Cellular uptake of β2M and AGE-β2M in synovial fibroblasts and macrophages

Kalisha D. O'Neill, Xuening (Neal) Chen, Mu Wang, Ross Cocklin, Yilong Zhang, Sharon Moe

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Background. Beta-2-microglobulin (β2M) amyloidosis is a destructive articular disease affecting dialysis patients. The amyloid deposits contain both β2M and β2M altered with advanced glycation end products (AGE-β2M). We have shown that β2M increases the expression of matrix metalloproteinase-1, vascular cell adhesion molecule-1 and cyclooxygenase-2 in human synovial fibroblasts, while the effect of AGE-β2M in this model is markedly reduced. Conversely, in human monocyte/macrophages, AGE-β2M stimulates cytokine release whereas β2M is less potent. Methods. To understand why the two forms of β2M produce variable responses in different cells, AGE-β2M was labelled with the fluorochrome Cy5, and β2M 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-β2M-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-β2M-Cy5 within these structures had increased. When a 20-fold excess of AGE-BSA was added to the synovial fibroblasts with the AGE-β2M-Cy5, the endosome-like particles were not seen, suggesting competitive inhibition of uptake through an AGE-receptor. In contrast, β2M-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-β2M-Cy5 Conclusion. Our results suggest that β2M and AGE-β2M 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.

Original languageEnglish
Pages (from-to)46-53
Number of pages8
JournalNephrology Dialysis Transplantation
Volume18
Issue number1
DOIs
StatePublished - Jan 1 2003

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Endosomes
Fibroblasts
Macrophages
Monocytes
Fluorescent Dyes
beta 2-Microglobulin
Matrix Metalloproteinase 1
Advanced Glycosylation End Products
Vascular Cell Adhesion Molecule-1
Amyloid Plaques
Amyloidosis
Cyclooxygenase 2
Endocytosis
Confocal Microscopy
Dialysis
Cell Culture Techniques
Joints
cyanine dye 5
Cytokines
Texas red

Keywords

  • β2 microglobulin
  • Advanced glycation
  • Dialysis amyloidosis
  • End products
  • Endocytosis
  • Macrophages
  • Synovial fibroblasts

ASJC Scopus subject areas

  • Nephrology
  • Transplantation

Cite this

Cellular uptake of β2M and AGE-β2M in synovial fibroblasts and macrophages. / O'Neill, Kalisha D.; Chen, Xuening (Neal); Wang, Mu; Cocklin, Ross; Zhang, Yilong; Moe, Sharon.

In: Nephrology Dialysis Transplantation, Vol. 18, No. 1, 01.01.2003, p. 46-53.

Research output: Contribution to journalArticle

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AU - Moe, Sharon

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N2 - Background. Beta-2-microglobulin (β2M) amyloidosis is a destructive articular disease affecting dialysis patients. The amyloid deposits contain both β2M and β2M altered with advanced glycation end products (AGE-β2M). We have shown that β2M increases the expression of matrix metalloproteinase-1, vascular cell adhesion molecule-1 and cyclooxygenase-2 in human synovial fibroblasts, while the effect of AGE-β2M in this model is markedly reduced. Conversely, in human monocyte/macrophages, AGE-β2M stimulates cytokine release whereas β2M is less potent. Methods. To understand why the two forms of β2M produce variable responses in different cells, AGE-β2M was labelled with the fluorochrome Cy5, and β2M 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-β2M-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-β2M-Cy5 within these structures had increased. When a 20-fold excess of AGE-BSA was added to the synovial fibroblasts with the AGE-β2M-Cy5, the endosome-like particles were not seen, suggesting competitive inhibition of uptake through an AGE-receptor. In contrast, β2M-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-β2M-Cy5 Conclusion. Our results suggest that β2M and AGE-β2M 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.

AB - Background. Beta-2-microglobulin (β2M) amyloidosis is a destructive articular disease affecting dialysis patients. The amyloid deposits contain both β2M and β2M altered with advanced glycation end products (AGE-β2M). We have shown that β2M increases the expression of matrix metalloproteinase-1, vascular cell adhesion molecule-1 and cyclooxygenase-2 in human synovial fibroblasts, while the effect of AGE-β2M in this model is markedly reduced. Conversely, in human monocyte/macrophages, AGE-β2M stimulates cytokine release whereas β2M is less potent. Methods. To understand why the two forms of β2M produce variable responses in different cells, AGE-β2M was labelled with the fluorochrome Cy5, and β2M 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-β2M-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-β2M-Cy5 within these structures had increased. When a 20-fold excess of AGE-BSA was added to the synovial fibroblasts with the AGE-β2M-Cy5, the endosome-like particles were not seen, suggesting competitive inhibition of uptake through an AGE-receptor. In contrast, β2M-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-β2M-Cy5 Conclusion. Our results suggest that β2M and AGE-β2M 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.

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