Extracellular vimentin is expressed at the rear of activated macrophage-like cells: Potential role in enhancement of migration and phagocytosis

Abstract

Macrophages have a vital role in the immune system through elimination of cell debris and microorganisms by phagocytosis. The activation of macrophages by tumour necrosis factor-α induces expression of extracellular cell-surface vimentin and promotes release of this vimentin into the extracellular environment. Vimentin is a cytoskeletal protein that is primarily located in the cytoplasm of cells. However, under circumstances like injury, stress, senescence and activation, vimentin can be expressed on the extracellular cell surface, or it can be released into the extracellular space. The characteristics of this extracellular vimentin, and its implications for the functional role of macrophages and the mechanism of secretion remain unclear. Here, we demonstrate that vimentin is released mainly from the back of macrophage-like cells. This polarisation is strongly enhanced upon macrophage activation. One-dimensional patterned lines showed that extracellular cell-surface vimentin is localised primarily at the back of activated macrophage-like cells. Through two-dimensional migration and phagocytosis assays, we show that this extracellular vimentin enhances migration and phagocytosis of macrophage-like cells. We further show that this extracellular vimentin forms agglomerates on the cell surface, in contrast to its intracellular filamentous form, and that it is released into the extracellular space in the form of small fragments. Taken together, we provide new insights into the release of extracellular cell-surface vimentin and its implications for macrophage functionality.

Keywords: activation; extracellular vimentin; macrophages; migration; phagocytosis; polarisation; vimentin secretion.

FIGURE 1

Polarised expression of extracellular vimentin on the surface of TNF-α–activated macrophages. (A) Representative HL-60 cells before (left) and after (right) differentiation with TPA. (B) Representative non-permeabilised fixed samples of macrophages (Differentiated) stained with the V9 anti-vimentin antibody, showing expression of vimentin on their surface after 1, 3, and 6 days of TNF-α activation. Top row: Phase contrast images. Middle row: Fluorescent images for vimentin (red). Bottom row: Overlay of phase contrast and fluorescent images. Scale bar, 10 µm. (C) Quantification of the proportion of macrophages expressing extracellular cell-surface vimentin in a polarised manner over the 6 days of TNF-α activation. n denotes the total number of cells analyzed, error bars correspond to standard deviation.

FIGURE 2

(A) Live cell imaging of activated macrophages using CSV antibody. (B) Comparison of permeablized vs. non permeablized activated macrophages labelled using beta actin antibody and anti vimentin V9 antibody (Nucleus, blue:actin, green: vimentin, magenta). (C) M1 macrophage classification using CD68 macrophage activation marker after TNF- α activation. Scale bar 10 µm.

FIGURE 3

Visualisation of surface vimentin on macrophages patterned on one-dimensional lines. (A) Representative three-dimensional projections of a TNF-α–activated macrophage (red, surface vimentin: green, cell membrane), demonstrated using IMARIS. Scale bar, 7 µm. (B) Illustration of the vimentin secretion sites (red) for the differentiated and TNF-α–activated macrophages using patterned lines. Yellow, nucleus; green, cell membrane. (C) Representative differentiated and TNF-α–activated macrophage attached and elongated along the pattern on a glass coverslip, revealing the site of vimentin secretion (yellow, nucleus; magenta, extracellular cell-surface vimentin). Scale bar, 10 μm.

FIGURE 4

Visualisation of surface vimentin on macrophages differentiated from GFP-vimentin HL60. (A) Maximum intensity projection and orthogonal views of differentiated and TNF-α–activated macrophage (red, membrane; green, surface vimentin; blue, nucleus), acquired with confocal microscopy. Scale bar, 10 µm. (B) Scanning electron microscopy images of vimentin secretion sites for the differentiated and TNF-α–activated macrophages. Bottom row: higher magnification of vimentin secretion site marked with yellow rectangle. This study was performed on 2D (glass coverslips) not on fibronectin coated 1D patterns. In order to have more residual cells after differentiation and activation, we used 1 day activation of both confocal LSM900 (A) and SEM imaging (B). (C) Confocal and SEM imaging using coverslips with grid. Top: Fluorescence images of vimentin (red; V9 antibody) stained in non-permeablized activated macropahges. Scale bar 10 μm. Bottom: SEM images of the same cell where the dot like structure can be seen at the same site of vimentin staining. Bottom (Right): magnified image of area marked in yellow of left. Scale bar 2 μm.

FIGURE 5

TNF-α–activated macrophages secrete vimentin into the extracellular space. (A) Representative total internal reflection fluorescence microscopy of secreted vimentin (magenta) around a TNF-α–activated macrophage (green, cell membrane). Scale bar is 10 µm. (B) Quantification of the relative fluorescence of vimentin in the media from activated macrophages expressing GFP-tagged vimentin (repeated 2 times with six replicates of each condition). Conditions: Control, blank; media, RMPI; Differentiation, media from differentiated macrophages; 1 day activation TNF-α; 3 days activation, media from macrophages activated for 3 days with TNF-α. * p < 0.05; ** p < 0.01 (Student’s t-tests).

FIGURE 6

Stimulation of migration and phagocytosis of macrophages by addition of recombinant (rh)Vimentin. (A) Migration tracks of TNF-α–activated macrophages visualised using TrackMate ImageJ plugin.(B) Quantification of migration speeds of differentiated macrophages without and with addition to the medium of 100 ng/ml rhVimentin and anti-cell-surface vimentin antibody, and of TNF-α–activated macrophages without and with addition of the anti-cell-surface vimentin antibody (CSV). (C) Visualisation of fluorescent beads (green) phagocytosed by a TNF-α–activated macrophage using fluorescence microscopy. Cell outline (yellow) defined from phase contrast image. (D) Phagocytic index determined by the fluorescence intensity within the differentiated macrophages without and with addition of 100 ng/ml rhVimentin and anti-vimentin antibody (V9), and of TNF-α–activated macrophages without and with addition of the anti-vimentin antibody (V9). *, p < 0.05; **, p < 0.01; ***, p < 0.001. n.s. not significant (Student’s t-tests). All experiments were repeated three times. The data is presented as box plots with whiskers drawn within the 1.5 IQR value. N indicates the total number of cells analyzed.All experiments were done three times.

FIGURE 7

Proposed model of vimentin secretion. (A) A macrophage with intracellular vimentin in the filamentous form. (B) Dissociation of filamentous vimentin at the cell membrane in the activated macrophage might lead to agglomeration at the surface of the macrophage. Then, the vimentin can be released into the extracellular space in the form of small fragments.