The Expression And Role Of IL-33in Amyloid-β Stimulated Retinal Pigment Epithelium Cells

Objective:1. To investigate IL-33expression after Aβ1-40stimulation in retinal pigmentepithelium (RPE) cells, and identify the signaling pathways involved inAβ1-40-associated IL-33production.2. To investigate the biological activities of IL-33on RPE cells in vitro andcharacterize signaling pathways with a role in IL-33-mediated inflammatory cytokinesecretion.Methods:1. Aβ1-40oligomers (0.001,0.01,0.1,0.3,1,5, and10μM) were used to stimulateD407cells for24h, or Aβ1-40oligomers (0.3,1μM) were used to stimulate D407cellsfor various times (3,6,12, or24h). The MTT reduction assay was used to examine theeffects of Aβ1-40on viability of confluent D407cells.2. The D407cells were treated with Aβ1-40(the oligomeric form) at0.1,0.3,1,5,10μM for24h in1ml of serum-free DMEM. Cells were pretreated with10μMspecific inhibitors of NF-κB, ERK1/2, JNK, p38MAPK or DMSO (vehicle) for1h,then were stimulated with Aβ1-40oligomers (0.3μM) for24h. IL-33mRNA expressionwere detected by real-time PCR and the protein level of IL-33was examined byELISA.3. The ST2L expression was measured by FCM under the stimulation of IL-33(100ng/ml) on D407cells surface and real-time PCR was used to detect the ST2Lexpression in D407cells which were stimulated with different concentrations of IL-33for16h.4. The D407cells were stimulated with different concentrations of IL-33for16hand cells were collected for analysis of proinflammatory factors such as IL-6, IL-8, IL-1β, TNF-α mRNA expression by real-time PCR and the protein level by ELISA.Cells were pretreated with10μM specific inhibitors of NF-κB, ERK1/2, JNK, p38MAPK or DMSO (vehicle) for1h. Proinflammatory factors mRNA expression weredetected by real-time PCR and the protein level was examined by ELISA.Results:1. All doses resulted in significant differences from cells grown in serum-freeDMEM, except at a concentration of0.001μM. Results also indicate that atime-dependent decrease in viability occurred in cells stimulated with Aβ1-40oligomersat0.3μM and1μM.2. IL-33mRNA levels and protein levels in cells were significantly higher whenAβ1-40oligomers were used at a concentration of0.3μM than in unstimulated cellsusing real-time PCR and ELISA (P <0.05). Aβ1-40oligomers-induced IL-33mRNAexpression was suppressed by ERK1/2and p38MAPK inhibitors (P <0.05). However,IL-33synthesis was only suppressed by the inhibitors of ERK1/2(P <0.05), with thep38MAPK inhibitor having no effect on IL-33protein levels.3. ST2L expression was detected on the cell surface of D407cells using flowcytometry. The results demonstrated that D407cells expressed ST2L on the surfaceand the mean fluorescence intensity (MFI) of ST2L on the surface of D407cells wasupregulated with rhIL-33stimulation. ST2L mRNA expression was significantlyincreased following rhIL-33stimulation and increased with increasing amounts ofrhIL-33used as demonstrated using real-time PCR.4. RhIL-33dose-dependently induced IL-6, IL-8, IL-1β and TNF-α mRNA andprotein expression in D407cells. Therefore, IL-33effectively induced inflammatorycytokine secretion in RPE cells. IL-6synthesis induced by IL-33was suppressed byinhibitors of p38MAPK (SB203580). In contrast, the production of IL-8wassuppressed by the inhibitors of ERK1/2(U0126), and IL-1β was inhibited by theinhibitors of NF-κB (BAY11-7082). Furthermore, TNF-α was suppressed by theinhibitors of JNK (SP600125) at the mRNA and protein levels. Therefore, IL-33 induces inflammatory cytokine secretion using various signaling mechanisms.Conclusion:1. Cell survival gradually dropped in retinal pigment epithelium cells stimulatedwith Aβ1-40oligomers at several doses and differernt time.2. IL-33was increased through Aβ1-40stimulation in RPE cells. ERK1/2MAPkinase plays a role in IL-33secretion with Aβ1-40stimulation and p38plays a role at thetranscriptional level.3. ST2L, the important component of the IL-33receptor, was significantlyincreased following recombinant human IL-33stimulation in RPE cells. RPE cells areone of the target cells of IL-33, and that IL-33can induce ST2L expression andsubsequently increase its biological effect.4. IL-33can effectively regulate inflammatory cytokines including IL-6, IL-8,IL-1β and TNF-α secretion in RPE cells and plays a role in the inflammatory reaction.5. IL-33induced IL-8production using the ERK1/2signaling pathway andregulated IL-6, IL-1β and TNF-α secretion via the p38MAPK, NF-κB and JNKsignaling pathways, respectively.