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Research on the Effect and the Mechanisms of IL-27on Differentiation and Maturation of Dendritic Cells

Author: ZhangZuo
Tutor: DanBaoEn; AiJun
School: Hebei Medical University
Course: Immunology
Keywords: interleukin-27 dendritic cell co-stimulatory molecule adhesion molecule chemokine receptor MAPKp38 P-STAT1 P-STAT3
CLC: R392
Type: Master's thesis
Year: 2012
Downloads: 113
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Abstract


Objective: To observe the effect of interleukin (IL)-27or IL-27synergicwith tumor ncrosis factor (TNF)-α on the differentiation, maturation andbiological function of human peripheral blood monocyte (PBMC)-deriveddendritic cells (DC), and to investigate the related mechanisms.Methods:1.15~20ml peripheral blood was collected from healthy adult and thePBMC were purified by Ficoll-hypaque method. The PBMC were incubatedwith granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-4for5days and immature DC were obtained. Then, the immature DC weredivided radomly into four groups by different treatment factors on the5th day:Negative group (no treatment factors), TNF-α group (TNF-α:20ng/ml), IL-27group (IL-27:20ng/ml) and TNF-α+IL-27group (co-cytokines group, TNF-α10ng/ml+IL-2710ng/ml).2. The morphous of DC from different groups were observed by invertedmicroscope on the1st,3rd,5th and7th day.3. The DC from different groups were sorted by immunomagnetic beads.4. The expressions of co-stimulatory molecules CD1a, CD83, CD80, CD86,and adhesion molecule ICAM-1on DC from different groups were analyzedby FACS (Fluorescence activating cell sorter, FACS).5. The mRNA expressions of chemokine receptors of CCR5and CCR7onDC from different groups were analyzed by RT-PCR.6. The effect on the proliferation of T cells stimulated by DC treated withMitomycin C from different groups was analyzed by MTS.7. The levels of cytokine IL-12and IFN-γ in the culture supernatant of DCfrom different groups were detected by ELISA.8. To investigate the expressions of signaling pathways, MAPKp38, P-STAT1and P-STAT3in DC from different groups were detected byWestern-blot.Results:1. By the optical microscope, the cells from different groups were uniformand showed a colony-like growth on the fifth day. We found that DC fromIL-27group and TNF-α+IL-27group had a typical morphologicalcharacteristic of mature DC on the7th day: the bodies of the cells were largewith pseudopodia and showed burr-like structure, there was no differenceamong TNF-α group, IL-27group and TNF-α+IL-27group, while the bodiesof the Negative group were small with few pseudopodia and the structure wasnot typical.2. The FACS analysis showed that, the expressions of co-stimulatorymolecules of CD1a and CD83on DC of Negative group, TNF-α group, IL-27group and TNF-α+IL-27group were23.29±4.49%,42.76±3.50%,35.75±4.10%and52.49±2.65%respectively, and the expressions of CD80andCD86were22.66±3.20%,45.56±2.47%,39.06±1.61%and54.10±0.46%respectively. There was no significant difference between IL-27group andTNF-α group (P>0.05) while there were significant differences amongTNF-α+IL-27group, IL-27group and TNF-α group (P<0.05). Theexpressions of adhesion molecule ICAM-1on DC of Negative group, TNF-αgroup, IL-27group and TNF-α+IL-27group were21.84±0.64%,27.66±1.03%,28.47±0.94%and35.11±1.32%. The positive rates in TNF-αgroup, IL-27group and TNF-α+IL-27group had significant differencescompared with Negative group (P<0.05), while that among the three groupshad no significant difference (P>0.05).3. The RT-PCR analysis showed that, the mRNA expressions of chemokinereceptor of CCR7in Negative group, TNF-α group, IL-27group and TNF-α+IL-27group were3.09±0.18,4.14±0.08,3.98±0.09and4.75±0.11respectively. The expressions of last three groups were higher compared withNegative group and there were significant differences between every twogroups(P<0.05). The expressions of CCR5in the four groups were1.23± 0.03,0.86±0.02,0.99±0.03and0.61±0.02respectively. The expressions inTNF-α group, IL-27group and TNF-α+IL-27group had significantdifferences compared with the Negative group(P<0.05). There was nosignificant difference between IL-27group and TNF-α group, while theexpression of CCR5was higher in TNF-α+IL-27group compared withTNF-α group and the result had statistical meaning (P<0.05).4. The MTS analysis showed that, DC from TNF-α group, IL-27group andTNF-α+IL-27group treated with Mitomycin C could stimulate theproliferation of T cells obviously. With the increase in proportion of DC and Tcells (1:100,1:50and1:10), the effect was still enhanced. Under the differentproportion of DC and T cells, the stimulatory effect in TNF-α group, IL-27group and TNF-α+IL-27group was up-regulated compared with theNegative group, and the result had statistical meaning (P<0.05). When theproportions of DC and T cells were1:100and1:50, there was no significantdifference among TNF-α group, IL-27group and TNF-α+IL-27group. Whenthe proportion of DC and T cells was1:10, the absorbance value in the TNF-α+IL-27group had statistical meaning compared with TNF-α group(P<0.05),while there was no significant difference between IL-27group and TNF-αgroup(P>0.05).5. The ELISA analysis showed that, the levels of IL-12in the supernatantfrom Negative group, TNF-α group, IL-27group and TNF-α+IL-27groupwere37.24±2.34pg/ml,45.83±1.09pg/ml,44.78±0.78pg/ml and49.82±0.76pg/ml respectively. The levels in TNF-α group, IL-27group and TNF-α+IL-27group were higher compared with that of Negative group. The result hadno significant difference between IL-27group and TNF-α group (P>0.05),while there was significant difference between TNF-α+IL-27group andTNF-α group(P<0.05). The levels of IFN-γ in the supernatant from the fourgroups were666.67±42.28pg/ml,831.90±69.88pg/ml,863.96±37.99pg/ml and1009.09±13.90pg/ml respectively. The levels in TNF-α group,IL-27group and TNF-α+IL-27group were higher compared with that ofNegative group. There was no significant difference between IL-27group and TNF-α group (P>0.05), while there was significant difference betweenTNF-α+IL-27group and TNF-α group(P<0.05).6. The western-blot analysis showed that, the proteins of signaling pathwayMAPKp38, P-STAT1and P-STAT3were up-regulated at different degrees inDC from TNF-α group, IL-27group and TNF-α+IL-27group. The tendencywas especially obvious in the TNF-α+IL-27group.Conclusion:1. The mature DCs were gained by using IL-27only or IL-27synergic withTNF-α.2. DC induced by IL-27could promote the proliferation of T cells and thelevels of IL-12and IFN-γ in the supernatant. These results suggested thatIL-27can increase the biological functions of DC.3. In vitro, IL-27could directly or synergically with TNF-α enhance thedifferentiation and maturation of DC. The mechanism might depend on theactivation of the signaling pathways of MAPKp38, P-STAT1and P-STAT3.4. By using the two cytokines in combination, the cytotoxicity was reduced,the function of DC was enhanced and anti-tumor function was higher. Theseconformed to the applied principle of clinical medicine and had potentialclinical value.

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