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Updated: Sep 28, 2026
Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue
Published on: February 24, 2023
Cellular uptake of beta2M and AGE-beta2M in synovial fibroblasts and macrophages
Kalisha D O'Neill1, Neal X Chen, Mu Wang
1Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
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.
Conclusion:
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.
Insights
Advanced glycation end products of beta-2-microglobulin (AGE-beta(2)M) and native beta-2-microglobulin (beta(2)M) are taken up differently by cells. This explains variable responses in dialysis patients with beta(2)M amyloidosis.
Area of Science:
- Biochemistry
- Cell Biology
- Medical Science
Background:
- Beta-2-microglobulin (beta(2)M) amyloidosis is a debilitating joint disease in dialysis patients.
- Amyloid deposits consist of beta(2)M and AGE-beta(2)M, which elicit different cellular responses.
- Native beta(2)M affects synovial fibroblasts, while AGE-beta(2)M impacts monocyte/macrophages.
Purpose of the Study:
- To investigate the distinct cellular uptake mechanisms of beta(2)M and AGE-beta(2)M.
- To understand the variable cellular responses to these two forms of beta(2)M.
Main Methods:
- Labeling AGE-beta(2)M with Cy5 and beta(2)M with Texas Red (TR).
- Utilizing live cell imaging and confocal microscopy to track uptake in human synovial fibroblasts and monocyte/macrophages.
- Employing competitive inhibition assays with AGE-BSA.
Main Results:
- AGE-beta(2)M was rapidly internalized into endosome-like structures in synovial fibroblasts, suggesting receptor-mediated uptake.
- Native beta(2)M showed minimal uptake in synovial fibroblasts, accumulating on the cell surface.
- Both AGE-beta(2)M and beta(2)M exhibited similar uptake patterns and kinetics in monocyte/macrophages.
Conclusions:
- Beta(2)M and AGE-beta(2)M utilize different endocytosis pathways in human synovial fibroblasts.
- Distinct cellular uptake mechanisms in monocytes/macrophages were observed.
- These findings provide a potential explanation for differential cell culture results in beta(2)M amyloidosis research.
