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. 2018 Mar 29;38(2):BSR20171217.
doi: 10.1042/BSR20171217. Print 2018 Apr 27.

Cell-cell Interactions Between Monocytes/Macrophages and Synoviocyte-Like Cells Promote Inflammatory Cell Infiltration Mediated by Augmentation of MCP-1 Production in Temporomandibular Joint

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Free PMC article

Cell-cell Interactions Between Monocytes/Macrophages and Synoviocyte-Like Cells Promote Inflammatory Cell Infiltration Mediated by Augmentation of MCP-1 Production in Temporomandibular Joint

Miho Ibi et al. Biosci Rep. .
Free PMC article

Abstract

Many inflammatory cells are known to be home to inflamed temporomandibular joint (TMJ) tissues by stimulation with cytokines and chemokines produced by inflammatory lesions in the TMJ. However, how the inflammatory cells affect the progression of inflammation in TMJ synovial tissues after their homing to inflamed TMJ site is still uncertain. Here, we isolated and cultured TMJ synoviocyte-like cells (TMJSCs) from murine TMJ tissues. We demonstrated that interleukin 1β (IL-1β) up-regulated expression of monocyte chemoattractant protein 1 (MCP-1) in TMJSCs. In addition, we found that IL-1β-treated TMJSCs strongly promoted migratory activity of mouse monocyte/macrophage RAW264.7 cells through secretion of MCP-1. On the other hand, IL-1β up-regulated expression levels of intracellular adhesion molecule 1 (ICAM-1), a leukocyte adhesion ligand in TMJSCs. In addition, IL-1β promoted cell-cell adhesion between TMJSCs and RAW264.7 cells. Intriguingly, we also found that cell-cell interactions mediated through soluble factors other than IL-1β and cell-cell adhesion molecules between IL-1β-stimulated TMJSCs and RAW264.7 cells synergistically augmented secretion of MCP-1 from these cells. Therefore, these results suggested that the IL-1β-induced recruitment of monocyte/macrophage lineage cells to inflamed synovial membranes in TMJ was further augmented by the cell-cell interaction-induced secretion of MCP-1 from the inflammation site, possibly resulting in prolonged inflammatory responses in TMJ synovial tissue.

Keywords: MCP-1; monocyte/macrophage; synoviocyte-like cells; temporomandibular joint.

Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. TMJSCs exhibit fibroblastic character but do not exhibit leukocyte character
(A) Morphology of cultured TMJSCs obtained from a 10-week-old EGFP mouse at passage 11 (phase contrast, left picture; EGFP, right picture). After the soft tissues were collected from around male EGFP mouse TMJ, TMJSCs were obtained by the outgrowth method, as described in ‘Materials and methods’ section. TMJSCs were cultured and passaged in 20% FBS in DMEM supplemented with rhFGF-2 (10 ng/ml). Scale bar, 200 μm. (B) Characterization of TMJSCs. TMJSCs, NIH3T3 cells, and RAW264.7 cells were cultured and grown to 80% confluence, then total RNA was isolated from each culture. mRNA expression patterns of colIα1 and vimentin (mesenchymal cells markers) or CD45 (leukocyte common antigen) in cultured TMJSCs, NIH3T3 cells, and RAW264.7 cells were evaluated by RT-qPCR. The mRNA expression levels of these genes were normalized to those of 18S rRNA, and the relative expression levels are shown as fold-increases or decreases relative to the level in NIH3T3 cells (colIα1 and vimentin) or RAW264.7 cells (CD45).
Figure 2
Figure 2. MCP-1 secreted from IL-1β-stimulated TMJSCs promotes migratory activity of RAW264.7 cells
(A) Dose-dependent response of MCP-1 mRNA expression by TMJSCs to IL-1β stimulation. TMJSCs were stimulated for 4 h with 0.01–1 ng/ml of IL-1β. The mRNA expression levels of MCP-1 were normalized to those of GAPDH and the relative expression levels are shown as fold-increases or decreases relative to the level in controls. (B) MCP-1 production from IL-1β-stimulated TMJSCs. TMJSCs were stimulated for 27 h with 0.01 ng/ml of IL-1β, and the conditioned medium was collected. The protein level of MCP-1 in the conditioned medium was measured by ELISA, as described in the ‘Materials and methods’ section. The relative production levels are shown as fold-increases or decreases relative to the level in controls. (A,B) Each value represents the mean ± S.D. (n=4). Similar results were obtained in two independent experiments. *P<0.05, **P<0.01, and ****P<0.0001 were considered significant compared with the control. (C) CCR2 mRNA expression in RAW264.7 cells and primary macrophages. The mRNA expression levels of CCR2 in C57BL/6J mouse splenocytes, resident peritoneal macrophages, RAW264.7 cells, TMJSCs, and NIH3T3 cells were evaluated by RT-qPCR. The levels were normalized to those of 18S rRNA and the relative expression levels are shown as fold-increases or decreases relative to the levels in C57BL/6J mouse splenocytes (data from C57BL/6J mouse splenocytes as a control were not shown). C57BL/6J mouse splenocytes and NIH3T3 cells were used as positive or negative controls, respectively. (D) Chemotaxis assay of RAW264.7 cells in response to rmMCP-1, as described in ‘Materials and methods’ section. Seven or eight different fields with migrated cells were recorded as photomicrographs, and the migrated cell numbers were counted for each field (n=8 for control; n=7 for rmMCP-1). Each value represents the mean ± S.D. Similar results were obtained in two independent experiments. **P<0.01 was considered significant compared with the control. (E) Effects of CCR2 selective antagonist, RS504393 on TMJSCs-secreted chemotactic factor-induced migration of RAW264.7 cells. This assay was carried out using transwell cell culture inserts, as described in ‘Materials and methods’ section. Ten different fields with migrated cells were recorded as photomicrographs, and the migrated cell numbers were counted for each field (n=10). Each value represents the mean ± S.D. Values of ****P<0.0001 were considered to be statistically significant.
Figure 3
Figure 3. IL-1β promotes adhesion of TMJSCs to RAW264.7 cells
(A) RAW264.7 cell adhesion assay for IL-1β-stimulated TMJSCs. RAW264.7 cells were layered over IL-1β-stimulated TMJSCs. Unbound cells were removed and immunostained with anti-CD45, anti-GFP, phalloidin, and DAPI (red, anti-CD45; green, anti-GFP or phalloidin; blue, DAPI). Scale bar, 50 μm. (B) Effect of IL-1β on the ability of TMJSCs to adhere to RAW264.7 cells. TMJSCs adhered to not less than three CD45-positive cells, which localized on GFP and phalloidin-positive areas, was counted in eight different fields. Each value represents the mean ± S.D. (n=8). Similar results were obtained in two independent experiments. **P<0.01 was considered significant compared with the control. (C) Dose-dependent response of mRNA expression of ICAM-1 in TMJSCs to IL-1β stimulation. TMJSCs were seeded in each well of 24-well plates and stimulated for 4 h with 0.01–1 ng/ml of IL-1β. The mRNA expression level of ICAM-1 was normalized to that of GAPDH. Each value represents the mean ± S.D. (n=4). ****P<0.0001 was considered significant compared with the control. (D) Immunostaining of ICAM-1 in TMJSCs stimulated with IL-1β. TMJSCs were seeded on 12-mm round coverslips in 24-well plates for 3 days. Then, after TMJSCs were starved, the cells were stimulated with 0.1 ng/ml IL-1β for 24 h. The cells were then fixed in methanol/acetone and immunostained with anti-ICAM-1 and anti-GFP (red, anti-ICAM-1; green, anti-GFP; and blue, DAPI). Scale bar, 50 μm. (E) CD18 mRNA expression in RAW264.7 cells and primary macrophages. The mRNA expression levels of CD18 in C57BL/6J mouse splenocytes, resident peritoneal macrophages, RAW264.7 cells, and NIH3T3 cells were evaluated by RT-qPCR. The levels were normalized to those of 18S rRNA and the relative expression levels are shown as increases or decreases relative to the level in C57BL/6J mouse splenocytes (data from C57BL/6J mouse splenocytes as a control were not shown). C57BL/6J mouse splenocytes and NIH3T3 cells were used as positive or negative controls, respectively.
Figure 4
Figure 4. Cell–cell interactions mediated through soluble factors and cell–cell adhesion molecules between IL-1β-stimulated TMJSCs and RAW264.7 cells synergistically augment secretion of MCP-1 from these cells
(A) Schema of the direct or indirect co-culture system. (B) MCP-1 production in conditioned medium from monocultures and direct or indirect co-cultures of IL-1β-stimulated TMJSCs and RAW264.7 cells. For preparation of the conditioned medium from IL-1β-treated TMJSC monoculture, TMJSCs were first stimulated for 24 h with 0.1 ng/ml of IL-1β. The IL-1β-stimulated TMJSCs were subsequently washed for the removal of IL-1β. Then, the fresh medium without IL-1β was added to each well to obtain conditioned medium. Then, the transwell devices were inserted into the individual wells of the plates. And then, the fresh medium without IL-1β was added on to the top of the membranes without seeding any RAW264.7 cells on to the top of the membranes. Then, the IL-1β-removed TMJSCs monoculture was maintained further for 24 h. On the other hand, for preparation of the conditioned medium from RAW264.7 cells monoculture, fresh medium with 0.1 ng/ml of IL-1β was added into each well in 24-well cell culture insert companion plates without seeding of any TMJSCs, and subsequently maintained for 24 h. After the washing of each well of the culture plates, the fresh medium without IL-1β was added to the each well. Then, the transwell devices were inserted into the individual wells of the plates. RAW264.7 cells (1 × 105 cells/well) were subsequently seeded on the top of the membranes and cultured further for 24 h. For the preparation of conditioned medium from the co-culture between TMJSCs and RAW264.7 cells without cell–cell contact, TMJSCs were first stimulated for 24 h with 0.1 ng/ml of IL-1β. The IL-1β-stimulated TMJSCs were subsequently washed for the removal of IL-1β. Then, the fresh medium without IL-1β was added to each well. Then, the transwell devices were inserted into the individual wells of the plates. RAW264.7 cells (1 × 105 cells/well) were seeded on the top of the membranes and cultured further for 24 h. On the other hand, for the preparation of conditioned medium from the co-culture between IL-1β-treated TMJSCs and RAW264.7 cells with cell-cell contact, TMJSCs were first stimulated for 24 h with 0.1 ng/ml of IL-1β. The IL-1β-stimulated TMJSCs were subsequently washed for the removal of IL-1β. Then, RAW264.7 cells (1 × 105 cells/well) were seeded and layered on to the IL-1β-stimulated TMJSCs monolayer and co-cultured for an additional 24 h. Each value represents the mean ± S.D. (n=4). *P<0.05 and ***P<0.001 were considered statistically significant.

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