibodies (mAbs) DH59B and TH14B (VMRD Inc., Pullman,Wash.), which recognize species-nonspecific epitopes on class II MHC molecules in human, dog, and other species [4]. The anti-Mac-1 mAb (Boehringer Mannheim Biochem., Indianapolis, Ind.), a speciesnonspecific [2] marker of macrophages/microglia,was also found to immunostain canine phagocytes, peritoneal macrophages, peripheral blood monocytes, and granulocytes, but not fibroblasts or endothelial cells (J. Frackowiak, unpublished results). Ferritin was detected with rabbit serum anti-human ferritin (DAKO Corp., Santa Barbara, Calif.); antiferritin antibodies are known to show interspecies cross-reactivity [1]. An indirect immunofluorescence method was performed to immunostain living cells with DH59B and TH14B, Mac-1 mAbs, and anti-ferritin serum. Cells fixed with acetone (-20~ 15 rain) were immunostained with anti-GFAP mAb (Accurate Chem. Sci. Corp., Westburg, N.Y.) and rabbit serum against human von Willebrand factor (DAKO corp.). Esterase cytochemistry was performed according to Mueller et al. [16], at 37 ~ for 30 rain.
Isolation of amyloid cores Amyloid cores were isolated by the procedure of Iqbal et al. [10], except that the detergent treatment was substituted by digestion with collagenase and dispase [7]. The amyloid core-enriched fraction admixed with neurofibrillary tangles obtained from 1.8 M to 2.2 M sucrose interphase was diluted sixfold with water, homogenized in a ground-glass homogenizer, and centrifuged at 50 g for 6 min.The supernatant was rehomogenized twice again and centrifuged as above. All three 50 g pellets were highly enriched in amyloid cores and contained only small amounts of neurofibrillary tangles. The pellets resuspended in RPMI were sonicated before adding to cultured cells.
Experimental procedure The secondary cultures, containing 92 %-98 % of microglia, were established for light microscopy on eight-chamber Permanox slides (Nunc, Inc. Naperville, Ill.), and for light and electron microscopy (EM) studies, on Millicell-CM culture Plate Inserts (Millipore Corp., Bedford, Mass.) previously precoated with collagen, flbronectin, and laminin. Amyloid was added to cultured microglia and after 24 h, the amyloid not ingested by cells was washed out. Samples for light and electron microscopy studies were taken 3, 6, and 24 h after amyloid was added, and 6, 12, 19, and 20 days after washing out the extracellular amyloid. Cultures not treated with amyloid were used as control. Cultures on Permanox slides and Millicell-CM Inserts were immunostained for light microscopy for the cell markers listed above, as well as for amyloid using 4G8 and 6E10 mAbs [13], by immunofluorescence and immunoperoxidase methods.
Results
Dog microglial cells cultured with isolated amyloid cores T h e s e c o n d a r y c u l t u r e s u s e d in t h e e x p e r i m e n t s cont a i n e d 92 % - 9 8 % o f cells p o s i t i v e for m i c r o g l i a / m a c r o phage markers. Low content of strongly esterasep o s i t i v e cells c o n f i r m e d a n e g l i g i b l e c o n t a m i n a t i o n o f cultures with monocytes derived from the bloodstream o r w i t h m e n i n g e a l m a c r o p h a g e s (Table 1). M i c r o g l i a l Table 1. Characterization of cultures of microglial cells isolated from dog brains Marker
% of positive cells
Class II MHC antigens Mac 1 Ferritin Nonspecific esterase a) weak reaction b) strong reaction GFAP Von Willebrand factor
95 +_ 3 92 +_ 4 93 _+ 3 82 + 5
