藥物調控B細胞上抑制性Fc受體的表現及探討其機轉並做為系統性紅斑性狼瘡之治療

Abstract

FcγRIIB是B細胞最重要的抑制性受體，在免疫系統中扮演調節抗體的主要角色。其作用機轉主要是透過抑制B-cell antigen receptor (BCR)，但近來的研究顯示FcγRIIB單獨活化能引致B細胞凋亡。FcγRIIB身為抑制性受體的重要可由基因惕除鼠呈現紅斑性狼瘡及系統性紅斑性狼瘡病人FcγRIIB在記憶B細胞表現量降低的得到印證及顯示其重要性。過去文獻對於FcγRIIB的研究主要針對其蛋白質功能以及訊息傳遞之探討，儘管有文獻提出當FcγRIIB的promoter出現特定的核酸多形性變化(SNP)時，會影響FcγRIIB基因的轉錄活性，進而改變其蛋白質的表現量，但對於可調節FcγRIIB基因表現的轉錄因子及其上游調控機制尚不清楚。因此，本研究透過利用螢光報導系統(FcγRIIB-Luc)篩選LOPAC藥庫，並篩選出42種可以調控FcγRIIB基因表現之藥物。更進一步地，本研究想找出具有治療紅斑性狼瘡的潛力藥物，因此從42個候選藥物中再挑出與治療自體免疫疾病相關但尚未被應用在治療紅斑性狼瘡之藥物，最終先選定resveratrol做為研究調控FcγRIIB基因表現之候選藥物。在給予resveratrol至BJAB細胞後，FcγRIIB之mRNA及其在細胞表面的蛋白質表現量確實顯著增加，並可以被SIRT1抑制劑 (Ex-527)預處理抑制，顯示resveratrol透過SIRT1增加FcγRIIB基因表現。另外，有文獻指出SIRT1會抑制轉錄因子NF-κB活性，因此本研究在BJAB 細胞過度表現p65和IKKβ，實驗結果發現兩者皆呈明顯增加FcγRIIB的表現。所以本研究推論，resveratrol會透過活化SIRT1進而促進FcγRIIB的表現。而NF-κB的角色及其他SIRT1下游調控因子須再驗證與釐清。最後，期望透過本研究之策略，在未來能透過研究resveratrol及其他可以調控FcγRIIB基因表現的藥物，找到治療系統性紅斑性狼瘡病人乃至其他自體免疫疾病之具有治療潛力之新藥物和新標靶供新藥研發。

FcγRIIB is the key inhibitory receptor in B cells. During the encounter of immune complexes, FcγRIIB inhibits BCR through phosphorylation of its immunoreceptor tyrosine-based inhibitory motif by Lyn to recruit SHIP for downstream inhibitory signals. Recently it has been shown that FcγRIIB itself can signal for apoptosis. The importance of FcγRIIB can be demonstrated by gene deficient mice that exhibit lupus-like disease. Consistent with this, FcγRIIB is down-regulated in memory B cells in patients with lupus. Previous studies have concentrated on protein function and signaling transduction of FcγRIIB without much known in transcriptional regulation. There are reports to indicate that the transcription activity of FcγRIIB gene can be altered by occurrence of single nucleotide polymorphism (SNP) on the FcγRIIB promoter, but the associated factors remain unclear. In this study we sought to screen LOPAC library using an FcγRIIB promoter linked luciferase assay and a total of 42 compounds showed significant modulatory effects on FcγRIIB gene expression. We next decided to select a candidate compound that is anti-inflammatory but has not yet been used in lupus for validation and further studies. Indeed, we found that resveratrol treatment to BJAB cells showed a dose-dependent increase of promoter activities, mRNA levels and surface expression. These effects were abolished by pre-treatment with Ex-527, a SIRT1 specific inhibitor, indicating that resveratrol up-regulates FcγRIIB gene expression through SIRT1. Since SIRT1 has been shown to be inhibitory to NF-κB activity for its anti-inflammatory effects, we over-expressed NF-κB p65 and IKKβ in BJAB cells and unexpectedly found a substantial increase of FcγRIIB expression, indicating that the effect of SIRT1 by resveratrol is not through NF-κB. Whether the effects of resveratrol to up-regulate FcγRIIB expression might be through mediators independent of NF-κB or in part independent of SIRT1 requires further investigation. In conclusion, our data indicates that resveratrol can up-regulate FcγRIIB expression through SIRT1 and distinct downstream targets. Our strategy to search for new compounds to treat autoimmune diseases by modulation of expression levels of FcγRIIB is novel. By further studying more candidate compounds, we should be able to discover new and effective drugs as well as identify new targets for the treatment of patients with lupus.