The Effects And Mechanisms Of TIMP-3on The Immune Function Of Dendritic Cells

Objective:1. To investigate the expression pattern of TIMP-3during the differentiation of human monocyte-derived dendritic cells and to determine the effect of maturation-promoting factor on the expression of TIMP-3. Then to exlpore the mechanism involved in TIMP-3expression by DCs during their differentiation.2. To study the effects of rhTIMP-3and gene silencing of TIMP-3by siRNA on the expression of co-stimulatory molecules and production of cytokines by LPS-matured DCs. Then to probe into the mechanisms involved in the inhibition of CD86expression and IL-12secretion by TIMP-3.3. To detect the effect of TIMP-3on the capacity of LPS-matured DCs to induce polarization of naive T cells. To analyze the influence of TIMP-3on T cell immune response. To provide an immunology theoretical basis for anti-tumor therapy based on TIMP-3.Methods:1. Evaluation of TIMP-3expression during the differentiation of dendritic cells from monocytesPBMCs were isolated from leukocyte-enriched buffy coats of healthy volunteers by centrifugation with Ficoll-Paque Plus. Monocytes were enriched from PBMCs by positive selection using anti-CD14-conjugated magnetic microbeads. Monocytes were cultured in complete RPMI medium containing1000U/ml GM-CSF and500U/ml IL-4for5days under37℃,5%humidified CO2to generate imDCs. To induce maturation,50ng/ml TNF-a or1μg/ml LPS was added in imDCs for48hours. TIMP-3expression was assayed by RT-PCR in monocytes and the cells following the induction for the1,3,5and7days. Monocytes and imDC were stained with DAPI (blue) and FITC-conjugated mAb (green) against TIMP-3and then examined by confocal laser scanning microscopy. The cell-free supernatants of imDCs were assayed by ELISA for TIMP-3secretion.2. Analysis of the mechanism underlining the induction of TIMP-3during DCs d i fferentiationMonocytes were differentiated by standard dose of GM-CSF (1000U/ml) in combination with gradient doses of IL-4(0,125,250,375, and500U/ml) or by standard dose of IL-4(500U/ml) in combination with gradient doses of GM-CSF (0,250,500,750, and1000U/ml). Five days later, the cells were collected and examined by RT-PCR for TIMP-3mRNA expression. Additionaly, for experiments using the signaling kinase inhibitors p38MAPK inhibitor SB203580, the JAK3inhibitor ZM39923hydrochloride, and the PI3K inhibitor wortmannin, monocytes were pre-incubated with the above indicated inhibitors for1h. Then the cells were washed extensively and incubated with GM-CSF and IL-4for3days, with TIMP-3mRNA examined by RT-PCR. DMSO was used as a background control. p38MAPK phosphorylation were also analyzed with lysates from monocytes cultured with or without IL-4(500U/ml) or GM-CSF (1000U/ml) for the indicated times (0,5,15,30,60min) using western-blot analysis. P38MAPK activity was detected with a commercial kinase assay kit.3. Si lencing TIMP-3gene expression by Iiposome-mediated siRNA transfection Day5-imDCs were harvested, washed with phosphate buffered saline (PBS) and then resuspended in serum-free RPMI1640medium with a mixture of Lipofectamine2000and TIMP-3siRNAs or negative control siRNAs previously incubated at room temperature for20min.10%FBS,1000U/ml GM-CSF,500U/ml IL-4and1μg/ml LPS were added after4h. Forty-eight hours later, the transfected cells and supernatants were collected. The silencing efficiency was monitored by western-blot.4. Assessing the effects of rhTIMP-3and gene silencing of TIMP-3by siRNA on the expression of co-stimulatory molecules and production of cytokines by LPS-matured DCs.rhTIMP-3or TIMP-3siRNA was applied during LPS-induced DC maturation. The expression of surface molecules HLA-DR, CD14, CD40, CD80, CD83and CD86was examined by flow cytometry. ImDCs were stimulated by LPS, TIMP-3or their combinations for maturation. Otherwise, LPS was supplemented to the TIMP-3-knockdowned imDCs (si-TIMP-3) or their corresponding control (si-Ctrl). Two days later, the cell-free supernatants of different cultures were collected and analyzed for IL-1β, IL-6, IL10, IL-12p70and TNF-a production by Bio-plex Protein Array system.5. Estimating the mechanisms underlining the downregulation of CD86and repression of IL-12by TIMP-3Various concentrations of wortmannin or DMSO were employed to treat imDC for1hour before their further stimulation with LPS or with LPS plus TIMP-3. Two days later, production of IL-12, IL-10and TNF-a in the cell-free supernatants was assayed by Bio-plex Protein Array system and the expression of CD86was examined by flow cytometry. The culture supernatants were collected at different time points (0,6,12,24and48hours) for IL-12assay by ELISA. Whole-cell lysates from imDCs with LPS±TIMP-3treatment for30min were subjected to Western blot analysis of phosphorylated Akt. Expression of MARCH1mRNA in DCs matured with TNF-a or LPS±TIMP-3for indicated times (2,4,8,16,24and48hours) were analyzed by RT-PCR.6. The impact of TIMP-3on the capacity of LPS-matured DCs to induce polarization of naive T cells.Naive human CD4+T cells for the allogeneic mixed lymphocyte reaction (MLR) were obtained from PBMCs using anti-CD4and anti-CD45RA MACS beads. To stimulate the naive T cells,4types of mDCs were applied:imDCs stimulated with LPS (Control); imDCs stimulated with LPS plus TIMP-3(TIMP-3); imDCs preincubated with500nM wortmamin for1h with the following maturation by LPS plus TIMP-3(wortmamin+TIMP-3); TIMP-3-konckdowned imDCs stimulated by LPS (si-TIMP-3). These mDCs were mixed with allogeneic naive T cells for6days to induce T cell polarization at a DC:T ratio of1:5. For intracellular analysis of cytokine production, CD4+T cells were re-stimulated with PMA and ionomycin in the presence of brefeldin A for5-6h. Cells were then fixed, permeabilized and stained for IFN-y and IL-4and analyzed by flow cytometry. Supernatants of the MLR co-culture were harvested and the production of IFN-y, IL-4, IL-5, IL-10and IL-13were detected by Bio-plex Protein Array system.Results:1. TIMP-3expression is induced during dendritic cells differentiation During DCs differentiation, the TIMP-3mRNA expression was found induced and strikingly up-regulated. It was also found that TIMP-3mRNA was markedly down-regulated in LPS or TNF-a-matured DCs. The confocal laser scanning microscopy images clearly revealed that TIMP-3was localized in both cytoplasm and nucleus in imDCs, but not or scarcely observed in monocytes. The cell products were then assayed by ELISA. However, TIMP-3was not detected in the supernatant of imDCs. Then the supernatants of imDCs which were incubated8h with heparin or extracted5min with Triton X-100or SDS were examined by ELISA, and found that these treatments caused an accumulation of TIMP-3in the cell culture.2. IL-4induces TIMP-3expression during DCs differentiation by activating p38MAPK s i gnali ng pathwayqRT-PCR results clearly showed that, while GM-CSF alone was not taking effect, the TIMP-3expression was dose-dependent on IL-4induction. On the contrary, in the presence of the fixed dose of IL-4(500U/ml), different doses of GM-CSF (0-1000U/ml) did not alter the expression of TIMP-3. Therefore, it was IL-4, but not GM-CSF that determined the TIMP-3expression during DC differentiation. We examined the possibilities of p38MAPK, JAK3and PI3K pathways using their selective inhibitors, and found that TIMP-3mRNA in monocytes was significantly blocked by p38MAPK inhibitor SB203580, regardless of the doses of IL-4employed. It was not GM-CSF but IL-4that enhanced the levels of phospho-p38MAPK. We investigated the enzymatic activity of the immunoprecipitated p38MAPK using GST-ATF-2as substrate. A significant phosphorylation of ATF-2was observed within60min of IL-4treatment, whereas the IL-4-induced p38MAPK kinase activity was inhibited by pretreatment with SB203580in monocytes.3. TIMP-3siRNA inhibits the expression of TIMP-3efficientlyWestern-blot showed that the TIMP-3expression was efficiency blocked by specific siRNA efficiently in DCs. The expression of TIMP-3protein was reduced by more than50%. 4. TIMP-3decreases the expression of co-stimulatory molecule CD86and the production of cytokine IL-12in LPS-matured DCs.When exogenous rhTIMP-3was supplemented to the LPS-matured DCs, there was a significant down-regulation of CD86, whereas other surface molecules (CD40, CD83, CD80, HLA-DR) remained unaltered. Consistent with this result, gene silencing of TIMP-3in DCs led to an obviously increasing level of CD86. We observed that imDCs spontaneously released relative low levels of IL-1β, IL-6, IL-10, IL-12and TNF-a. Exposure of imDCs to rhTIMP-3alone did not change cytokines production significantly. As expected, LPS stimulation induced a significant up-regulation of all the cytokines. Interestingly, when rhTIMP-3was added simultaneously with LPS, there was a dramatic decrease in the production of TNF-a and IL-12, whereas the levels of IL-10, IL-1β and IL-6were not markedly modulated by rhTIMP-3. Consistently, knockdown of endogenous TIMP-3boosted LPS-induced TNF-a and IL-12production in DCs, and loss of TIMP-3had no apparent influence on IL-10, IL-1β and IL-6production.5. TIMP-3inhibits IL-12production in LPS-matured DCs by further activating the PI3K signaling pathway and enhances ubiquitination of CD86through restraining MARCH1reduction induced by LPSPretreatment of imDCs with500nM wortmannin could significantly augment LPS-induced IL-12production. Clearly, the inhibitory effect of rhTIMP-3on IL-12production in LPS-induced DCs was completely reversed by wortmannin. By contrast, the production of IL-10and TNF-a was not strikingly altered in wortmannin-pretreated DCs. Additionally, the effect of wortmannin was dose-dependent:the efficient reversing function was observed at the concentrations higher than100nM. IL-12production by DCs reached a plateau at6hours after LPS stimulation and maintained a high level up to48hours. In addition, western blotting revealed that the level of phosphorylated Akt was significantly up-regulated within30min in imDCs without altering total amounts of Akt under TIMP-3stress. Unfortunately, we found that CD86was further down-regulated rather than reversed by wortmannin under TIMP-3stress. Then the dynamic expression pattern of MARCH1during DCs maturation was comprehensively examined. The MARCH1transcription in DCs was significantly down-regulated on LPS-induced maturation (16h and24h). However, it tended to restrain the reduction of MARCH1as the addition of TIMP-3, suggesting that CD86ubiquitination may be a potential mechanism underlying TIMP-3-mediated CD86suppression.6. TIMP-3modulates LPS-matured DCs to programme naive T cells toward a Th2profileAllogeneic naive T cells were stimulated with differently conditioned DCs. Six days later, cytokine production pattern in T cells was measured by intracellular cytokine staining and Bio-plex Protein Array system. The intracellular staining assay revealed that DCs matured by LPS in the presence of rhTIMP-3programmed naive T cells toward a Th2profile with a markedly reduced number of IFN-y-producing cells and a strikingly increased number of IL-4-producing cells. Preincubation of imDCs with wortmannin largely reversed the effect of TIMP-3:the Thl profile was restored, suggesting that the PI3K pathway was functionally involved in TIMP-3-induced DC modulation. Additionally, siRNA silencing of TIMP-3led DCs to generate a slightly increased number of IFN-y producing T cells. Consistent with the intracellular cytokine staining, TIMP-3rendered DCs to stimulate naive T cells to produce less IFN-y, but more IL-4. Although the TIMP-3-caused pattern of other Th2cytokine (IL-5, IL-10and IL-13) production was not as clearly as IL-4, when we calculated the ratio of Th2cytokine to IFN-y, there were significantly higher values for all the Th2cytokines.The ratios could be reversed by the PI3K inhibitor wortmannin.Conclusions:1. TIMP-3was not detectable in freshly isolated monocytes, but gradually induced during their differentiation towards DCs by IL-4through activating p38MAPK signaling pathway. TIMP-3mRNA was markedly decreased in both LPS-matured DCs and TNF-a-matured DCs.2. TIMP-3decreased the expression of co-stimulatory molecule CD86and the production of cytokine IL-12in LPS-matured DCs. This suggested that the capacity of LPS-matured DCs to induce polarization of naive T cells could be modulated by TIMP-3.3. TIMP-3inhibited IL-12production in LPS-matured DCs by further activating the PI3K signaling pathway 4. TIMP-3downregulated the expression of CD86by enhancing ubiquitination of CD86through restraining MARCH1reduction induced by LPS5. TIMP-3modulated LPS-matured DCs to programme naive T cells toward a Th2profile. Thus, the efficacy of anti-tumor therapy based on TIMP-3requires further evaluation.