Cellular uptake of beta2M and AGE-beta2M in synovial fibroblasts and macrophages.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
O'Neill
Kalisha D
KD
Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Chen
Neal X
NX
Wang
Mu
M
Cocklin
Ross
R
Zhang
Yilong
Y
Moe
Sharon M
SM
D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001692:Biological Transport; D002478:Cells, Cultured; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D008854:Microscopy, Electron; D009000:Monocytes / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D010003:Osteoarthritis / Q000473:pathology / Q000601:surgery; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013583:Synovial Membrane / Q000473:pathology* / Q000648:ultrastructure; D001613:beta 2-Microglobulin / Q000302:isolation & purification / Q000378:metabolism*
D017127:Glycation End Products, Advanced; D001613:beta 2-Microglobulin; D001120:Arginine; C062187:pentosidine; D008239:Lysine