Beta-2-microglobulin (beta(2)M) amyloidosis is a destructive articular disease affecting dialysis patients. The amyloid deposits contain both beta(2)M and beta(2)M altered with advanced glycation end products (AGE-beta(2)M). We have shown that beta(2)M increases the expression of matrix metalloproteinase-1, vascular cell adhesion molecule-1 and cyclooxygenase-2 in human synovial fibroblasts, while the effect of AGE-beta(2)M in this model is markedly reduced. Conversely, in human monocyte/macrophages, AGE-beta(2)M stimulates cytokine release whereas beta(2)M is less potent.
To understand why the two forms of beta(2)M produce variable responses in different cells, AGE-beta(2)M was labelled with the fluorochrome Cy5, and beta(2)M was labelled with the fluorochrome Texas Red (TR) and the uptake of 50 microg/ml of each was examined through live cell imaging at different time points using confocal microscopy.
In human synovial fibroblasts, the AGE-beta(2)M-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-beta(2)M-Cy5 within these structures had increased. When a 20-fold excess of AGE-BSA was added to the synovial fibroblasts with the AGE-beta(2)M-Cy5, the endosome-like particles were not seen, suggesting competitive inhibition of uptake through an AGE-receptor. In contrast, beta(2)M-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-beta(2)M-Cy5 and beta(2)M-TR had similar patterns of distribution and kinetics of uptake.
Our results suggest that beta(2)M and AGE-beta(2)M 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.