Cellular uptake of ß2M and AGE-ß2M in synovial fibroblasts and macrophages
Kalisha D. O'Neill1,
Neal X. Chen1,
Mu Wang2,
Ross Cocklin2,
Yilong Zhang2 and
Sharon M. Moe1,3,
1 Department of Medicine and
2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN and
3 Roudebush Veterans Affairs Medical Center, Indianapolis, IN, USA
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Abstract
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Background. Beta-2-microglobulin (ß2M) amyloidosis is a destructive articular disease affecting dialysis patients. The amyloid deposits contain both ß2M and ß2M altered with advanced glycation end products (AGE-ß2M). We have shown that ß2M increases the expression of matrix metalloproteinase-1, vascular cell adhesion molecule-1 and cyclooxygenase-2 in human synovial fibroblasts, while the effect of AGE-ß2M in this model is markedly reduced. Conversely, in human monocyte/macrophages, AGE-ß2M stimulates cytokine release whereas ß2M is less potent.
Methods. To understand why the two forms of ß2M produce variable responses in different cells, AGE-ß2M was labelled with the fluorochrome Cy5, and ß2M was labelled with the fluorochrome Texas Red (TR) and the uptake of 50 µg/ml of each was examined through live cell imaging at different time points using confocal microscopy.
Results. In human synovial fibroblasts, the AGE-ß2M-Cy5 could be seen in endosome-like structures inside cells by 45 min. After 3.5 h the distribution of endosome-like structures had become perinuclear in nature and the concentration of AGE-ß2M-Cy5 within these structures had increased. When a 20-fold excess of AGE-BSA was added to the synovial fibroblasts with the AGE-ß2M-Cy5, the endosome-like particles were not seen, suggesting competitive inhibition of uptake through an AGE-receptor. In contrast, ß2M-TR progressively concentrated along the surface of synovial fibroblasts with minimal cellular uptake indicated by faint endosome-like structures seen only after 8 h. Interestingly, in a different model, human and mouse monocyte/macrophages, the AGE-ß2M-Cy5 and ß2M-TR had similar patterns of distribution and kinetics of uptake.
Conclusion. Our results suggest that ß2M and AGE-ß2M are endocytosed via different mechanisms in human synovial fibroblasts and monocytes/macrophages. These results may offer a potential explanation of differences observed in cell culture experiments.
Keywords: advanced glycation; ß2 microglobulin; dialysis amyloidosis; end products; endocytosis; macrophages; synovial fibroblasts
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Introduction
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Beta-2-microglobulin (ß2M) amyloidosis is a destructive osteoarticular disease that affects patients undergoing long-term dialysis. The predominant manifestations are articular, including carpal tunnel syndrome, bone cysts and spondylolisthesis [1]. Pathological analysis reveals earliest deposition of an acellular amyloid containing ß2M fibrils in the cartilage and synovium [2,3]. Later in the disease course, macrophage infiltration appears [4,5]. The pathogenesis of the disease remains elusive. However, evidence supports the overall retention of ß2M with time on dialysis as a prerequisite for the development of the disease. Unfortunately, based on radiolabelled experiments in animals, the only known site of ß2M clearance is the kidney [6]. In the proximal tubule, ß2M is endocytosed in a classic receptor-mediated endocytic pathway with recycling of the receptor and subsequent degradation in lysosomes [7].
Studies in the last few years have demonstrated the presence of advanced glycation end products (AGEs) in ß2M amyloid deposits [2,3]. AGEs are a heterogeneous group of proteins with glucose or carbohydrate adducts, and are felt responsible for many complications of diabetes and ageing [8]. In vitro experiments with monocytes/macrophages have demonstrated that AGE-ß2M is more potent than ß2M in inducing chemotaxis and the release of interleukin 1ß (IL-1ß), interleukin-6 (IL-6), tumour necrosis factor alpha (TNF
) and transforming growth factor ß (TGFß), mediated through the receptor for advanced glycation end products (RAGE) [911]. Conversely, in synovial fibroblasts, we have found that ß2M, but not AGE-ß2M, leads to increased protein production of matrix metalloproteinase-1 (MMP-1) but not its inhibitor tissue inhibitor metalloproteinase-1 (TIMP-1), vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2) [1214]. By northern blot analysis, mRNA expression for MMP-1 and VCAM-1 was increased in the presence of ß2M. Alteration of the ß2M with AGE markedly diminished this response, although it remained greater than control conditions [12,13]. Similarly, Owen et al. [15] found AGE-ß2M inhibited collagen synthesis in synovial fibroblasts through a RAGE mediated process. Thus, alteration of ß2M with AGE appears to increase its activity in monocyte/macrophages, but decrease or negate its activity in synovial fibroblasts.
These different responses of ß2M and AGE-ß2M led us to hypothesize that the cellular uptake of these proteins differs in synovial fibroblasts and monocyte/macrophages. To test this hypothesis, we examined the cellular uptake of ß2M and AGE-ß2M using fluorescent labelled proteins and live cell imaging. This technique is useful for determining the pattern of cellular uptake via endocytosis. Once endocytosed, two pathways are available. In one, the receptorligand complex is transported to early endosomes where receptors and their ligands are sorted, with subsequent recycling of the receptor back to the cell membrane and transport of the ligand to lysosomes. The other potential pathway is transport fluid phase uptake and then to lysosomes where degradation occurs [16].
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Subjects and methods
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Isolation of human synovial fibroblasts
Human synovial fibroblasts were isolated from human synovium obtained from discarded tissue of osteoarthritis patients undergoing total hip or total knee replacements, as described previously [1214] and approved by the local Institutional Review Board. Briefly, synovial tissue was minced and digested by collagenase (Wako, Richmond, VA) followed by trypsin (Sigma, St Louis, MO). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Rockville, MD) with 20% fetal bovine serum (FBS; Sigma) and only passages four to six were utilized, as previous studies demonstrated pure fibroblast population by these passages [13]. For some experiments, human foreskin fibroblasts (gift of Dr Dan Spandau, Indiana University Department of Dermatology) were utilized.
Isolation of human monocyte/macrophages
Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) by the adherence method. Diluted blood (10 ml) was layered onto 5 ml of Histopaque®-1077 (Sigma) and centrifuged at 400 g for 30 min. The buffy layer was isolated and washed with 1xPBS. The PBMCs were then re-suspended in serum-free Roswell Park Memorial Institute media (RPMI-1640) (ATCC, Manassas, VA) and plated at 2x106 cells per cover slip Petri dish and incubated for 1 h in a humidified 37°C, 5% CO2 incubator. The media and non-adherent cells were aspirated and the wells were washed twice with warm serum-free RPMI, and 10% FBS-RPMI was then added to the remaining monocytes (
25% of the PBMCs). The cells were allowed to differentiate into macrophage-like cells for 7 days, confirmed by their appearance with phase contrast microscopy [11]. For some experiments, mouse (J2) and human (U937) macrophage cell lines (ATCC) were utilized.
Isolation and purification of ß2M and AGE-ß2M
ß2M was isolated and purified as described previously [1214]. Briefly, ultrafiltrate was collected during haemofiltration with an F-80 polysulfone hollow fibre dialyzer (Fresenius, Ogden, UT) from a chronic (25 years) haemodialysis patient with documented ß2M amyloidosis. The ultrafiltrate was concentrated, desalted and then separated by isoelectric focusing. The purity was confirmed on silver stained polyacrylamide gel electrophoresis (SDSPAGE), and the protein run over a Toxigel column (Pierce, Rockford, IL) to remove lipopolysaccharide. The purified ß2M was divided into two fractions. One was incubated in glycating buffer (1xPBS, 200 mM D-glucose, 1.5 mM PMSF, 1 mM EDTA, all from Sigma) at 37°C for 60 days and represents glycated ß2M (AGE-ß2M). The other fraction was incubated with 1.5 mM PMSF and 1 mM EDTA at 4°C for 60 days and represents non-glycated ß2M. At the end of 60 days, the fractions were dialyzed separately to remove glycating buffer and/or preservatives.
Identifying AGEs
The ß2M was found to not contain AGE protein by western blot to an anti-AGE antibody (Altean, Inc., Ramsen, NJ) and lacked the typical fluorescence pattern of AGE proteins as described previously [13]. In addition, bands for ß2M and AGE-ß2M were cut from an SDSPAGE gel and a MassPrep Proteome Workstation (Packard Instruments) was used to robotically destain, reduce, alkylate, digest, extract and spot the proteins onto a matrix-assisted laser desorption/ionization mass spectrometry (MALDI) plate for analysis. The peptides were extracted to confirm the presence of AGEs in our AGE-ß2M sample. Once confident of the protein identification, the measured peptide masses along with theoretical peptide masses were re-evaluated with a simple Perl script. The Perl script matches and sends to an output file any measured mass that corresponds to a theoretical peptide plus the additional mass of different AGEs, Amadori product or Schiff base (+162 Da). The pyrraline (+108 Da), imidazolone B (+142 Da), imidazolone A (+144 Da), AFGP (antifreeze glycoprotein, +270 Da), pentosidine (+59 Da), ALI (arginine-lysine imidazole, +90 Da), FFI [2-furoyl-4(5)-(2-furanyl)-1H-imidazole, +96 Da] and crossline (+253 Da) forms of AGE products were considered when searching for potential sites. The ß2M and AGE-ß2M spectra were then compared to see if there was a significant difference in peptide peak intensity between the samples. The MALDI analysis confirmed the presence of significant amounts of pentosidine, ALI, FFI, and crossline AGEs in our AGE-ß2M sample than in the ß2M sample (Table 1
), with ALI and FFI the most abundant. There were no matches for imidazolone A or B, AFGP or pyrraline in the AGE-ß2M sample.
Fluorescent labelling of ß2M and AGE-ß2M and live cell imaging
ß2M was labelled with Texas Red-X dye (TR) using a Texas Red®-X Protein Labeling Kit (Molecular Probes, Eugene, OR). AGE-ß2M was labelled with FluoroLinkTM Cy5 monofunctional dye (Amersham Pharmacia Biotech, Piscataway, NJ). The solutions were then dialyzed to remove excess dye. The final concentrations were determined by competitive RIA and integrity of ß2M confirmed on SDSPAGE.
For the experiments, the live cells were removed from the incubator, rinsed and placed at 37°C for 10 min for equilibration in M2 media (150 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 5 mM KCl, 1 mM MgCl2 and 25 mM glucose, at pH 7.4). This media was aspirated and replaced with either 50 µg/ml ß2M-TR solution in M2 or a 50 µg/ml AGE-ß2M-Cy5 solution in M2. The cells were consistently kept at 37°C while images were captured with MRC-1024 laser scanning confocal microscopy (Bio-Rad, Hercules, CA). The cells were imaged for a maximum of 60 min, to ensure continued viability. For longer experiments, parallel experiments were performed using cells incubated with fluorochrome at 37°C, 5% CO2 in an incubator, and then removed at specific time points. For some experiments, Dextran-TR and Transferrin-TR (gifts from Dr Ken Dunn, Indiana University) and FITC labelled anti-MHC-I antibody (PharMingen, San Diego, CA) were utilized.
Electron microscopy
Synovial fibroblasts were isolated and cultured as described above and seeded on 18 mm circle glass disks in a 12-well culture plate. Cells were then incubated with a 50 µg/ml ß2M-TR solution in M2 for 60 min at 37°C and 5% CO2. Cells were fixed with 4% paraformaldehyde, then rinsed with 1xPBS. Photo-oxidation was performed as described previously by Sundin et al. [17]. Appropriate excitation filters were positioned directly over cells immersed in DAB substrate solution (Vector Laboratories, Burlingame, CA) at 4°C. A mercury lamp suspended 30 cm above the filter cube was used to excite the ß2M-TR sample with
568 nm light for 3 min three times. After each excitation, fresh 4°C DAB solution was added to the sample wells. Reactive species produced by photo-oxidation of the fluorophores react with DAB to form a dense electron microscope-observable reaction product wherever ß2M-TR is present. Samples were fixed with 1% glutaraldehyde for 1 h, washed with PBS four times, post-fixed with 0.14% osmium tetroxide/0.04% tannic acid/0.50% K3Fe(CN)6 in PBS, dehydrated with graded ethanol, infiltrated with graded solutions of propylene oxide/Spurr's resin to 100% Spurr's resin (incubated for 2x1 h at 100%), and then polymerized overnight at 65°C [17]. Sections 80 nm in thickness were mounted on nickel grids and stained with 2% aqueous uranyl acetate. The cells were viewed at 80 kV in a Philips CM 120 electron microscope (FEI Co., Hilsboro, OR) and digital images were captured with a Gatan 791 Mulit-Scan Camera (Gatan Inc., Pleasanton, CA).
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Results
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Live cell imaging of synovial fibroblasts
Live cell imaging of synovial fibroblasts demonstrated that AGE-ß2M becomes internalized by 3545 min in a punctate pattern consistent with endocytosis (Figure 1A
, arrow). In contrast, the ß2M without alteration by AGE localized around the cells (Figure 1B
, arrow). Reversing the fluorochrome labels had no effect on the uptake pattern, indicating that the fluorescent moiety was not responsible for the different uptake patterns of ß2M and AGE-ß2M. Experiments with primary skin fibroblasts showed a similar pattern of uptake (data not shown).

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Fig. 1. Endocyte uptake of ß2M and AGE-ß2M in synovial fibroblasts. Live human synovial fibroblasts were incubated for 45 min in the presence of 50 µg/ml AGE-ß2M-Cy5 (A) or ß2M-TR (B) and imaged by confocal microscopy. The faint white colour is the media, and the dark areas are cells. An outline of a cell is indicated by dashed line in box in (A). The uptake pattern of AGE-ß2M-Cy5 revealed punctate structures consistent with endocytosis (A, arrow). In contrast, these structures were not seen in the cells incubated with ß2M-TR where the fluorochrome lined up in a pattern consistent with the outline of the cell surface (B, arrow).
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To determine the effect of time on uptake, synovial fibroblasts were incubated in parallel in the presence of ß2M-TR or AGE-ß2M-Cy5 and different dishes removed at periodic intervals for imaging. The ß2M became progressively concentrated at or adjacent to the plasma membrane or cell surface, although gradually over 8 h, more endosome-like particles were observed in some cells (Figure 2A
C). In contrast, the AGE-ß2M became progressively concentrated over time in a perinuclear pattern (Figure 2D
F).

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Fig. 2. Effect of time on endocyte uptake of ß2M and AGE-ß2M in synovial fibroblasts. Live synovial fibroblasts were incubated in the presence of 50 µg/ml ß2M-TR (AC) or 50 µg/ml AGE-ß2M-Cy5 (DF), and imaged by confocal microscopy at 3.5, 6 and 8 h using parallel experiments. Similar to observations at 45 min, the ß2M-TR remained localized along the perimeter of the cell (AC, closed arrow) until 68 h when some intracellular uptake was noted (C, open arrow). In contrast, the AGE-ß2M-Cy5 showed uptake into punctate structures (DF, arrow) that became progressively concentrated in a perinuclear pattern by 68 h (E and F, arrow).
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To confirm that the ß2M remained clustered or concentrated along the plasma membrane, electron microscopy of synovial fibroblasts was performed using photo-oxidation techniques described previously [17]. These studies demonstrated that the ß2M remained concentrated or clustered at or near the cell surface (black rim around the apical side of cell; Figure 3
, arrow). There are no confirmed cell membrane receptors for ß2M, but exogenous ß2M (MHC-I light chain) is known to freely exchange with the heavy chains of the MHC-I complex in lymphocytes [18]. To determine if ß2M added to the media combined with MHC-I, co-localization experiments were performed utilizing ß2M-TR and a fluorescently labelled anti-MHC-I antibody. The results demonstrated co-localization as evidenced by a yellow pattern (combined red and green fluorescence). However, both antibodies stained so intensely and covered the entire cell surface when individually imaged, that true co-localization could not be clearly differentiated from identical staining patterns (data not shown).

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Fig. 3. Electron microscopy of ß2M uptake in synovial fibroblasts. Synovial fibroblasts were incubated with ß2M-TR for 1 h, followed by photo-oxidation and imaging with electron microscopy. The black reaction indicates the presence of ß2M. In synovial fibroblasts, the ß2M was localized along the apical cell surface and not intracellularly (arrow, 5600x).
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To determine if AGE-ß2M was taken up by synovial fibroblasts into late endosomes or lysosomes, co-localization experiments were performed with fluorescently labelled transferrin or dextran. Protein uptake can occur via a receptor-mediated transferrin-like pathway, with endocytosis into early endosomes, followed by release of iron from transferrin at the lower pH of the early sorting endosomes and recycling of the receptor and transferrin to the cell surface from early/recycling endosomes. Alternatively, endocytosis can occur via a fluid phase or dextran-like pathway with uptake being followed by transit of dextran with eventual deposition in lysosomes through the endocytic pathway within minutes [16]. Dextran concentrates in lysosomes due to an inability to be degraded and thus serves as a marker for lysosomes. The pattern of AGE-ß2M staining in synovial fibroblasts was consistent with that observed with the fluid phase/lysosomal marker dextran-TR (Figure 4A and D
). In contrast, its staining pattern was different from the early endosome marker transferrin-TR (Figure 4B and E
), confirming trafficking of the AGE-ß2M to lysosomes.

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Fig. 4. Mechanism of AGE-ß2M endocytic uptake. To determine the mechanism of AGE-ß2M uptake in synovial fibroblasts, live cells were simultaneously incubated with AGE-ß2M-Cy5 without (A) or with dextran-TR (D), a marker of lysosomes. The punctate-receptor pattern of AGE-ß2M-Cy5 matched that of dextran, confirming lysosomal uptake. In contrast, when AGE-ß2M-Cy5 was co-incubated without (B) or with transferrin-TR (E), the patterns were not similar suggesting no role for endosomal recycling. The uptake of AGE-ß2M-Cy5 was also incubated without (C) or with 20x AGE-BSA (F), which blocked the endosomal uptake, indicating this was via AGE-specific mechanism. The differences in the background colour of the figures are due to normal variability between experiments.
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To determine if the AGE modification induced differential uptake and trafficking of AGE-ß2M in synovial fibroblasts, competitive inhibition by AGE-BSA was performed. Synovial fibroblasts were incubated with a 20-fold excess of AGE-BSA together with AGE-ß2M-Cy5, or ß2M-TR. The AGE-ß2M uptake could no longer be visualized in the presence of excess AGE-BSA (Figure 4C and F
), suggesting similar uptake pathways of the two AGE-modified proteins. However, the binding of ß2M around the cell surface was unaffected by AGE-BSA.
Live cell imaging of monocyte/macrophages
Uptake of ß2M and AGE-ß2M was also examined in human and mouse monocytes/macrophages. In human monocytes/macrophages, the AGE-ß2M-Cy5 is rapidly taken up, and then concentrated near the nucleus (Figure 5A and C
). The ß2M not altered with AGE was also taken up, but at what appeared to be a slower rate (Figure 5B and D
). However, this technique does not allow quantification. Similar to these human primary cells, experiments with mouse (J2) and human (U937) transformed monocyte/macrophage cell lines showed both AGE-ß2M and ß2M were endocytosed and trafficked similarly (data not shown).

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Fig. 5. Endocytic uptake of ß2M and AGE-ß2M in monocytes/macrophages. Live human monocytes/macrophages were incubated with AGE-ß2M-Cy5 and ß2M-TR, and uptake determined. An outline of a monocyte/macrophage is depicted in the upper corner. Compared with images at 5 min (A), the AGE-ß2M was rapidly taken up and heavily concentrated in the cell by 60 min (C, arrow). Similarly, the ß2M-TR was not visualized intracellularly or around the cell perimeter at 5 min (B), but could be observed in a perinuclear pattern by 60 min (D, arrow).
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Discussion
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Experimental studies have demonstrated discordant effects of AGE-ß2M and ß2M in synovial fibroblasts and monocytes/macrophages, suggesting differences in uptake, which was confirmed in the present study using live cell imaging. In vivo AGE-modified proteins consist of many glycated/oxidated alterations, which cannot be exactly replicated in vitro. However, our MALDI analysis shows that our in vitro modified AGE-ß2M, compared with ß2M, does have significantly greater quantities of several AGE compounds commonly observed in vivo, including pentosidine, FFI, ALI and crossline AGEs. In synovial fibroblasts, ß2M without alteration by AGE progressively concentrated at or near the cell surface within 45 min, and only after 8 h were a few endosome-like particles observed. Previous studies have shown that human skin fibroblasts do not rapidly internalize their Class I MHC, taking hours rather than minutes to internalize. Once eventually in the cell, the MHC-I-ß2M are transported to lysosomes for degradation [19]. Similar to our findings by live cell and electron microscopy in synovial fibroblasts, Huet et al. [19] found ß2M localized along the cell surface in skin fibroblasts by electron microscopy. This sticking of ß2M on the cell surface may still induce cell signalling, as the non-covalent binding of ß2M to the heavy chains of MHC-I led to changes in intracellular calcium and phosphatidylinositol-3-kinase activity in lymphocytes [20]. We have similarly found that ß2M induced increases in intracellular calcium in synovial fibroblasts [14]. In addition, inhibitors of intracellular calcium and phosphatidylinositol-3-kinase could block VCAM-1, and COX-2 expression in synovial fibroblasts in response to ß2M [14]. Thus, given the absence of a known alternative cell surface receptor for ß2M, and evidence that exogenous ß2M can attach to the heavy chains of MHC-I [18,21], it is very plausible that in synovial fibroblasts binding of exogenous ß2M to MHC-I could trigger cell signalling pathways leading to the release of inflammatory like mediators.
In contrast, live cell imaging of synovial fibroblasts demonstrated that AGE-ß2M was endocytosed by 3545 min and completely transported near the nucleus by 8 h. We demonstrated that the pattern of AGE-ß2M staining was similar to that observed with dextran, a lysosomal marker, but not similar to transferrin, a marker of early endosomal receptor recycling. With competitive inhibition by AGE-BSA, we were no longer able to visualize uptake of AGE-ß2M. This suggests that AGE-ß2M is taken up by an AGE-receptor, presumably through RAGE, which has been identified in synovial fibroblasts [15]. In contrast, there was no inhibition of the unmodified ß2M with AGE-albumin, confirming distinct mechanisms of cellular uptake. Thus, in synovial fibroblasts, the alteration of ß2M with AGE negates or decreases the ß2M-induced release of MMP-1, VCAM-1, COX-2 and collagen synthesis [1215]. The transport of AGE-ß2M to lysosomes for degradation, but continued cell signalling by ß2M that is bound to cell surface MHC-I, may explain these observations.
In monocytes/macrophages, both AGE-ß2M and ß2M were taken up, and then concentrated near the nucleus. This pattern was similar to that observed in the renal proximal tubule, where anionic receptors appear to mediate the uptake of ß2M to endosomes and then lysosomes, as demonstrated by biochemical and semi-quantitative morphological methods [7]. In monocytes/macrophages, AGE proteins have been shown to be taken up via both RAGE [22] and a distinct scavenger receptor [23,24]. This uptake could then lead to the release of inflammatory molecules seen in the monocyte/macrophage response to exposure to AGE-ß2M in vitro. The ß2M that was not altered with AGE appeared to be taken up more slowly, suggesting, but not confirming, different uptake mechanisms than AGE-ß2M, and possibly explaining the relative increased potency of AGE-ß2M over ß2M in experimental models [911]. However, live cell imaging cannot quantify such a potential difference in the uptake of ß2M or AGE-ß2M. It was clear, however, that the ß2M did not concentrate at the cell surface in monocytes/macrophages as was observed in synovial fibroblasts. If our hypothesis that ß2M-induced activity is mediated by its association with the heavy chains of MHC-I is correct, the lack of this cell surface pattern of ß2M in monocytes/macrophages would be consistent with the reduced effect of ß2M compared with AGE-ß2M in vitro [911].
These findings in monocytes/macrophages are also consistent with previous reports of ultrastructural findings in pathological specimens from patients with ß2M amyloidosis. Macrophages generally appear late in the course of ß2M amyloidosis, after initial deposition of ß2M amyloid in the synovium and capsule [25]. However, macrophages appear to be important in the process. Niwa et al. [5,26,27] found AGE in endosome-like structures in macrophage-like cells surrounding amyloid deposits. Presumably, in the early endosomes, the ß2M and AGE are separated, with transport of the AGE to lysosomes and possible recycling of ß2M via endosomes. Indeed, by electron microscopy, Garcia-Garcia [5] found ß2M localized in both endosomes and lysosomes in macrophages around ß2M amyloid tissue by immunoelectron microscopy. Similarly, Brancaccio [27] found that in electron micrograph sections of ß2M amyloid specimens, ß2M and AGE were co-localized by immunostaining in similar compartments, but staining for ß2M and AGE were also found in different subcellular compartments.
In summary, the cellular uptake pattern of ß2M and AGE-ß2M differed in synovial fibroblasts and monocytes/macrophages. Taken together, one could hypothesize that the initial process involved in the formation of ß2M amyloid may be a response of synovial fibroblasts to high synovial concentration of ß2M, which binds to the heavy chains of MHC-I. This leads to the production of COX-2 [14], metalloproteinases [12] and adhesion molecules [13], resulting in degradation of the joint surface where ß2M amyloid deposits by an, as of yet, undefined process. Once deposited, the ß2M becomes glycated, leading to an infiltration of macrophages, which attempt to phagocytose the AGE-ß2M, but in the process of doing so also release cytokines. Perhaps the late onset of clinical symptoms is a result of this macrophage-induced release of cytokines. Clearly, much work remains to be conducted to fully understand the pathogenesis of this debilitating disease.
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Acknowledgments
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This work was presented in part at the American Society of Nephrology Annual Meeting 2000, and was supported by a Veteran's Administration Merit Review Award. The authors wish to thank Dr David Sundin for his helpful comments and Ms Anni Hine for her excellent secretarial assistance.
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Notes
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Correspondence and offprint requests to: Sharon M. Moe, MD, Associate Professor of Medicine, Assistant Dean for Research Support, Indiana University School of Medicine, 1001 West 10th Street, OPW 526, Indianapolis, IN 46202, USA. Email: smoe{at}iupui.edu 
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Received for publication: 14. 2.02
Accepted in revised form: 16. 8.02