Monocyte chemoattractant protein 1 and fractalkine play opposite roles in angiogenesis via recruitment of different macrophage subtypes

AIM： To explore the interaction between macrophages and chemokines [monocyte chemoattractant protein 1（MCP-1/CCL2） and fractalkine/CX3CL1] and the effects of their interaction on neovascularization.METHODS： Human peripheral blood mononuclear cells, donated by healthy volunteers, were separated and cultured in RPMI-1640 medium containing 10% fetal bovine serum, then induced into macrophages by stimulation with 30 μg/L granulocyte macrophage-colony stimulating factor（GM-CSF）. The expression of CCR2 and/or CX3CR1 in the macrophages was examined using flow cytometry. Macrophages were then stimulated with recombinant human CCL2（rh-CCL2） or recombinant human CX3CL1（rh-CX3CL1）. The expression of angiogenesis-related genes, including VEGF-A, THBS-1 and ADAMTS-1 were examined using real-time quantitative polymerase chain reaction（PCR）. Supernatants from stimulated macrophages were used in an assay of human retinal endothelial cell（HREC） proliferation. Finally, stimulated macrophages were cocultured with HREC in a migration assay.RESULTS： The expression rate of CCR2 in macrophages stimulated by GM-CSF was 42%±1.9%. The expression rate of CX3CR1 was 71%±3.3%. Compared with vehicle-treated groups, gene expression of VEGF-A in the macrophages was greater in 150 mg/L CCL2-treated groups（P〈0.05）, while expression of THBS-1 and ADAMTS-1 was significantly lower（P〈0.05）. By contrast, compared with vehicle-treated groups, expression of VEGF-A in 150 mg/L CX3CL1-treated groups was significantly lower（P〈0.05）, while expression of THBS-1 and ADAMTS-1 was greater（P〈0.05）. Supernatants from CCL2 treated macrophages promoted proliferation of HREC（P〈0.05）, while supernatants from CX3CL1-treated macrophages inhibited the proliferation of HREC（P〈0.05）. HREC migration increased when co-cultured with CCL2-treated macrophages, but decreased with CX3CL1-treated macrophages（P〈0.05）.CONCLUSION： CCL2 and CX3CL1 exert different effects in regulation of macrophage in expression of angiogenesisrelated factors, including VEGF-A, THBS-1 and ADAMTS-1. Our findings suggest that CCL2 and CX3CL1 may be candidate proteins for further exploration of novel targets for treatment of ocular neovascularization.

Effects of Sanchi extract on the activation and proliferation of murine lymphocytes and NO secretion by peritoneal macrophages in vitro

The aim of this study is to elucidate the molecular and cellular mechanisms underlying the immunosuppressive effect of Sanchi extract （SE） via investigating the effects of SE on the activation and proliferation of murine lymphocytes and NO secretion by peritoneal macrophages in vitro. ConA was used to activate lymphecytes, and expression of CD69 on T cells and CFSE labeled cell division were detected by flow cytometry. Murine peritoneal macrophages were stimulated with LPS or lymphocytes culture supernate （LCS） and the concentration of NO was determined by Griess reagent assay. After 6 h of culture, SE ranging from 50 to 100μg/ml downregulated CD69 expression on ConA-activated T cells, while SE ranging from 12.5 to 100μg/ml inhibited the proliferative response of lymphocytes to ConA. Additionally, SE （12.5-100μg/ml） inhibited secretion of NO by peritoneal macrophages stimulated by LPS or LCS. This study reveals that SE inhibits the activation and proliferation of routine lymphocytes and NO secretion by peritoneal macrophages.

Effects of GTW treatment on proteinuria and acute glomerular immune lesion in experimental mesangial proliferative glomerulonephritis

To examine the effect of multi-glycoside of Tripterygium wilfordii Hook. f. （GTW） on proteinuria and acute glomerular immune lesion in experimental mesangial proliferative glomerulonephritis （MsPGN） induced by anti-Thyl. 1 monoelonal antibody （mAb） 1-22-3. The reversible model of MsPGN with anti-Thyl. 1 mAb 1-22-3 was established. After 7 days of oral treatment with GTW （ 100 mg/kg per day） and vehicle （distilled water, 5 ml/kg per day）, its effects on proteinuria, renal functions, mesangial morphological change, glomerular macrophage accumulation, and mRNA expressions of cytokines （PDGF-BB and MCP-1 ） were evaluated by light microscope （LM）, immunofluorescence （IF）, and reverse transcription polymerase chain reaction （RT-PCR）. It was found that GTW ameliorated proteinuria （on day 3 and day 7）, mesangial proliferation （total cell number, matrix expansion, a-smooth muscle actin expression, and collagen type Ⅰ expression） and macrophage accumulation （ED3^＋ ） in experimental MsPGN. In addition, GTW significantly suppressed the increased mRNA expressions for MCP-1 （67.6% to eontrol group, P 〈 0.01） together with the tendency to reduce the expression of PDGF （24.44% to control group） on day 7. It is concluded that GTW can not only decrease proteinuria, but also ameliorate acute mesangial alterations and glomerular activated macrophage accumulation probably by reduction of cytokines. These data indicate that GTW is an effective agent for early MsPGN.