Nephrol Dial Transplant (2003) 18: 46-53
© 2003 European Renal Association-European Dialysis and Transplant Association
Cellular uptake of ß2M and AGE-ß2M in synovial fibroblasts and macrophages
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
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
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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