Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis.
Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis.
复制标题
Kveim试剂的蛋白质组学分析鉴定了结节病中细胞免疫的靶标。
DOI：
10.1371/journal.pone.0170285
复制
发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Lalvani A
中科院分区：
文献类型：
--
作者：
Eberhardt C;Thillai M;Parker R;Siddiqui N;Potiphar L;Goldin R;Timms JF;Wells AU;Kon OM;Wickremasinghe M;Mitchell D;Weeks ME;Lalvani A
Kveim试剂（Kv）皮肤试验是诊断结节病的历史方法。皮内注射治疗的结节病脾组织导致4-6周注射部位的肉芽肿反应。先前的工作表明蛋白质可能是这种反应的触发剂。我们的目的是确定Kv特异性蛋白质，并研究来自结节病、结核病和健康对照患者的外周血单核细胞（PBMC）在用Kv和选定的Kv特异性蛋白质刺激时的离体反应。Kv提取物经1D-SDS-PAGE和2D-DIGE分离后进行质谱鉴定。首先用Kv刺激结节病和对照PBMC，然后用从蛋白质组学分析鉴定的三种选择的重组蛋白候选物刺激。随后通过多重细胞因子测定来测量PBMC分泌的细胞因子。我们观察到结节病患者Kv刺激的PBMC分泌的IFN-γ和TNF-α显著高于健康志愿者的PBMC（IFN-γ：207.2 pg/mL vs. 3.86 pg/mL，p = 0.0018; TNF-α：2375 pg/mL vs. 42.82 pg/mL，p = 0.0003）。通过蛋白质组学方法，我们确定了74个结节病组织特异性蛋白。其中，3种蛋白质（波形蛋白，微管蛋白和α-辅肌动蛋白-4）进行了鉴定，使用1D-SDS-PAGE和2D-DIGE。数据可通过ProteomeXchange获得，标识符为PXD 005150。随后在结节病PBMC与结核病PBMC的波形蛋白刺激下观察到细胞因子分泌增加（IFN-γ：396.6 pg/mL vs 0.1 pg/mL，p = 0.0009; TNF-α：1139 pg/mL vs 0.1 pg/mL，p<0.0001）。与来自健康对照的PBMC相比，在结节病PBMC的波形蛋白刺激中也观察到这一发现（IFN-γ：396.6 pg/mL vs. 0.1 pg/mL，p = 0.014; TNF-α：1139 pg/mL vs 42.29 pg/mL，p = 0.027）。当用微管蛋白或α-辅肌动蛋白-4刺激时，结节病和对照PBMC之间的细胞因子分泌没有差异。Kveim试剂和波形蛋白的刺激诱导结节病PBMC分泌特异性促炎细胞因子。对Kveim特异性蛋白的细胞免疫应答的进一步研究可能会发现新的生物标志物，以帮助诊断结节病。
Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4–6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins. Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay. We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4. Stimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis.