Title: Novel Grooved Substrata Stimulate Macrophage Fusion, CCL2 and MMP-9 Secretion

Moon, Haisle (OBMS, UBC)
Cremmel, Clement V.M. (ETH Zurich)
Kulpa, Alina (ECE, UBC)
Jaeger, Nicolas A.F. (ECE, UBC)
Spencer, Nicholas D. (ETH Zurich)
Waterfield, John D. (OBMS, UBC)
Brunette, Donald M. (OBMS, UBC)

Introduction

The monocyte-derived macrophage is one of the first cell types found on newly implanted devices and is known to play a role in inflammation, immunoregulation and wound healing depending on its functional phenotype. Classically activated (M1) macrophages are associated with classical inflammatory responses whereas alternatively activated (M2) macrophages are involved in wound healing or immunoregulatory behavior. They also undergo fusion to form multinucleated giant cells (MGC) in various conditions. This study examines the effect of a novel topography, grooved surfaces, fabricated by anisotropic-etching of Si (110) crystals, on gene expression and cyto-/chemokine secretion of RAW 264.7 macrophages and its association with cell fusion.

Materials and Methods

Novel grooved surfaces G1 and G2 were fabricated by anisotropic-etching technique of Si (110) crystals. G2, produced by more vigorous etching than used in the production of G1, had a less ordered pattern than G1. Murine macrophage-like RAW 264.7 cells were cultured on polished (Pol), G1 and G2 for 1 and 5 days. Scanning electron microscopy (SEM) was used to study the cell morphology. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the gene expression and cyto-/chemokine antibody microarray and ELISA were used to quantify the cyto-/chemokine secretion pattern of the macrophage cells. To study multinucleated cell formation, DAPI staining was used.

Results

Preliminary results showed elongated morphology of cells cultured on Pol with the presence of IL4 (Pol + IL4—M2 phenotype control) for 24hrs. Cells cultured on G1 and G2 exhibited elongated morphology. Another morphological finding was in greater number of multinucleated cells (MC) on both G1 and G2 substrata than on Pol on Day5. In addition, a significant increase in gene expression of RAW 264.7 on G1 vs. Pol surface was detected on Day1 for CCL2/MCP-1 and CCL4/MIP-1β, whereas on Day5, CCL2/MCP-1, CCL4/MIP-1β, CCL5/RANTES, and CCL7/MCP-3, but not CCL3/MIP-1α, increased significantly. At the protein level, Day1 secretion profiles of RAW264.7 cultured on Pol, G1, and G2 surfaces showed higher secretion of CCL3/MIP-1α and CCL4/MIP-1β on both G1 and G2 surfaces relative to the Pol surface. In addition, on Day1, a higher secretion level of the anti-inflammatory cytokine IL-1ra was observed on Pol surface, but on Day5, IL-1ra secretion level was increased on G1 surface. In contrast, pro-inflammatory chemokine IP-10/CXCL10 secretion level increased on Pol surface on Day5 compared to that of G1. Also, on Day5, CCL2/MCP-1 and CCL4/MIP-1β secretion level increased on G2. Further quantitative analysis of CCL2/MCP-1 and CCL4/MIP-1β secretion level was done by sandwich ELISA. Macrophages cultured on G2 showed higher secretion level of CCL2/MCP-1 and CCL4/MIP-1β on both Day1 and Day5. Also, the quantitative analysis of MMP-9 using ELISA showed increased secretion level of MMP-9 on G2 compared to Pol on both Day1 and Day 5.

Discussion and Conclusion

The SEM micrographs exhibited an increased proportion of cells with an elongated morphology on G1 and G2 surfaces. In particular this elongated morphology was similar to the cell shape observed when the macrophages were treated with IL-4, a known inducer of the wound healing M2 phenotype. Macrophages exhibited enhanced cell fusion on the G1 and G2 surfaces relative to the Pol surface. This increase in cell fusion on G2 could be related to the increase in the secretion level of CCL2/MCP-1 observed on G2 surface: CCL2/MCP-1 being the critical participant in macrophage fusion. Therefore, it was of interest to analyze the secretion level of MMP-9, which is characteristic of foreign body giant cells (FBGC), on cells cultured on the novel grooved surfaces using ELISA. ELISA showed increased secretion level of MMP-9 on G2 surface compared to Pol surface. The increased gene expression of macrophage chemoattractants-CCL2/MCP-1, CCL3/MIP-1, CCL4/MIP-1β, and CCL7/MCP3-on G1 surface compared to Pol surface suggested that there was a tendency for increased expression of chemokines that are implicated in wound healing. This tendency was further explored using microarray and ELISA technologies to determine the amounts of cyto-/chemokines secreted by the macrophages on G1, G2 and control Pol surfaces. An increase in the secretion of macrophage-attractant chemokines CCL3/MIP-1 and CCL4/MIP-1β on both G1 and G2 for Day1 and an increase in CCL2-MCP-1 and CCL4/MIP-1β on G2 for Day5 was observed. However the profiles on the grooved surfaces were not identical to that found for the M2 phenotype. Thus, the novel grooved topographies appeared to induce changes in gene/protein expression, in particular an increase in the chemokines that are involved in recruitment and fusion of macrophages. More bi-nucleated or multinucleated cells reminiscent of FBGCs were seen on the grooved surfaces.


Figure1. SEM Micrographs of novel grooved surfaces fabricated by anisotropic-etching of Si (110) crystals without the use of photolithography: G1 with smooth walled v-shaped grooves (left) and G2 with less ordered grooved patterns (right)


Figure 2. Fluorescence images of topographic effects on multinucleated cell (MC) formation in RAW264.7 macrophages cultured on G1 (a) and G2 (b) on Day5 and analysis of multinucleated cell formation of RAW264.7 macrophages cultured on G1 (c) and G2 (d) for 5 days


Figure 3. Quantitative analysis of CCL2 secretion by RAW 264.7 macrophages cultured on Pol, G1 and G2 surfaces on Day1 and Day5 and quantitative analysis of MMP-9 secretion by RAW 264.7 macrophages cultured on Pol and G2 surfaces on Day1 and Day5

Acknowledgements

Supported by CIHR operating grant to Dr. Don Brunette

References

1. Barth KA, Waterfield JD, Brunette DM. The effect of surface roughness on RAW 264.7 macrophage phenotype. J Biomed Mater Res A. 2013;101(9):2679–88 2. Kyriakides TR, Foster MJ, Keeney GE, et al. The CC chemokine ligand, CCL2/MCP1, participates in macrophage fusion and foreign body giant cell formation. Am J Pathol. 2004;165(6):2157–66 3. MacLauchlan S, Skokos EA, Meznarich N, et al. Macrophage fusion, giant cell formation, and the foreign body response require matrix metalloproteinase 9. J Leukoc Biol. 2009;85(4):617–26

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