The Mechanisms Of Interleukin-6-induced C-C Chemokine Receptor Type 7 Suppression In Human Monocyte-derived Dendritic Cells

Background:DCs are very critical for the balance in immunity and for protective fighting against pathogens and tumours. Understanding of tumor in recent years, the tumour immune escape is considered to be one of the main reasons for occurrence and development of tumours.And the relationship between dendritic cells and tumour immune escape is a hotspot of current research. After recognizing,acquiring and processing tumor antigen,mature dendritic cells can activate CD8 + T cells which can remove the tumour cells. In this process, the dendritic cells must express high levels of costimulatory molecules(CD86,CD80,CD83,CD40,etc), MHC class I and II molecules,and C-C chemokine receptor type 7(CCR7). Costimulatory molecules and Major Histocompatibility Complex(MHC) are essential for the activation of T cells,and if lack of their expression,T cells can not be activated.And the CCR7 is the most important chemokine receptor for regulating DCs migration function. In the body, DCs are widely distributed in lymphoid organs and peripheral nonlymphoid organs(liver, skin, kidney, stomach,etc), and once stimulated,DCs upregulate the levels of CCR7.After CCR7 combines with ligand CCL19 or CCL21, It can make mature DCs migrate to lymphoid organs along the afferent lymphatic vessels,and in lymphoid organs,mature DCs can active cells via costimulatory molecules and MHC. From what has been discussed above, the ability of DCs migration and the ability to stimulate T cells are both key of the induction of an antigen-specific immune response.In the early stage of tumour development, the dendritic cells usually can effectively activate T cells to kill tumour cells. But as the further development of tumours,DCs often become anergy. Tumor cells interact with the other stromal cells,and secrete immunosuppressive cytokines,and change their living environment to create the tumor microenvironment(TME) which can promote tumor growth, suppress the anti-tumour immune response. In the tumour microenvironment, The differentiation,maturation and migration of DCs is suppressed.In the physiological conditions,interleukin-6(IL-6) is important for the biological function of DCs.But in the tumor microenvironment,high levels of immunosuppressive cytokines IL-6 is proved to be an important negative factor for the differentiation,maturation and migration of DCs.In the microenvironment, IL-6 can suppress the levels of costimulatory molecules and MHC on DCs surface, and inhibit differentiation of the DCs from CD34+ progenitor cells. Dendritic cells in the tumor microenvironment usually express lower levels of CCR7, and higher levels of CCR6, CCR2 and CCR1 and CCR5 and CXCR4 which are expressed on the surface of imDCs and tolerogenic DCs,that are both immunosuppressive.It has been proved that the lack of CCR7 will inhibit the migration of DCs, eventually lead to the immune defects. Recently, some scholars reported that the tumour cells-derived IL-6 can obviously inhibit the CCR7 expression of human monocyte-derived dendritic cells(moDCs),and impair the migration ability of moDCs, but the molecular mechanism is unclear. In certain types of cancer specimen,such as cervical cancer,dendritic cells in the tumor stroma express high levels of CD83 which is the maturation symbol of DCs, but express low levels of CCR7 or no CCR7.So IL-6 prevents DCs migrate to lymphoid organs through impairing the CCR7 expression,which may be one of the reason for the immune defects of the tumour-bearing hosts. So we hope that by investigating the mechanisms of CCR7-expressing of human monocyte-derived dendritic cells inhibited by IL-6,can find interfering molecular targets of moDCs migration dysfunction, and correct tumor patients’ immunodeficiency.Method:1. CD14+ monocytes were separated from human peripheral blood via Magnetic-Activated Cell Sorting(MACS),and were cultured in 10%RPMI-1640 with rhGM-CSF(800U/ml),rhIL-4(500U/ml),penicillin and streptomycin, to induce the DCs differentiation.The monocytes were cultured in the incubator with 5%CO2 and 37℃.Every three days,the half of culture medium was replaced by fresh 10%RPMI-1640 with rhGM-CSF(800U/ml),rhIL-4(500U/ml).IL-6 with different doses was added from the third day of culture. The final concentration of IL-6 in the culture medium is 50ng/ml,100ng/ml and 150ng/ml.LPS(100ng/ml) was added on the sixth day.After moDCs were stimulated by LPS for 48 h, Flow Cytometry was used to assesses the levels of CD86、CD83 and HLA-DR on moDCs,and qRT-PCR was used to analyse the CCR7 mRNA levels in mo DCs.2. On the basis of the first part of the experiment,the mechanisms of CCR7-expressing of moDCs inhibited by IL-6 would be investigated.Because IL-6 triggers two main signaling pathways:the gp130 Tyr759-SHP-2/ERK pathway and gp130 YXXQ JAK/STAT3 pathway, so we used the western blotting to detect the levels of p-STAT3 and p-ERK1/2 in IL-6-treated moDCs.3. On the basis of the second part of the experiment,we use JSI-124,which selectively inhibit the activation of JAK2 and STAT3,and PD98059,which selectively inhibit the activation of ERK1/2,to inhibit the the p-STAT3 and p-ERK in the IL-6-treated moDCs respectively.qRT-PCR was used to analyse the levels of CCR7 mRNA in the IL-6-treated moDCs added JSI-124 or PD98059 respectively.Flow cytometry was used to analyse the levels of CCR7 on the IL-6-treated moDCs added JSI-124.Transwell migration experiment was used to analyse the migration ability of the IL-6-treated moDCs added JSI-124.Results:1. We harvested the human monocyte-derived dendritic cells with typical dendritic morphology via rh GM-CSF and rh IL-4 combined treatment,and got mature dendritic cells stimulated by LPS. The consequence of Flow analysis indicated that IL-6 with the different final concentration 50ng/ml,100ng/ml,150ng/ml respectively,can’t inhibit the levels of phenotypic maturation molecular CD86,CD83,HLA–DR on the mo DCs.In IL-6(50ng/ml)+LPS group,CD86/CD83/HLA-DR expression rate were 90.1± 1.2, 81.2 ± 1.5,94.6±1.8 respectively. In IL-6(100ng/ml)+LPS group,CD86/CD83/HLA-DR expression rate were 96.2±1.6,86.9±0.9,87.7±1.3 respectively. In IL-6(150ng/ml)+LPS group, CD86/ CD83/HLA-DR expression rate were 91.3±1.2,91.0±1.6,87.0±1.7 respectively.And all were not significantly different compared with LPS–treated group(92.6±1.7,82.0±1.2,93.4±1.4)(P > 0.05). And the consequence of q RT-PCR indicated that IL-6 with the different final concentration 50ng/ml,100ng/ml,150ng/ml significantly inhibited CCR7 m RNA expression in the mo DCs,and the relative expression of CCR7 m RNA was 0.760± 0.079, 0.440± 0.029,0.435±0.059 respectively,and was significantly different compared with LPS– treated group(1.246±0.102)(P < 0.05).2. The consequence of western blotting indicated that the levels of p-STAT3 andp-ERK1/2 in IL-6-treated mo DCs were obviously higher than that in LPS-treated mo DCs.The relative expression of p-STAT3 in the mo DCs treated by IL-6 with different final concentration 50ng/ml,100ng/ml,150ng/ml was 1.240±0.092,1.438±0.105,1.875±0.127 respectively, and was significantly different compared with LPS–treated group(0.724±0.086)(P < 0.05). The relative expression of p-ERK in the mo DCs treated by IL-6 with the different concentration 50ng/ml,100ng/ml,150ng/ml was 1.085± 0.067, 1.071± 0.082,0.856±0.039 respectively,and was significantly different compared with LPS–treated group(0.359±0.024)(P < 0.05)3. The consequence of western blotting indicated that JSI-124 and PD98059 respectively could inhibit the levels of p-STAT3 and p-ERK1/2 activated by IL-6. In IL-6(100ng/ml)+JSI-124 group,the relative expression of p-STAT3 was 0.841±0.018,and was lower than that in IL-6(100ng/ml) group(1.388±0.090)(P<0.05). In the IL-6(100ng/ml)+PD98059 group,the relative expression of p-ERK was 0.389±0.076,and was lower than that in IL-6(100ng/ml)group(0.688±0.082)(P<0.05).The consequence of q RT-PCR indicated that JSI-124 could reverse the low levels of CCR7 m RNA in the mo DCs inhibited by IL-6,but PD98059 didn’t. In IL-6(100ng/ml)+JSI-124+LPS group,the relative expression of CCR7 m RNA was 0.640±0.101,and was obviously higer than that in IL-6(100ng/ml)+LPS group(0.219±0.018)(P<0.05). In IL-6(100ng/ml)+PD98059+LPS group,the relative expression of CCR7 m RNA was 0.260±0.038,and was not significantly different with that in IL-6(100ng/ml)+LPS group(P>0.05). The consequence of Flow analysis indicated that SI-124 could reverse the low levels of CCR7 on the mo DCs inhibited by IL-6. In IL-6( 100ng/ml) +JSI-124+LPS group,the expression rate of mo DCs CCR7 was 83.0±1.2,and was obviously higer than that in IL-6(100ng/ml)+LPS group(15.4±0.9)(P<0.05).The consequence of Transwell migration experiment indicated that JSI-124 could reverse the impaired migration of mo DCs caused by IL-6. In IL-6(100ng/ml)+JSI-124+LPS group,the migration rate of mo DCs was 74.5±3.3,and was obviously higer than that in IL-6(100ng/ml)+LPS group(26.2±2.1)(P<0.05).Conclusions:1. IL-6 can not inhibit the expression of phenotypic maturation molecule CD86, CD83, HLA-DR in mo DCs,but obviously inhibit the exression of CCR7 m RNA in mo DCs.2. IL-6 can obviously active the STAT3 pathway and ERK pathway in mo DCs.3. After the activation of p-STAT3 inhibited by JSI-124,It is reversed that IL-6 suppresses the CCR7-expressing of mo DCs,and impairs the migration of mo DCs,But PD98059 can not. It indicate that IL-6 suppresses the CCR7-expressing of mo DCs,and impairs the migration of mo DCs via STAT3 pathway,not ERK pathway.STAT3 may become the interfering moleculer target of correcting the migration dysfunction of mo DCs.and it can provide theoretical support for correcting the immune deficiency of the tumour host.