ABSTRACT
Rheumatoid arthritis (RA) is an autoimmune disease with a complex etiology. Monocyte-derived macrophages (MDMs) infiltration are associated with RA severity. We have reported the deletion of G-protein-coupled receptor kinase 2 (GRK2) reprograms macrophages toward an anti-inflammatory phenotype by recovering G-protein-coupled receptor signaling. However, as more GRK2-interacting proteins were discovered, the GRK2 interactome mechanisms in RA have been understudied. Thus, in the collagen-induced arthritis mouse model, we performed genetic GRK2 deletion using GRK2f/fLyz2-Cre+/- mice. Synovial inflammation and M1 polarization were improved in GRK2f/fLyz2-Cre+/- mice. Supporting experiments with RNA-seq and dual-luciferase reporter assays identified peroxisome proliferator-activated receptor γ (PPARγ) as a new GRK2-interacting protein. We further confirmed that fms-related tyrosine kinase 1 (Flt-1), which promoted macrophage migration to induce angiogenesis, was inhibited by GRK2-PPARγ signaling. Mechanistically, excess GRK2 membrane recruitment in CIA MDMs reduced the activation of PPARγ ligand-binding domain and enhanced Flt-1 transcription. Furthermore, the treatment of mice with GRK2 activity inhibitor resulted in significantly diminished CIA pathology, Flt-1+ macrophages induced-synovial inflammation, and angiogenesis. Altogether, we anticipate to facilitate the elucidation of previously unappreciated details of GRK2-specific intracellular signaling. Targeting GRK2 activity is a viable strategy to inhibit MDMs infiltration, affording a distinct way to control joint inflammation and angiogenesis of RA.
ABSTRACT
Objective: To investigate the role of M2-type macrophages in migration and invasion of lung adenocarcinoma A549 cells, and to explore the possible mechanism and the related signaling pathway. Methods: PMA (phorbol 12-myristate 13-acetate) was used to induce THP-1 cells into M2- type macrophages. The non-contacting co-culture model of M2-type macrophages and A549 cells was established by Transwell method, then the co-culture medium was collected. The migration and invasion abilities of A549 cells after treatment with co-culture medium (named as CO-A549 cells) were detected by the wound-healing assay and Transwell chamber invasion assay, respectively. The expression levels of vascular endothelial growth factor receptor 3 (VEGFR3) and vascular endothelial growth factor-C (VEGF-C) in CO-A549 cells were detected by Western blotting. After treatment with VEGFR3 inhibitor MAZ51, the migration and invasion abilities of CO-A549 cells were detected by wound-healing assay and Transwell invasion assay again, and the expression level of phospho-extracellular signal-regulated protein kinase (p-ERK) in CO-A549 cells was detected by Western blotting. Finally, the expression level of matrix metalloproteinase 2 (MMP-2) mRNA in CO-A549 cells after treatment with MAZ51 or U0126, an inhibitor of mitogen-activated protein kinase (MAPK)/ERK pathway, was detected by RT-PCR. Results: M2-type macrophages were induced for from THP-1cells by PMA. After treatment with the co-culture medium, the migration and invasion abilities of A549 cells were significantly enhanced (both P < 0.01), the expression level of VEGFR3 was significantly improved (P < 0.01). After inhibition of VEGFR3, the migration and invasion abilities of CO-A549 cells were decreased (P < 0.01, P < 0.05), and the expression level of p-ERK was down-regulated (P < 0.05). The expression of MMP-2 mRNA was down-regulated in CO-A549 cells after treatment with MAZ51 or U0126 (both P < 0.05). Conclusion: M2-type macrophages which were induced by PMA from THP-1 cells can promote the migration and invasion of A549 cells by up-regulating the expression of VEGFR3. The mechanism may be related to the activation of the MAPK/ERK signaling pathway.
ABSTRACT
Background Choroidal neovascularization (CNV) is one of the primary causes leading to visual damage in many fundus diseases.Many evidences indicate that macrophage activation and monocyte chemoattractant protein-1 (MCP-1) play important roles in CNV.However, the dynamic expression of macrophage and MCP-1 in the initial stage of CNV is not clear.Objective This study was to investigate the dynamic changes of F4/80 and MCP-1 expressions in retina-choroid tissue with experimental CNV.Methods Laser-induced CNV models were monocularly established in 105 SPF 8-week-old male wild type C57BL/6 mice.The mice were sacrificed at 6,12,24, 48 and 72 hours after photocoagulation, respectively, and the retina-choroid tissue sections and choroidal flatmounts were prepared.The histopathological examination was carried out to observe the changes of morphology and structure as well as inflammatory response in CNV.The expression and distribution of F4/80 and MCP-1 protein in retinachoroid were detected by double immunofluorescence technique.The expression and distribution of F4/80 in choroid were examined by immunofluorescence.The relative expression levels of F4/80 mRNA and the content of MCP-1 protein in RPE-choroid complex were assayed using real-time quantitative PCR and ELISA,respectively.The use and care of the mice complied with the Regulation for the Administration of Affair Concerning Experimental Animals by Ethic Committee of Experimental Animals of Nanjing Medical University.Results The rupture of Bruch membrane, RPE, outer nuclear layer and choroid was exhibited under the optical microscope 6 hours after photocoagulation.Infiltration of inflammatory cells and tissue edema were seen as the lapse of photocoagulation time, and proliferation of vascular endothelial cells was found 72 hours after photocoagulation.F4/80 was expressed in photocoagulation area 6 hours later, and MCP-1 was expressed around the area.With the lapse of photocoagulation time,the expression intensity of MCP-1 weakened and that of F4/80 enhanced.The contents of MCP-1 protein in RPE-choroid complex were (31.25±4.73), (276.31 ±4.20), (331.95 ±5.86), (221.24±4.42), (179.89 ± 4.10) and (130.80 ± 5.90) pg/mg in the normal control group, photocoagulation 6-, 12-, 24-, 48-and 72-hour groups, respectively,with a significant difference among the groups (F=1 416.46 ,P＜0.01).The contents of MCP-1 protein peaked at 12 hours after photocoagulation and then gradually declined.The expression levels of MCP-1 protein in different time groups were higher than those in the normal control group (all at P＜0.01).A significant difference in F4/80 mRNA expression in RPE-choroid complex was also found among the groups (F =762.72, P＜0.01, and a gradually raising tendency was seen over time, showing evidently increase in comparison with the normal control group (all at P＜0.01).Conclusions Inflammatory response occurs in the early stage of experimental CNV.MCP-1 responds to the CNV at early stage,and the accumulation and activation of macrophage play an important role in the development of CNV.
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Objective To elaborate the role of hepatic inherent macrophages and monocyte-derived macrophages in acute liver injury.Methods A model of acute liver injury in mice was induced via intraperitoneally injection of CCl 4 .Af-ter CCl4 injection, analysis of the expression of CD45, F4/80, Ly6C and CD11b on the hepatic macrophage surface was performed by flow cytometry at 24, 48 and 72 h before the hepatic inherent macrophages (CD45+F4/80hi) and monocyte-de-rived macrophages ( CD45+F4/80 lo ) were sorted at the same time .The relative expression of cytokines in the two popula-tions of macrophages was detected by qRT-PCR.Results Compared with control , the number of total F4/80 +cells in the liver was markedly increased after CCl 4 injection, especially at 72 h.The number of CD45+F4/80lo cells increased signifi-cantly after CCl4 injection 24 h.The mRNA levels of MCP-1, TNF-α, TGF-β1, matrix metalloproteinase(MMP)-12 and MMP-13 were elevated significantly both in F 4/80 hi and F4/80 lo macrophages .IL-12βand IL-6 mRNA levels increased significantly only in F4/80hi macrophages, while the level of MMP-9 mRNA increased markedly only in F4/80lo macropha-ges.When compared with F4/80lo macrophages, MCP-1 and MMP-12 mRNA levels were elevated significantly , while the level of TNF-αmRNA decreased significantly in F4/80hi macrophages.Conclusion In acute liver injury, hepatic inherent macrophages and monocyte-derived macrophages both express inflammatory cytokines , promoting inflammation response and leading to liver damage .The ability to recruit inflammatory monocytes into the liver is much stronger ,the expression of in-flammatory cytokines ( IL-12βand IL-6 ) and MMP-12 is higher , but the expression of inflammatory cytokines ( TNF-α) MMP-9 is lower in hepatic inherent macrophages than in monocyte-derived macrophages .