The CC chemokine ligand, CCL2/MCP1, participates in macrophage fusion and foreign body giant cell formation

Abstract

The foreign body reaction (FBR) develops in response to the implantation of almost all biomaterials and can be detrimental to their function. The formation of foreign body giant cells (FBGC), which damage the surface of biomaterials, is considered a hallmark of this reaction. FBGC derive from blood-borne monocytes that enter the implantation site after surgery in response to the release of chemotactic signals. In this study, we implanted biomaterials subcutaneous (s.c.) in mice that lack the monocyte chemoattractant CC chemokine ligand 2 (CCL2) and found that biomaterials were encapsulated despite reduced FBGC formation. The latter was due to compromised macrophage fusion rather than migration. Consistent with the reduction in FBGC formation, biodegradable biomaterials sustained reduced damage in CCL2-null mice. Furthermore, blockade of CCL2 function by localized gene delivery in wild-type mice hindered FBGC formation, despite normal monocyte recruitment. The requirement for CCL2 in fusion was confirmed by the ability of both a CCL2 inhibitory peptide and an anti-CCL2 Ab to reduce FBGC formation from peripheral blood monocytes in an in vitro assay. Our findings demonstrate a previously unreported involvement of CCL2 in FBGC formation, and suggest that FBGC are not the primary determinants of capsule formation in the FBR.

Figure 1

CCL2-null mice display reduced FBGC formation. Sponges and filters were implanted s.c. in mice for a period of 2 and 4 weeks, respectively. Representative images are shown of sections stained with H&E from filters (F), (A and B) and sponges (S), (C and D), implanted in wild-type (A and C) and CCL2-null mice (B and D). Arrows indicate the presence of FBGC. Magnification, ×400 (A and B), ×200 (C and D). E: The number of FBGC (cells with three or more nuclei) per high-power field was estimated by examination of H&E- and Mac-3-stained sections (n = 50). The number of FBGC per unit length surrounding filters was also estimated because capsules did not occupy an entire high-power microscopic field (see A and B). (F) Reduced number of nuclei in CCL2-null FBGC. The number of nuclei per FBGC (from sponges and filters) was determined by microscopic examination of over 100 FBGC per genotype. The location of nuclei within FBGC was similar between the two groups. The data represent mean ± SEM. *, n = 8; statistical significance, P ≤ 0.05, when compared with wild-type using Student’s t-test.

Figure 2

Increased presence of cells of the monocyte lineage in CCL2-null capsules. Representative sections of capsules, implanted s.c. for 4 weeks and stained with anti-F4/80 (A and B) and anti-Mac3 (C and D) Ab from wild-type (A and C) and CCL2-null (B and D) mice are shown. Ab stains were visualized with the peroxidase reaction (brown color). Arrows indicate the presence of FBGC (A and C) and macrophages (B and D). FBGC are not immunoreactive with F4/80 (A) but can be detected with Mac3 (C). The abundance of mononuclear macrophages in CCL2-null sections is evident. Nuclei are visualized with methyl green counterstain. Original magnification, ×400.

Figure 3

Reduced biomaterial degradation in CCL2-null mice. Representative sections are shown of scaffold implanted s.c. for 4 weeks in CCL2-null (A) and wild-type (B) mice and stained with Masson’s trichrome. In CCL2-null mice, scaffolds underwent minimal degradation and collagen deposition. The majority of the cells present within the scaffold were mononuclear inflammatory cells (arrows), and the scaffold was not subjected to fibrovascular invasion. In wild-type mice (B), scaffolds were invaded by a fibrovascular response, evident by the presence of collagen (bright blue color) and blood vessels (yellow arrows). In addition, numerous FBGC were observed. Scaffold remnants have a grayish blue color appearance. Images were taken from the center area of the scaffolds. Original magnification, ×200. C: The average percentage of a high-power field (0.04 μm²) occupied by scaffold 4 weeks following implantation was estimated with the aid of imaging software. A total of three scaffolds per genotype were used to obtain images and a total of 24 images per scaffold were analyzed. The data represent mean ± SEM. *, Statistical significance, P ≤ 0.05, when compared with wild-type using Student’s t-test.

Figure 4

CCL2 is present in FBGC. Sections of a filter (A) and sponges (B–D) implanted s.c. in wild-type mice for 4 and 2 weeks, respectively, were stained with anti-CCL2 Ab (A–C) or control Ab (D) and visualized with the peroxidase reaction (brown color). Arrows indicate the presence of FBGC in wild-type mice (A–B, D) and CCL2-null mice (C). F and S denote areas occupied by filter and sponge, respectively. The FBGC in A and B are positive for CCL2, whereas those in C and D are negative. Nuclei are visualized with methyl green counterstain. Original magnification, ×400.

Figure 5

Reduced FBGC formation in vivo by localized blockade of CCL2. Representative sections are shown of pMT-7ND GAM- (A and B) and pcDNA3 GAM- (C) coated filters implanted s.c. for 4 weeks in wild-type mice and stained with H&E (A) or the macrophage marker Mac3 and visualized with the peroxidase reaction (B and C). In A–C, arrows indicate individual macrophages, and arrowheads indicate FBGC. Magnification, ×400 (A–C). (D) The number of FBGC (cells with three or more nuclei) per unit length of filter was estimated by the examination of Mac3-stained sections. The data represent mean ± SEM, n = 75; *, statistical significance, P ≤ 0.05. Representative sections are shown of pMT-7ND GAM- (F) and pcDNA3 GAM- (E) coated filters stained with an anti-FLAG FITC-conjugated Ab. Arrows in F indicate FLAG-expressing cells in the capsule. Magnification, ×200 (E–F).

Figure 6

Reduced fusion following treatment of monocytes with a CCL2 inhibitory peptide. Monocytes were induced to fuse in the presence of an inactive, control peptide MCP-1 (9–76-4A) (A) or active, inhibitory peptide MCP-1 (9–76) (B). A total of 10 fields per well and five wells per group were analyzed. Cells with three or more nuclei were classified as FBGC (examples denoted by arrows in A and B). Magnification, ×200. The average number of nuclei per giant cell is shown (C). Percentage fusion was determined by estimating the number of nuclei in FBGC in relation to the total number of nuclei (D). The data represent mean ± SEM. *, Statistical significance, P ≤ 0.05, when compared with control peptide treatment using Student’s t-test.

Figure 7

Reduced fusion following treatment of monocytes with a function-blocking anti-MCP-1 Ab. Monocytes were treated as described in the text and a total of 10 fields per well and five wells per group were analyzed. Cells with three or more nuclei were classified as FBGC (A). Percentage fusion was determined by estimating the number of nuclei in FBGC in relation to the total number of nuclei (B). The data represent mean ± SEM. *, Statistical significance, P ≤ 0.05, when compared with isotype control IgG treatment using Student’s t-test.