Background

Macrophages may participate in amyloid fibril formation by processing the protein precursor. Although this theory seems to apply for amyloidosis, in which proteolytic cleavage is a prerequisite for amyloid fibril formation, it has not been demonstrated for 2-microglobulin (₂m) amyloidosis. We aimed to establish the role played by macrophages in ₂m amyloidosis.

Methods

We used a double immunogold electron microscopy technique, including mouse antihuman CD68, rabbit antihuman ₂m, amyloid P component, and lysosome-associated membrane protein (LAMP-1) antibodies. Differential density labeling studies of ₂m and amyloid P component were performed extra- and intracellularly to assess protein processing by macrophages.

Results

The cells surrounding amyloid fibrils were found to be mostly CD68 positive, suggesting that they were of monocyte–macrophage lineage. Intracellular accumulation of amyloid fibrils was also observed; these fibrils were constantly surrounded by LAMP-1–linked gold particles, demonstrating that intracellular ₂m was almost exclusively lysosomal. The rough-surface endoplasmic reticulum was not labeled by ₂m antibody, suggesting that there was no active synthesis of ₂m by the cells. As a marker of endocytosis, protruded cytoplasmic processes in close relation with the intracellular accumulations of ₂m amyloid fibrils were observed. No difference in density labeling (extracellular vs. intracellular) was observed for ₂m, whereas intracellular P component labeling was significantly decreased.

Conclusions

All of these data are strongly suggestive of phagocytosis and not synthesis of amyloid fibrils by macrophages. Further, they demonstrate an impaired lysosomal processing specific for ₂m, as other compounds of the amyloid fibrils (P component) are significantly cleared.

METHODS

Patients and samples

Amyloid deposits were obtained from carpal tunnel of three men and one woman, who were 65 4 years old, and had dialysis-related amyloidosis. Their renal diseases were chronic glomerulonephritis (three patients) and autosomal-dominant polycystic renal disease (the remaining one). None of the patients had systemic diseases known to be associated with amyloidosis.

Sample preparation and immunohistochemistry

Congo red staining.

Unfixed amyloid deposits surgically obtained were immersed in O.C.T. compound (Miles Inc., Diagnostics Division, Elkhart, IN, USA), frozen, and stored at -70°C. Cryostat sections of 10 m were transferred onto gelatin-coated (0.3%) glass slides and were stained with Congo red (Searle Scientific Services, High Wycombe Bucks, UK) following a slight modification of Puchtler's method¹³. Briefly, sections were dehydrated in 70% ethanol for five minutes and were then treated with an alkaline-saturated salt solution. The stained sections were mounted in a medium compatible with organic solvents and were observed under polarized light in a Zeiss microscope (Oberkochen, Germany).

Immunogold methods.

Blocks of approximately 1 mm³ of amyloid deposits were fixed in 4% (vol/vol) paraformaldehyde and 0.1% (vol/vol) glutaraldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4, for two hours at 4°C.

Single immunolabeling.

Samples were progressively dehydrated in graded ethanols and were finally embedded in Lowicryl K4M (Chemische Werke Lowi, Wald-Kraiburg, Germany) as previously described¹⁴. Ultrathin sections (60 to 90 nm) were obtained using an Ultracut E system (Reichert-Jung, Wien, Austria) and were mounted on formvar-coated and etched gold grids. Before labeling, sections were rinsed twice with 0.1 M glycine-PBS for five minutes and were incubated with 2% ovalbumin in PBS for 30 minutes to block unspecific antibody–antigen complexes. The grids were then incubated with polyclonal anti-₂m (dilution 1:500; Nordic, Tilburg, The Netherlands) or anti-amyloid P component (Dako, Glostrup, Denmark) antibodies in ovalbumin-PBS for two hours. After three 15-minute rinses in glycine-PBS, bound antibodies were visualized with 10 nm protein A gold (provided by Dr. J.W. Slot, University of Utrecht, Utrecht, The Netherlands). The sections were finally rinsed with PBS and were double distilled in water prior to counterstaining with aqueous uranyl acetate and lead citrate.

Double immunolabeling.

For double immunolabeling, the samples were fixed as for single labeling, cryoprotected by infusing 2.1 M sucrose in PBS, snap freezing, and storing in liquid nitrogen until analysis. Ultrathin cryosections were obtained using an FC-40 cryosystem (Reichert-Jung) and transferred to formvar-coated and etched gold grids in a drop of 2.3 M sucrose in PBS. Before labeling, sections were rinsed twice with 0.1 M glycine-PBS for 10 minutes and were incubated with 2% ovalbumin in PBS for 20 minutes. For immunolabeling, sections were first incubated with anti-2-microglobulin antibodies for 30 minutes and were labeled with 15 nm protein A gold after three glycine-PBS rinses. Then the sections were stabilized for five minutes with 0.5% glutaraldehyde in PBS, blocked with 150 mM NH₄Cl in PBS, and incubated with a rabbit anti-human lysosome-associated membrane protein (LAMP-1) antibody (dilution 1:120; provided by Dr. S.R. Carlsson, University of Umea, Umea, Sweden) or monoclonal anti-CD68 antibody (dilution 1:100; Dako) followed by unlabeled rabbit antimouse Ig (dilution 1:50; Dako). The ultrathin sections were rinsed with PBS and double-distilled water, were then counterstained with uranyl acetate, and were embedded in methyl cellulose¹⁵.

Controls were performed with the omission of the primary and/or the secondary antibodies, and/or the omission of the rabbit antimouse Ig. Examination was performed in a Hitachi H-600 AB and a Philips EM-300 transmission electron microscopes.

Morphology studies

Semithin sections of 1 m were obtained from small blocks of amyloid deposits embedded in Lowicryl K4M resin. Sections were transferred onto glass slides and were stained with 0.5% methylene blue 0.5% Borax for 30 seconds at 90°C. The stained sections were then mounted in DPX medium and were observed in a Polyvar 2 optical microscope (Reichert-Jung) under immersion oil.

For ultrastructural studies, small pieces of amyloid deposits were fixed as for Lowicryl embedding and were postfixed in 2.5% glutaraldehyde (vol/vol) in PBS. Then the samples were progressively treated in graded acetone-resin solutions, postfixed with 0.1% osmium tetroxide, and embedded in Spurr (Agar Scientific LTD, Stansted, UK). Ultrathin sections were obtained as for Lowicryl embedded samples using an Ultracut E system and were then counterstained with uranyl acetate and lead citrate before examination in the transmission electron microscope.

Quantitative evaluation of labeling density

Label density was estimated as the number of gold particles per area of amyloid deposit by stereological methods¹⁶. Differences were checked using the Student's t-test. A P of less than 0.05 was considered significant.

RESULTS

Amyloid diagnosis

The surgically obtained deposits were of amyloid origin, as they were positively stained with Congo red and displayed apple-green birefringence under polarized light. An example of Congo red staining without and with polarized light is given in Figure 1 a and b, respectively.

Figure 1.

Amyloid criteria: Congo red staining of a 2-microglobulin amyloid deposit observed under light microscopy (A) and under polarized light (B). The publication of this figure in color was made possible by a grant from Gambro Renal Care R&D, Lund, Sweden.

Full figure and legend (192K)

Deposits and related cells

Amyloid deposits were observed as nodules, consisting of intercellular amorphous material and long-shaped cells as well as macrophages. The long-shaped cells contained in the nodules presented an enlarged perinuclear region with long and narrow cytoplasmic processes extending to the amyloid material Figure 2. Electron microscopy showed that the extracellular material had a heterogeneous composition consisting of amyloid clumps and some scattered collagen fibers. The macrophage-like cells had an irregular surface with numerous invaginations that were usually occupied by the amyloid clumps (Figure 2 a, b). The cytoplasm had a granular appearance and showed a well-developed Golgi compartment, endoplasmic reticulum, and vesicle compartments. The vesicles seemed generated by endocytosis of the fibrillar clumps Figure 2c. The extracellular amyloid clumps were heavily labeled with anti-₂m antibody Figure 2b.

Figure 2.

Amyloid deposit location and 2-microglobulin distribution. (Inset) Methylene blue staining showing the cell distribution around the amyloid fibrils. (a) Electron microscopy showing a cell engulfing an amyloid deposit (short thick arrows). Cellular processes are indicated by thin arrows (18,000). Immunogold labeling with anti-2-microglobulin antibody showing an extracellular (b) and intracellular (c) location of an amyloid deposit (27,000).

Full figure and legend (368K)

Amyloid fibrils immunoreactive for anti-₂m antibody were also observed within the cytoplasm of macrophage-like cells. These intracellular fibrils were usually contained in vesicles, and cytoplasmic amyloid clumps were seldom devoid of membrane Figure 2c and 3. Intracellular labeling of ₂m was clearly not associated with the compartments related to the synthesis and secretory pathways of cells (endoplasmic reticulum or the Golgi compartment; Figure 3b).

Figure 3.

Intracellular immunolocalization of 2-microglobulin. No labeling with anti-₂-microglobulin antibody was observed in endoplasmic reticulum (er) (a, 33,000) nor in the Golgi compartment (g) (b, 33,000). (c) Double immunolabeling for 2-microglobulin (15 nm) and lysosome-associated protein (LAMP-1; 10 nm) showing that amyloid fibrils are within the lysosomes (40,000). (d) Double immunolabeling for 2-microglobulin (15 nm) and CD68 (10 nm) confirming that amyloid fibrils are within the lysosomes and that the cells involved are of macrophage-monocytic lineage (40,000).

Full figure and legend (304K)

Two series of studies of double immunolabeling were performed using the LAMP-1 or KP1 in addition to ₂m and amyloid P component antibodies, to characterize the cytoplasmic membranes containing amyloid fibrils. When anti-₂m antibody was incubated along with a polyclonal antihuman LAMP-1 antibody (lysosome marker), both antibodies colocalized on the same vesicular structures Figure 3c. Some vesicles labeled with anti-₂m antibody were only weakly labeled with LAMP-1 antibody. According to their location (marginal, close to the plasma membrane) and to their immunolabeling pattern (near constant LAMP-1 label), these vesicles had an endocytic character (that is, early endosomes or endocytic vesicles; Figure 3c).

When anti-₂m antibody was incubated along with KP1 antibody, their labeling again colocalized within the same vesicles. Because KP1 antibody is a lysosomal marker specific for monocytes/macrophages that is directed against the CD68 antigen, these experiments confirmed both the lysosomal character of the vesicles containing amyloid fibrils and the monocyte–macrophage lineage of the cells associated with the amyloid deposits.

Density labeling of ₂m and amyloid P component

The presence of numerous lysosomes filled with ₂m fibrils suggests a failure in degrading the engulfed material by the macrophages. To verify this hypothesis, differential density labeling studies between intracellular and extracellular material were performed for ₂m. Furthermore, the same studies were performed assessing the extracellular and intracellular density labeling of amyloid P component. Amyloid P component is a common constituent of most types of amyloidoses, irrespective of the characteristics of their major protein component. Extracellular density labeling for ₂m was not significantly different from the intracellular one (Figure 4 a, b). On the contrary, extracellular density labeling for amyloid P component was significantly stronger than the intracellular one (Figure 4 c, d), suggesting that the engulfed amyloid P component is processed by the cells, whereas there is no cellular processing of ₂m. The analysis of the density labeling is summarized in Table 1.

Figure 4.

Density labeling studies of 2-microglobulin and amyloid P component (APC): Comparison of intracellular and extracellular protein density. It can be observed that extracellular labeling of 2-microglobulin (a, 27,000) was similar to that observed intracellularly (b, 27,000), whereas a clear decrease in intracellular APC labeling (d, 33,000) was observed when comparing with extracellular APC labeling (c 37,800).

Full figure and legend (311K)

Table 1 - Intracellular and extracellular density labeling with anti-2-microglobulin and anti-amyloid P substance antibodies.

Full table (16K)

DISCUSSION

Our study shows, by immunogold labeling, that most of the cells around and between ₂m amyloid deposits were monocyte–macrophages, presenting cytoplasmic extension processes surrounding extracellular ₂m fibrils. Some cells, also positive for macrophage markers, contained intracellular ₂m amyloid fibrils. Double immunogold labeling allowed us for the first time to localize precisely intracellular ₂m fibrils within the lysosomes: LAMP-1 antibody was distributed on and underneath the membranes of the vesicles containing ₂m amyloid fibrils. These results were fully confirmed by KP1 antibody immunostaining. Finally, and more importantly, the analysis of differential density labeling of ₂m and amyloid P component showed that monocyte–macrophage cells are unable to degrade ₂m once it is in the lysosomes and that this impaired lysosomal function is specific for ₂m.

The studies aiming to characterize the cells participating in amyloid formation and/or persistence resulted in the identification of a variety of cells with a common feature: their monocyte–macrophage lineage^2,5,12,17,18. We have previously established, by specific antigen typing, the frequency of the different leukocyte cell types surrounding the amyloid deposits¹². However, there were no previous immunohistochemical studies at the ultrastructural level confirming the identity of the cells.

Because in most of the different amyloidoses the amyloid proteins result from partial proteolytic digestion of their precursors, proteolysis has been considered for a long time as a prerequisite for amyloid fibril formation¹. Because monocytes and macrophages are enriched in lysosomal structures that may proteolyze the different amyloid precursors, these cells have been thought to be responsible for the amyloid fibril formation^1,2,4,192021. However, the opposite may also hold true, and macrophages could participate in the reaction processes of amyloidosis by trying to remove the amyloid fibrils as they clear other exogenous or endogenous material^22,23. If this were the case, extracellular amyloid deposition would take place when the rate of amyloid production overcomes the clearing capacity of macrophages.

Interestingly, there are a few exceptions to the proteolysis rule in amyloidosis. The first known exception has been ₂m amyloidosis. Although a few reports suggest that the amyloidogenic ₂m may be proteolyzed in its N-terminal side²⁴, several groups have shown that the amyloid deposits in ₂m amyloidosis are mainly constituted of intact ₂m^25,26. Other examples of unproteolyzed precursor proteins have been reported subsequently by Buxbaun for the AL type of amyloidosis²⁷ and by Ericsson et al for the AA type of amyloidosis²⁸. Finally, atrial natriuretic factor is frequently found in intact form in amyloid fibrils of the senile type of amyloidosis²⁹.

Our findings are very much supportive of a phagocytosis role for monocyte macrophages and render less likely the participation of these cells in the synthesis of amyloid fibrils. (a) We found the extracellular amyloid fibrils frequently surrounded by cellular extensions at different stages of endocytosis. (b) We observed intracellular ₂m unevenly distributed and located the amyloid fibrils within the lysosomes, and (c) we observed no label for ₂m in the synthesis pathways of the cells.

Because the amyloidogenic ₂m is intact in its N-terminal side, it seems unlikely that macrophages with lysosomes filled with ₂m participate in synthesis of amyloid fibrils.

A second line of evidence supporting the phagocytic role for the cells surrounding amyloid fibrils comes from Alzheimer's disease. Cataldo et al reported the up-regulation of cathepsin D synthesis and the accumulation of hydrolase-laden lysosomes in pyramidal neurons, indicating that the endosomal-lysosomal system is abnormally activated in Alzheimer's disease³⁰. On the other hand, the addition of inhibitors of lysosomal proteinases does not block the formation of amyloid fibrils in the in vitro setting³¹³²³³. In other words, the endosomal-lysosomal system is activated, but its blockade does not result in preventing the formation of amyloid fibrils. Thus, such activation would be a consequence rather than the cause of amyloid fibril formation. In keeping with the phagocytic hypothesis, no detection of messenger RNA of amyloid protein precursor (APP) has been found by in situ hybridization in plaque-associated microglia, suggesting no synthesis of the amyloid precursor³⁴.

Once the ultrastructural studies enabled us to conclude that amyloid fibrils are contained in lysosomes, we aimed to step further and assessed the degradation of the different proteins of these amyloid fibrils. We compared the density labeling of the extracellular amyloid fibrils in regard to the intracellular ones. It came out clearly from our studies that macrophages were able to degrade the amyloid P component through phagocytosis and lysosomal processing. On the contrary, ₂m, after being internalized by the same cells, was not processed further. Therefore, the lysosomes of macrophages were unable to pursue the processing of ₂m. Whether the lack of processing of ₂m is due to the conformational situation of the molecule within amyloid fibrils or to an intrinsic characteristic of ₂m is not known. Very recently, Inoue et al have nicely shown the arrangement of ₂m, amyloid P component, and chondroitin sulfate proteoglycan within amyloid fibrils³⁵. According to these elegant ultrastructural studies, ₂m is associated with the surface of the amyloid fibrils³⁵ and would be accessible to lysosomal proteases. Therefore, the structure of ₂m, comprising of two -pleated sheets of three and four strands, respectively, may render it resistant to degradation. Whether the accumulated ₂m behaves like a cell toxin remains to be demonstrated.

In summary, although the biochemical analysis of the different types of amyloidosis is quite well established, the cellular participation has been less assessed. There is some controversy as to whether the cells surrounding the amyloid deposits synthesize the amyloid fibrils or are there to degrade them. Previous studies on AL and AA amyloidoses suggested a synthesis role. Our analysis of ₂m amyloid deposits showed that the phagocytic cells frequently observed in amyloid deposits have cytoplasmic extension processes in regards to the ₂m amyloid fibril-enriched zones. Further, it showed that the intracellular ₂m is preferentially and almost exclusively intralysosomal, and there were no signs of synthesis of ₂m or amyloid fibrils. We have previously reported the identification of the major serum antiprotease 2-macroglobulin in ₂m amyloid deposits³⁶ and proposed a role for antiproteases in the pathogenesis of amyloidosis³⁷. These data represent an extension from the protein to the cellular level on the assessment of the catabolism of amyloid fibrils. We hypothesize that failure of lysosomes to degrade intact ₂m may be one of the key points in the pathogenesis of ₂m amyloidosis. Our data are supportive of a phagocytic role for macrophages and provide some evidence that lysosomal function is not effective in processing and clearing ₂m, suggesting a putative ₂m toxicity on these cells.

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Acknowledgments

We thank Baxter Healthcare Corporation for contributing to the funding of this work through its Extramural Grant Program. The Centre Hospitalier Universitaire of Montpellier also funded this study. M.G. was a recipient of a grant from the CIRIT—Direcció General de Recerca, Generalitat de Catalunya. We thank Almudena García and the staff of Serveis Científico-Tècnics (Universitat de Barcelona) for technical assistance. The publication of Figure 1 in color was made possible by a grant from Gambro Renal Care R&D, Lund, Sweden. A part of the results included in this study was orally presented at the XXXIII^rd ERA-EDTA meeting in Amsterdam.