| Objective:1.To investigate the expression pattern of RGC-32 during the differentiation and polarization of macrophages and determine the effect of LPS or IFN-y in M1 macrophages and IL-4 in M2 macrophages on the expression of RGC-32.2. To study the effects of siliencing RGC-32 and over-expression of RGC-32 on the production of cytokines in macrophages. Then to reveal the mechanisms involved in the regulation of cytokines by RGC-32.Methods:1. Evaluate the expression of RGC-32 during the differentiation of THP-1 cells into macrophages and macrophage polarization.To generate PMA-treated macrophages, 1x106 THP-1 cells were seeded into complete growth medium supplemented with 100ng/ml PMA for 48 h. To generate Ml-polarized THP-1 macrophages, THP-1 cells were cultured with 100ng/ml PMA for 6 h and then treated with PMA plus 100ng/ml LPS and 20ng/ml IFN-y for 42 h. To generate M2-polarized THP-1 macrophages, THP-1 cells were cultured with 100ng/ml PMA for 6 h and then treated with PMA plus 20ng/ml IL-4 for 42 h. RGC-32 expression was detected by quantitative RT-PCR and western-blot in THP-1 cells and the cells following the induction of for 6, 12, 24, 48h. THP-1 cells and macrophages were stained with TRITC-conjugated mAb (red) against RGC-32 and DAPI (blue). RGC-32 expression in M1- and M2-polarized THP-1 macrophages was confirmed through western-blot.2. Evaluate the expression of RGC-32 during the differentiation of human monocytes into macrophages and macrophage polarization.Human peripheral blood mononuclear cells were isolated from leukocyte-enriched buffy coats from healthy donors using density gradient centrifugation and were positively selected by MACS CD14 microbeads. The purity of the isolated CD 14+monocytes was 98%, as determined by flow cytometry. Monocytes were differentiated into macrophages (MO) in RPMI using 10% FBS containing 100ng/ml human rhM-CSF for 7 days. M1 and M2 macrophages were obtained by culturing MO cells for an additional 48 h with 100ng/ml LPS containing 20ng/ml IFN-y (for M1 polarization) or 20ng/ml IL-4 (for M2 polarization), respectively. Human TAMs were obtained from the ascetic fluid of colon adenocarcinoma. CD14+ macrophages were isolated through Ficoll gradient cell separation and subsequent magnetic cell sorting using CD 14 microbeads. RGC-32 expression in M1 and M2 macrophages was confirmed through quantitative RT-PCR.3. The mechanisms of the induction of RGC-32 during macrophage polarizationPMA-treated macrophages were polarized to Ml using a standard dose of LPS (100ng/ml) in combination with graded doses of IFN-y (0, 5, 10 and 20ng/ml) for 42 h. PMA-treated macrophages were stimulated using a standard dose of IFN-γ (20ng/ml) in combination with graded doses of LPS (0, 25, 50 and 100ng/ml) for 42 h. PMA treated macrophages were polarized to M2 using graded doses of IL-4 (0, 5, 10 and 20ng/ml) for 42 h. RGC-32 mRNA were determined by quantitative RT-PCR.4. Silencing RGC-32 by liposome-mediated siRNA transfection and over-expression of RGC-32 by retroviral transfection.A suspension of THP-1 cells was diluted with an equal volume of serum-free RPMI containing HiPerFect Transfection Reagent and scrambled or RGC-32 siRNA. After 12 h, the cells were induced to differentiate by the addition of PMA. Transfected cells were maintained in culture for 48 h, and subsequently used for the detection of RGC-32 expression.Supernatants containing pBMN-GFP or pBMN-GFP-RGC-32 were collected, filtered and subsequently used to infect THP-1 cells. Transfected cells were treated with PMA for 48h, and subsequently used for the detection of RGC-32 expression.5. The effect of RGC-32 siliencing and overexpression of RGC-32 on the secretion of cytokines by macrophages.THP-1 cells transfected by RGC-32 siRNA or PBMN-GFP-RGC-32 were induced to differentiate into macrophages. Total RNA from transfected cells was extracted for quantitative RT-PCR. The transfected cells were washed thrice and cultured for 48h. Culture supernatants were collected from macrophages. Cytokine levels were measured using TNF-a, IL-1β, IL-6 and TGF-β ELISA kits.6. A study of the mechanism of RGC-32 expression on the production of IL-6 by macrophages.LY294002 or DMSO was employed to treat si-control and si-RGC-32 THP-1 cells for 60min, before their further stimulation with PMA. Two days later, production of TNF-a, IL-1β, IL-6 and TGF-β was determined by ELISA. Whole-cell lysates from si-control and si-RGC-32 macrophages were subjected to western-blot analysis of phosphorylated AKT.Results1. RGC-32 expression is induced during differentiation of THP-1 cells into macrophages and differently expressed in Ml and M2-polarized THP-1 macrophages. During macrophage differentiation, Treatment of THP-1 cells with PMA significantly up-regulated RGC-32 mRNA expression in a time dependent manner. The expression of RGC-32 protein was also evaluated through western blotting and immunofluorescence staining experiments. RGC-32 protein was greatly increased in cells treated with PMA. Consistent with these data, RGC-32 was barely detectable in THP-1 cells, whereas PMA-treated THP-1 cells contained high levels of cytoplasmic RGC-32. The M1-or M2-polarized THP-1 macrophage phenotype is induced by LPS and IFN-y or IL-4, respectively. RGC-32 mRNA expression was considerably higher in PMA-treated and M2-polarized THP-1 macrophages than in M1-polarized THP-1 macrophages. Consistent with RGC-32 mRNA expression, RGC-32 protein was increased in PMA-treated and M2-polarized THP-1 macrophages. To further analyze RGC-32 mRNA expression in human primary cells, human M1 and M2 macrophages were generated. As expected, the results indicated that RGC-32 was expressed at high levels in M2 macrophages.2. LPS and IL-4 modulated RGC-32 expression during macrophage polarization.Four different doses of LPS or IFN-y in combination with a standard dose of the other cytokine counterpart were applied to identify the contributions of these cytokines to the regulation of RGC-32 expression. LPS significantly decreased RGC-32 mRNA expression in a dose-dependent manner, whereas IFN-y alone did not show any effect. In addition, the results also showed that RGC-32 mRNA expression was dependent on the dose of IL-4 in PMA and IL-4-treated THP-1 cells. Therefore, these results suggest that LPS and IL-4, but not IFN-y, influence the expression of RGC-32 during macrophage polarization.3. Increased expression of RGC-32 in TAMsTAMs originate from blood monocytes recruited to the tumor site by molecules produced by tumors and stromal cells. CD14+TAMs isolated ex vivo from colon adenocarcinoma ascetic fluid expressed higher levels of RGC-32 than did CD 14+monocytes isolated from human peripheral blood. The increased expression of RGC-32 mRNA in TAMs prompted us to examine whether tumor cells influence the expression of this protein in monocytes. The results showed that colon adenocarcinoma ascitic fluid promoted strong up-regulation of RGC-32 mRNA, confirming that tumor cells release factors that up-regulate RGC-32 expression in monocytes.Given that RGC-32 expression is induced by M-CSF and IL-4, we tested whether both cytokines contributed to RGC-32 induction in monocytes by tumor-derived ascetic fluids. The blocking Abs anti-M-CSF and anti-IL-4 reduced the induction of RGC-32 by colon adenocarcinoma ascitic fluid by 46% and 66%, respectively. However, a synergistic effect was not observed when neutralizing Abs against M-CSF and IL-4 were added to the conditioned medium. Therefore, these results suggest that the increased expression of RGC-32 in TAMs is dependent on tumor-associated M-CSF and IL-4.4. RGC-32 modulated cytokine production in macrophagesTHP-1 cells were transfected with control or RGC-32 siRNA before being differentiated with PMA for 48 h. western blot showed that RGC-32 expression was effectively reduced using the specific siRNA. RGC-32 was over-expressed in THP-1 cells. These cells were induced into macrophages. We observed increased production of the key pro-inflammatory cytokine IL-6 and decreased production of the anti-inflammatory cytokine TGF-β in RGC-32-silenced THP-1 macrophages. Complementary experiments in which RGC-32 was over-expressed in THP-1 macrophages resulted in significant inhibition of IL-6.5. A crucial role for the PI-3K signaling pathway in RGC-32-mediated IL-6 inhibitionPI-3K is involved in the positive regulation of IL-6 production. To determine whether the PI-3K-mediated pathway is activated in PMA-treated THP-1 cells, we analyzed the phosphorylation of AKT. Western blot analysis showed that AKT phosphorylation gradually increased and peaked at 48 h after treatment with PMA. To determine the mechanisms underlying RGC-32-regulated IL-6 production, we examined the effects of LY294002, a PI3K inhibitor, in RGC-32-silenced macrophages on the up-regulation of IL-6 production.The pre-treatment of THP-1 cells with lOμM LY294002 significantly inhibited PMA-induced IL-6 production. Clearly, the positive effect of RGC-32 silencing on IL-6 production was completely blocked by LY294002. By contrast, the production of IL-1β, TNF-a and TGF-β was not affected in LY294002-pretreated macrophages. In addition, western blot analysis revealed that phosphorylated AKT was up-regulated after 48 h in RGC-32-silenced macrophages, whereas the total amount of AKT was unaltered. To test whether RGC-32 binds to AKT in THP-1 cells and PMA-treated macrophages, we performed coimmunoprecipitation. The results showed that RGC-32 physically interacts with AKT. Taken together, these results suggest that the PI-3K pathway is critically involved in the regulation of IL-6 production by RGC-32 in macrophages.Conclusion1. RGC-32 was up-regulated during the differentiation of monocyte into macrophages. RGC-32 expression was down-regulated by LPS in Ml macrophages and up-regulated by IL-4 in M2 macrophages.2. RGC-32 modulated IL-6 production by PI-3K signaling pathway in macrophages. |
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