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. 2010 Feb 18;115(7):1444-52.
doi: 10.1182/blood-2009-04-218735. Epub 2009 Nov 6.

Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis

Céline Cougoule  1 , Véronique Le Cabec, Renaud Poincloux, Talal Al Saati, Jean-Louis Mège, Guillaume Tabouret, Clifford A Lowell, Nathalie Laviolette-Malirat, Isabelle Maridonneau-Parini
Affiliations

Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis

Céline Cougoule et al. Blood. .

Erratum in

  • Blood. 2010 Sep 23;116(12):2195

Abstract

Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck(-/-) mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow-derived macrophages (BMDMs), were affected in Hck(-/-) BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain-of-3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocyte-limited expression with a role in 3D migration, could be a target for new anti-inflammatory and antitumor molecules.

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Figures

Figure 1
Figure 1
Phagocyte recruitment in the peritoneal cavity is inhibited in Hck−/− mice. (A) Total inflammatory cell recruitment in wt and Hck−/− mice after thioglycollate injection in the peritoneal cavity (n = 4-10 mice per group). (B) Recruitment of macrophages and neutrophils is decreased in Hck−/− mice 24 and 72 hours after thioglycollate injection (n = 3). At 0 hours, neutrophils are absent in the peritoneal cavity and resident macrophages are more numerous in wt mice. (C) Immunohistochemistry of macrophages (F4/80 antibodies) performed before and 72 hours after thioglycollate injection. Micrographs show a representative experiment of 3; macrophages are accumulating in peritoneal tissue of Hck−/− mice and no apoptotic cell is noticed. Original magnification ×100 (insets: ×400). Asterisks indicate areas of macrophage accumulation. (D) Quantification of macrophage and neutrophil distribution in peritoneal tissue from wt and Hck−/− mice (expressed as the number of immunostained cells/mm2 of peritoneal tissue). *P < .05; **P < .01; ***P < .001.
Figure 2
Figure 2
Hck is required for 3- but not 2-dimensional macrophage migration. (A) The gates used for flow cytometry sorting of wt or Hck−/− CD11b+ and F4/80+ BMDMs. Hck−/− BMDMs (gray open histograms) express the same levels as wt (black open histograms) of the differentiation markers CD11b and F4/80 antigens. Closed histograms represent cells alone. (B) Log10 expression levels of genes in resting wt and Hck−/− BMDMs in M1 (□) or M2 (■) polarization. Results are expressed as the ratio of the expression level in Hck−/− BMDMs versus wt BMDMs. (C-D) Defective migration of Hck−/− BMDMs through Matrigel matrix. (C) BMDMs were cultured on commercial Matrigel transwells for 24 hours and cells that reached the lower face of the membrane were counted. Mean value of migrating wt cells (860 ± 723 cells; n = 3) was set arbitrarily to 100%. (D) Quantification of 3D cell migration experiments performed in triplicate. Mean value of migrating wt cells (38% ± 14.3%) was set arbitrarily to 100% (n = 4). (E-F) BMDMs from wt and Hck−/− mice have similar 2D-migration capacity tested with 2 in vitro assays. (E) Scraped area replenishment assay. Replenishment of the scraped area was measured at the indicated time points using the ImageJ software. (F) wt and Hck−/− BMDM 2D-migration through uncoated or fibronectin-coated transwells. BMDMs that reached the lower face of transwells were counted. Mean value of migrating cells (n = 3) was set arbitrarily to 100%; *P < .05; ***P < .001.
Figure 3
Figure 3
Ectopic expression of Hck provides fibroblasts the capacity to migrate through Matrigel. (A) Tet-Off MEF-3T3 cells expressing Hck isoforms together or separately that migrated through Matrigel transwells were counted as in the legend of Figure 2C. Results are expressed as mean ± SD of 3 experiments; parental cells were Hck-negative Tet-Off MEF-3T3 cells. (B) Photomicrograph showing nuclei (DAPI [4,6 diamidino-2-phenylindole]: blue), p59/61Hckca-GFP (green), and F-actin (phalloidin–tetramethyl rhodamine isothiocyanate: red); arrows indicate podosome rosettes located at the pore exit (dotted circles) of the transwell. p59/61Hckca-MEF-3T3 Tet-Off cells had more podosome rosettes after transmigration (24 hours, cells examined at the bottom face of the 8-μm pore transwells) than before (0 hours, cells examined at the top of transwells). (C) The cell migration capacity through Matrigel is correlated to the rate of podosome rosettes. The percentage of cells with podosome rosettes was quantified in 2 p59/61Hckca-MEF-3T3 Tet-Off cell clones (clones A and B; left panel) and invasion assays were performed in triplicate in 4 separate experiments (right panel); ***P < .001.
Figure 4
Figure 4
Hck−/− BMDMs do not form large podosome rosettes. BMDMs were seeded on fibronectin-coated coverslips for 24 hours, fixed, and permeabilized. F-actin was stained with Texas red–coupled phalloidin; vinculin, with primary and FITC-coupled secondary antibodies; and nuclei, with DAPI, and cells were examined by fluorescence microscopy. (A top 2 panels) Characteristic podosomes are present in wt and Hck−/− BMDMs with an actin core surrounded by a ring of vinculin (insets). (Bottom 2 panels) wt BMDMs form a large podosome rosette (arrowhead on the Merge image); Hck−/− BMDMs do not form large, but small, podosome rosettes (arrowhead; scale bar represents 10 μm; magnification ×100). (B) Quantification of podosome rosettes in wt and Hck−/− BMDMs. Rosettes were counted in at least 100 cells in duplicate (n = 4). (C) Quantification of small and large rosettes in wt and Hck−/− BMDMs. Hck−/− BMDMs preferentially form small podosome rosettes (n = 4); **P < .01; ***P < .001.
Figure 5
Figure 5
Podosome rosette–associated ECM degradation is a protease-dependent process altered in Hck−/− BMDMs. (A) Podosome rosettes exhibit ECM degradation activity. BMDMs were seeded on FITC-coupled gelatin-coated coverslips. After 24 hours, cells were fixed and stained for F-actin and microscopically examined. Large degradation area is formed underneath large podosome rosettes (i). In contrast, small degradation area is observed underneath small podosome rosettes (ii). Measurements of fluorescent intensities along the white dashed line depicted in subpanels i and ii in the merged image shows increase of intensities of F-actin (red line) correlating with loss of fluorescent intensity of the gelatin (green line). (B) Podosome rosette is a site for LysoTracker accumulation. (C) MT1-MMP and MMP2 localize at sites of gelatin-FITC degradation. BMDMs were subjected to cell surface labeling with MT1-MMP and MMP2 antibodies. (D-E) BMDMs were seeded on FITC-coupled gelatin-coated coverslips and incubated overnight in the presence or absence of a protease inhibitor cocktail, then fixed and stained for F-actin and nuclei and microscopically examined for quantification. (D top 2 panels) wt BMDMs form large podosome rosettes associated with large areas of gelatin-FITC degradation (arrowheads). Hck−/− BMDMs form small podosome rosettes and degrade small gelatin areas (arrowheads). (Bottom 2 panels) The same experiments performed in the presence of protease inhibitors (PI), which significantly block matrix degradation (scale bar represents 10 μm; magnification ×40). (E) Quantification of FITC-coupled gelatin degradation by BMDMs in the presence and in the absence of protease inhibitors. The percentage of degradation corresponds to the number of pixels of degradation for 100 pixels of cell surface. Results are expressed as mean ± SEM (n = 8) of FITC-gelatin degradation areas. (F) wt and Hck−/− BMDMs express similar levels of MMP2, MMP9, and MT1-MMP. Immunoblot analysis performed on BMDM cell lysates for MMP9 (92 kDa), MMP2 (72 kDa), MT1-MMP (64 kDa), and actin (46 kDa). (G) Hck−/− BMDMs are not defective in MMP9 and MMP2 activity and release. Gelatin zymograph of BMDM cell lysates or conditioned media collected from cells adhering on fibronectin.
Figure 6
Figure 6
Protease-dependent macrophage migration through Matrigel. (A) Scanning electron microscopy images showing wt and Hck−/− BMDMs (green) on thick Matrigel (brown) transwells in the presence or absence of protease inhibitors. Matrigel remodeling (arrows) is more important with wt than Hck−/− BMDMs and is inhibited by protease inhibitors. Images are showing BMDMs infiltrating the matrix and are representative of 3 experiments (scale bar represents 10 μm). (B) Protease inhibitors inhibit both wt and Hck−/− BMDMs migration through Matrigel. Results are expressed as the percentage of cells that migrate through Matrigel (mean ± SEM; n = 4). ***P < .001.
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