Macrophage-Mediated Phagocytosis and Dissolution of Amyloid-Like Fibrils in Mice, Monitored by Optical Imaging

Abstract

Light chain-associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore. After injecting this material subcutaneously in mice, optical imaging was used to quantitatively monitor phagocytosis and dissolution of fibrils concurrently. Histologic evaluation of the residual fibril masses revealed the presence of CD68⁺, F4/80⁺, ionized calcium binding adaptor molecule 1^- macrophages containing Congo red-stained fibrils as well as neutrophil-associated proteins with no evidence of intact neutrophils. These data suggest an early infiltration of neutrophils, followed by extensive phagocytosis of the light chain fibrils by macrophages, leading to dissolution of the mass. Optical imaging of this novel murine model, coupled with histologic evaluation, can be used to study the cellular mechanisms underlying dissolution of synthetic amyloid-like fibrils and human amyloid extracts. In addition, it may serve as a test bed to evaluate investigational opsonizing agents that might serve as therapeutic agents for light chain-associated amyloidosis.

Figure 1

Amyloid-like fibrils composed of rVλ6Wil are phagocytosed by murine macrophages in culture. A: The structure and varied aggregation states of rVλ6Wil amyloid-like fibrils were determined by electron microscopy. B and C: Incubation of murine RAW 264.7 cells (B) with rVλ6Wil fibrils (20 µg; 20% w/w pHrodo red-labeled, C) in culture results in 27% of the cell population staining positive for uptake of fibrils into the acidified phagolysosome. Scale bars: 200 nm (A); 1.2 μm (A, arrow). AU, arbitrary unit.

Figure 2

Phagocytosis of rVλ6Wil amyloid-like fibrils in mice monitored by optical imaging. A: Representative (mouse 2) optical images of fluorescence from a mouse with a s.c. rVλ6Wil fibrilloma containing 20% w/w pHrodo red–labeled fibrils. Images were captured from day 1 (D1) to day 18 (D18) after injection. B: Optical image of fibrilloma (arrowhead) harvested at 18 days after injection, associated with the skin tissue (arrow). C: Quantification of pHrodo red fluorescence intensity as background-corrected mean raw density. Individual data points are shown at each time point. D: Changes in background-corrected mean raw density pHrodo red fluorescence for each mouse over 18 days after injection. Data are expressed as means ± SD (C). ^∗P < 0.05.

Figure 3

Histologic evaluation of residual fibrilloma shows macrophages with intracellular amyloid. Representative photomicrographs of rVλ6Wil fibrilloma, with 15% w/w pHrodo red–labeled fibrils, harvested 18 days after injection. A: Hematoxylin and eosin (H&E)–stained tissue section of skin showing panninculus carnosus muscle (Mus.) and amyloid fibril–containing layer (Amy). B–D: H&E-stained tissue sections showing macrophages with apparent intracellular amyloid (arrows) and multinucleated giant cells (arrowheads). E: Congo red–stained s.c. amyloid viewed under cross-polarized filters showing green-gold birefringence associated with amyloid fibrils. F: Congo red–stained s.c. amyloid viewed under bright-field (BF) illumination showing congophilia associated with amyloid fibrils. G: Congo red–stained s.c. amyloid viewed with epifluorescent illumination (using Texas red emission filter) showing fluorescence of amyloid-like fibrils. H: Merged image of F and G indicating that all fluorescent amyloid-like fibrils appear intracellular. I–L: Immunostaining of human λ6 light chain, CD68, F4/80, and ionized calcium binding adaptor molecule 1 (Iba-1) in s.c. amyloid. M–P: Immunostaining of lysosomal associated membrane protein 1 (LAMP1), neutrophil elastase, Ly6G (using the RB6 monoclonal antibody clone), and myeloperoxidase (MPO) in s.c. amyloid. Red arrows indicate the presence of intracellular immunostained amyloid-like fibril masses. Scale bars: 50 μm (A and E–H); 20 μm (B–D and I–P).

Figure 4

Dual-fluorophore imaging showing phagocytosis and dissolution of rVλ6Wil fibrilloma in vivo. A: Optical imaging of fluorescence from mice with a s.c. rVλ6Wil fibrilloma (15% w/w Dylight800 and 15% w/w pHrodo red–labeled fibrils) in each subject on days 1 (D1), 7 (D7), and 15 (D15) after injection. Dylight800 data were scaled to the D1 image, and pHrodo red data were scaled to the D7 images. B–D: Quantification of the Dylight800 fluorescence intensity for the cohort (B), for each mouse in the study (C), and for the pHrodo red fluorescence (D) was determined by region-of-interest analyses and expressed as background-corrected mean raw density. B and D: Individual data points are shown at each time point. Data are expressed as means ± SD (B and D). ^∗P < 0.05, ^∗∗P < 0.01.