肠粘膜淋巴细胞对葡聚糖硫酸钠诱导小鼠结肠炎的作用研究
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摘要
炎症性肠病(IBD)是一组累及胃肠道的慢性非特异性炎症性疾病,主要包括溃疡性结肠炎(UC)和Crohn病(CD)。大量研究表明免疫反应尤其是自身免疫反应参与IBD的发病。IBD患者对肠腔抗原失去耐受,产生免疫应答,体内对肠腔抗原的抗体水平增高。因此,研究口服耐受对治疗IBD具有重要的指导意义。多次低剂量口服抗原主要通过调节性T细胞分泌抑制性细胞因子来介导免疫抑制。已有报道口服五剂低剂量结肠炎提取蛋白(CEP)或正常结肠蛋白(NCEP)对三硝基苯磺酸(TNBS)诱导结肠炎有预防及治疗作用,但正常结肠蛋白的作用稍差,耐受诱导是由抑制性细胞因子如TGF-β1、IL-4、IL-10等所介导。
γδT细胞倾向性分布于上皮组织。日益增多的证据表明γδT细胞能发挥多种免疫调节功能。它们诱导对感染或转化细胞的溶细胞效应,支持粘膜IgA生成,维持上皮的自稳。γδT细胞亦在口服耐受中发挥关键的调节作用。然而,关于γδT细胞在炎症过程中有利及有害的作用均有报道,在IBD患者及动物模型中的作用多年来存在争议,而且关于肠粘膜γδT细胞在口服耐受干预结肠炎过程中所起的作用尚未明确。CD8+T细胞在肠粘膜中相当丰富,尤其是上皮层。已有报道CD8+调节性T细胞对IBD的抑制效应。口服耐受能诱导产生脾CD8+调节性T细胞。但对肠粘膜CD8+T细胞在口服耐受中的作用研究甚少。
因此,本课题以多次低剂量口服CEP对葡聚糖硫酸钠(DSS)诱导BALB/c小鼠结肠炎的预防模型为基础,利用流式细胞术检测T细胞亚群CD8α+和TCRγδ+T细胞在肠相关淋巴组织(GALT)及脾中的改变,探讨T细胞亚群在DSS诱导结肠炎中的作用以及T细胞亚群在口服耐受干预结肠炎中的作用,同时过继转移来自未处理小鼠或口服耐受处理小鼠的小肠上皮内淋巴细胞(SI-IEL)及经磁性活化细胞分选技术(MACS)分选的SI-γδIEL,观察受体鼠结肠炎的症状及组织学病变,并初步探索细胞作用的机制,为IBD的发病机制及临床治疗提供新的依据。
第一部分小鼠结肠炎的改善与结肠粘膜中CD8α+和TCRγδ+T细胞增多相关
为建立BALB/c小鼠结肠炎,予7周龄雄性小鼠自由饮用4%DSS,每日监测疾病活动指数(DAI),7日后取脾曲至肛门的肠段用于组织学评分。结果表明结肠炎小鼠的第7日疾病活动指数和组织学评分均较未处理对照鼠显著升高。
取结肠炎诱导7日后小鼠的结肠标本,制备结肠炎提取蛋白(CEP)。以每只200μl 1.25 mg/ml CEP灌胃6周龄小鼠,隔日1次,共5次。以等浓度等量牛血清白蛋白(BSA)灌胃为对照。于末次灌胃后休息3天开始用4%DSS诱导结肠炎,评估两组小鼠(每组12只)的疾病活动指数及病理学改变。从结肠炎诱导第4日至第7日,口服CEP结肠炎小鼠的DAI显著低于口服BSA结肠炎小鼠。两组的组织学评分具有显著性差异(33.6±2.8 vs 25.4±5.0,p<0.01),表明口服五次低剂量CEP能缓解DSS诱导的结肠炎。Spearman分析显示组织学评分与第7日疾病活动指数相关(r=0.78,p<0.01)。
收集未处理小鼠、结肠炎小鼠、口服CEP结肠炎小鼠、口服BSA结肠炎小鼠的血清,ELISA法检测血清中TGF-β1和IFN-y的浓度。结果示结肠炎小鼠血清中的TGF-β1浓度低于未处理鼠,与口服BSA对照鼠相比,口服CEP结肠炎小鼠血清中的TGF-β1有2至3倍的增加。然而,血清IFN-y浓度在各组小鼠之间未见明显差异。
然后,我们重建结肠炎模型、口服CEP模型及对照组(每组8只小鼠),提取小肠(SI)和大肠(LI)的上皮内淋巴细胞(IEL)、固有层淋巴细胞(LPL)、脾细胞、派氏集合淋巴结(PP)及肠系膜淋巴结(MLN)的淋巴细胞,利用流式细胞术检测各组小鼠中CD3+、TCRγδ+, CD8a+和CD8a+TCRγδ+T细胞的改变。
与未处理组相比,结肠炎小鼠SI-IEL和SI-LPL的TCRγδ5+和CD8a+TCRγδ+T细胞的比例增加,相应的TCRγδ-T细胞和CD8a+TCRγδ-T细胞的比例降低。与口服BSA结肠炎对照组相比,口服CEP结肠炎小鼠LI-IEL和LI-LPL的TCRγδ+T细胞、CD8α+TCRγδ+T细胞、CD8α+T细胞和CD8α+TCRγδ-T细胞的比例均增加,相应的TCRγδ- T细胞和CD8αTCRγδ-T细胞的比例降低。第7日的DAI与大肠粘膜中CD8α+T细胞或TCRγδ+T细胞的百分比之间呈负相关(p均小于0.05)。
口服CEP结肠炎小鼠的结果表明结肠炎的改善伴随大肠粘膜淋巴细胞中CD8α+T细胞和TCRγδ+T细胞的增多,因此我们检测DSS处理终止5日的修复期小鼠的肠粘膜淋巴细胞。与未处理对照组相比,结肠炎修复期小鼠LI-IEL和LI-LPL的TCRγδ+T细胞、CD8α+TCRγδ+T细胞、CD8α+T细胞和CD8α+TCRγδ-T细胞的比例均增加,相应的TCRγδ- T细胞和CD8α-TCRγδ-T细胞的比例降低。
口服CEP结肠炎小鼠脾淋巴细胞中CD8α+T细胞的比例高于口服BSA结肠炎小鼠。PP的CD3+T细胞在结肠炎小鼠中的比例高于未处理鼠,CD8α+T细胞的比例亦增多,口服CEP结肠炎小鼠的PP中CD3+T细胞和CD8α+T细胞的比例均低于口服BSA结肠炎小鼠。与PP中细胞的变化模式相反,MLN的CD3+T细胞在结肠炎小鼠中的比例低于未处理鼠,CD8α+T细胞的比例亦减少,口服CEP结肠炎小鼠的肠系膜淋巴结中CD3+T细胞和CD8α+T细胞的比例均高于口服BSA结肠炎小鼠。
总之,口服多次低剂量CEP能缓解DSS诱导的结肠炎,口服抗原致结肠炎的改善与大肠粘膜淋巴细胞中CD8α+T细胞和TCRγδ+T细胞的比例增多相关,修复期粘膜的修复也伴随大肠粘膜淋巴细胞中CD8α+T细胞和TCRγδ+T细胞比例的增多,提示肠粘膜CD8α+T细胞和TCRγδ+T细胞在DSS诱导结肠炎中具有保护性调节作用。口服耐受诱导脾淋巴细胞中CD8α+T细胞增多。PP与MLN中的T细胞在DSS诱导结肠炎及口服耐受中发挥不同的作用。
第二部分小肠上皮内γδT细胞的提纯
γδT细胞在外周血及大多数淋巴器官中比例较少,但富集于上皮组织如肠上皮内,该部分主要建立BALB/c小鼠小肠上皮内γδT细胞的提纯过程。
取BALB/c小鼠的小肠,外翻并振荡,以离散上皮组织。在一部分实验中,细胞悬液直接用40%／70%不连续Percoll离心,在另一部分实验中,离散上皮首先用10ml的30%Percoll离心,去除大部分上皮细胞,然后用40%／70%不连续Percoll离心。利用两步Percoll离心获取SI-IEL的总过程耗时约2小时,而一步提纯法则缩短半小时左右时间。对每组8份提取细胞的分析显示：一步40%／70%Percoll离心后纯度约88.8%,两步30%/40%/70% Percoll离心后纯度约93.2%,两组之间存在显著性差异,污染的细胞主要为上皮细胞。然而在细胞量方面未见明显差异,一步Percoll离心后细胞产量为0.81-1.48×107不等,平均1.141×107细胞,两步Percoll离心后细胞产量为0.92-1.34×107不等,平均1.123×107细胞。利用三色流式细胞术检测SI-IEL中CD3+、TCRγδ+、CD8a+和CD8a+TCRγδ+T细胞的比例。SI-IEL主要由T细胞构成,而且绝大多数是CD8a+T细胞。CD3+T细胞中,TCRyγδ+T细胞占了较高的比例,大约占30-50%, TCRγδ+T细胞中大约90%是CD8a+的。两种Percoll提纯的细胞也被用于流式检测,结果表明两组之间细胞亚群的构成无显著差异。
利用MACS分选两步Percoll提纯的SI-IEL中的γδT细胞。在这个过程中,抗体和微珠的量需要经过优化处理,以获得足够的纯度和产量。实验结果显示1.5×107细胞用3μg PE-conjugated anti-TCRδ-chain抗体染色,45μl anti-PE微珠分选能获得95%以上的纯度。经包被的anti-CD3ε抗体刺激,SI-IEL比分选的γδT细胞分泌更多的IFN-y和TGF-β1,γδT细胞产生中等量的TGF-β1和极少量的IFN-γ。
总之,我们建立的SI-IEL分离程序是可依赖的简单的方法,获得的细胞纯度高及细胞量多。利用一步40%/70% Percoll提纯法可以少费力,足以用于细胞亚群的表型分析。先用10 ml的30% Percoll离心再用40%/70% Percoll提纯法获得的细胞适合用MACS技术分选出γδT细胞,从而为表型及功能研究奠定基础。
第三部分小肠上皮内淋巴细胞对葡聚糖硫酸钠诱导小鼠结肠炎的保护作用
为进一步研究SI-IEL及γδT细胞对DSS诱导小鼠结肠炎的作用,来自未处理鼠、结肠炎鼠、口服CEP结肠炎鼠及口服BSA结肠炎鼠的SI-IEL 3×106或SI-γδIEL 9×105重悬在150μl PBS中,通过尾静脉注射至受体鼠(每组8只),以注射PBS作为空白对照组,然后受体鼠自由饮用4%DSS 7日以诱导结肠炎,评估受体鼠的DAI和组织学评分。结果显示来自未处理小鼠及结肠炎小鼠的SI-IEL或分选的γδT细胞的转移均能缓解受体鼠的结肠炎,来自口服CEP及BSA结肠炎小鼠的小肠上皮内淋巴细胞或分选的γδT细胞的转移均能缓解受体鼠的结肠炎,来自口服CEP结肠炎鼠的SI-IEL的保护效果强于来自口服BSA结肠炎小鼠的SI-IEL.转移各种SI-IEL的受体结肠炎鼠的DAI及组织学评分在第7日与转移SI-γδIEL的受体结肠炎鼠相比均有所减少,但未达到统计学意义。
经包被的anti-CD3ε抗体刺激,来自口服CEP结肠炎鼠的SI-IEL和分选的γδT细胞比来自口服BSA结肠炎鼠的细胞分泌更多的TGF-β1。来自未处理鼠与结肠炎鼠之间、来自口服CEP与口服BSA结肠炎鼠之间的SI-IEL和γδT细胞分泌的IFN-γ无显著差异。细胞转移的受体鼠用于检测血清中TGF-β1和IFN-y的水平。与PBS对照组相比,细胞的转移包括来自未处理鼠、结肠炎鼠、口服CEP结肠炎鼠、口服BSA结肠炎鼠的SI-IEL或分选的γδT细胞,均引起受体鼠血清中TGF-β1水平提高2-3倍。血清TGF-β1的浓度与结肠炎体重下降百分比或组织学评分呈负相关(r=-0.762和r=-0.770,p=0.002和p=0.002)。转移口服CEP结肠炎鼠SI-IEL的受体鼠比转移口服BSA结肠炎鼠SI-IEL的受体鼠拥有更高浓度的TGF-β1 (p=0.04).血清IFN-γ在细胞转移受体鼠与PBS对照组之间均无明显差异。体外培养显示SI-IEL对anti-CD3s抗体和ConA刺激均无明显增殖。
总之,SI-IEL及分选的γδT细胞对DSS诱导的BALB/c小鼠结肠炎具有保护作用,口服耐受加强了SI-IEL中调节细胞亚群的抑制效应,包括γδT细胞。TGF-β1最可能介导这一抑制效应。口服免疫耐受、调节性T细胞与免疫抑制因子对人类IBD的治疗潜能是下一步研究的重点。
Inflammatory bowel diseases (IBD) include ulcerative colitis (UC) and Crohn's disease (CD), which are the major chronic inflammatory diseases of the gastrointestinal tract in humans. Although the causes of these disorders remain unknown, various features strongly suggest the involvement of immune responses, particularly autoimmune reactions, in the pathogenesis of IBD. Tolerance to normal flora seems to be disrupted in IBD patients, suggesting an active immune response against luminal antigens. IBD patients have elevated serum antibodies against dietary antigens. Therefore, the study of oral tolerance may have a significant clinical impact on IBD management. Several low doses of antigens induce tolerance through the negative immunoregulatory cytokines secreted by regulatory T cells. It was reported that oral colitis-extracted proteins (CEP) or normal colon-extracted proteins (NCEP) with five low doses can prevent experimental colitis induced by 2,4, 6-trinitrobenesulfonic acid (TNBS) in rodent animals. Tolerance induction was mediated by immunosuppressive cytokines such as TGF-β1, IL-4, IL-10 and so on.
γδT cells are preferentially localized in the epithelial tissues. Multiple functions have been ascribed toγδT cells. They induce cytolysis of infected or transformed intestinal epithelial cells, support mucosal IgA production and maintain homeostasis of the intestinal epithelium through an intranet withαβT and epithelial cells.γδT cells may also play an important regulatory role in oral tolerance induction. However, both the beneficial and detrimental roles ofγδT cells in the inflammatory process are evident. The role ofγδT cells in IBD patients and animal models is still controversial. The relative contributions of intestinal mucosal y8 T cells in the protective effect of oral tolerance on IBD also remain unclear. The CD8+T cells are abundant in the intestinal mucosa, especially in the intraepithelial compartment. It was reported that CD8+ regulatory T cells could play suppressive effects on IBD. Splenic CD8+Treg are induced upon oral antigen feeding. Despite of much recent interest in the intestinal mucosal CD8+T cells, the immunological function of these cells in oral tolerance is less understood.
Therefore, we evaluated the changes of CD8+T cells and TCRγδ+T cells in gut-associated lymphoid tissues (GALT) and spleens in dextran sulfate sodium (DSS)-induced colitis mice and CEP-fed DSS-induced colitis mice by flow cytometry (FCM). Then we evaluated the roles of SI-IEL or SI-γδIEL sorted by magnetic activated cell sorting (MACS) technology in DSS-induced colitis by adoptive transfer of SI-IEL or SI-y8 IEL from untreated, colitis and CEP-fed colitis mice.
Part I Improvement of colitis is associated with increases of CD8α+and TCRγδ+T cells in large intestinal mucosa
Acute colitis was induced in BALB/c mice by treatment with 4% DSS in drinking water for 7 days. The daily disease activity index (DAI) was evaluated and the large intestine from the anus to the splenic flexure area was used to estimate histological scores. DAI and histological scores in DSS-treated mice were significantly higher than those in untreated mice.
The colons were removed from DSS-induced colitis mice and used for preparation of oral antigen. CEP was orally administered into mice using a feeding atraumatic needle,250μg/mouse in 200μl, every other day for five doses. Bovine serum albumin (BSA) was used in parallel with a protocol identical to that used with CEP. After three days of rest, mice were given 4% DSS for 7days. The daily monitored disease activity index was reduced in CEP-fed colitis mice (n=12) compared with that of BSA-fed colitis mice (n=12) from day 4 to 7. There is significant difference in histological scores between two groups (33.6±2.8 vs 25.4±5.0, p<0.01).The relationship between histological scores and DAI of the last day correlated well (r=0.78,p<0.01).
Serum TGF-β1 and IFN-y levels were measured in CEP-fed colitis mice, colitis mice, and untreated mice by enzyme-linked immunosorbent assay (ELISA). Compared to untreated mice, colitis mice demonstrated significantly lower levels of serum TGF-β1. The feeding of CEP induced a two-to three-fold increase in serum TGF-β1 levels, compared to the BSA-fed colitis mice. However, no significant change was found in serum IFN-y levels.
Then percentages of CD3+, TCRγδ+, CD8α+and CD8α+TCRγδ+T cells in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) of small intestine (SI) and large intestine (LI), Peyer's patches (PP), the spleens and mesenteric lymph nodes (MLN) from model mice (n=8 each group) were determined by flow cytometry.
The proportion of TCRγδ+T cells and CD8a+TCRγδ+T cells in SI-IEL and SI-LPL from colitis mice was significantly higher compared to untreated mice. Accordingly, TCRγδ- T cells and CD8a+TCRγδ- T cells were significantly reduced in SI-IEL and SI-LPL from colitis mice. The proportion of TCRγδ+T cells, CD8α+TCRyγδ+T cells, CD8α+T cells and CD8a+TCRγδ- T cells in LI-IEL and LI-LPL from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice. Accordingly, TCRγδ-T cells and CD8αTCRγδ-T cells were significantly reduced. The relationship between DAI of the last day and percentages of TCRγδ+T cells/CD8α+T cells in large intestinal mucosa was also negatively correlated well (p<0.05).
From the results of CEP-fed DSS mice, it seemed that improvement of colitis was accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. Hence, the percentages of these cells in repair-period mice five days after termination of DSS treatment were also investigated. The proportion of TCRγδ+T cells, CD8α+TCRγδ+T cells, CD8α+T cells and CD8α+TCRγδ- T cells in LI-IEL and LI-LPL from repair-period mice was significantly higher compared to untreated mice. Accordingly, TCRγδ- T cells and CD8α-TCRγδ- T cells were significantly reduced.
The proportion of CD8α+T cells in spleens from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice. The proportion of CD3+T cells in PP from colitis mice was significantly higher than that from untreated mice. The proportion of CD8α+T cells was also increased. The proportion of CD3+T cells and CD8α+T cells from CEP-fed colitis mice was significantly lower compared to BSA-fed colitis mice. The proportion of CD3+T cells in MLN from colitis mice was significantly lower than that from untreated mice. The proportion of CD8α+T cells was also reduced. The proportion of CD3+T cells and CD8α+T cells from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice.
In conclusion, oral administration of colitis-extracted proteins could alleviate experimental colitis. Improvement of colitis resulted from oral administration of antigen was accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. Mucosal repair in repair-period mice was also accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. These results support protective regulatory role of intestinal mucosal CD8α+T cells and TCRγδ+T cells in DSS-induced colitis. Oral tolerance induced an increase of CD8α+T cells in spleens. PP and MLN may play differential roles not only in DSS-induced colitis but also in oral immune regulation.
PartⅡIsolation of murine small intestinal intraepithelialγδT cells
γδT cells represent a small leukocyte subpopulation in the peripheral blood and most lymphoid organs. However, they are enriched at epithelial barriers such as the gastrointestinal mucosa. The experiments presented here describe a purification procedure for small intestinal intraepithelialγδT lymphocytes in BALB/c mice.
Each small intestine was removed from BALB/c mice, everted and shaken to individualize epithelial content. In some experiments, the discrete epithelium was directly centrifuged using discontinuous 40%-70% Percoll gradients. In other experiments, the discrete epithelium was first centrifuged in 10 ml of 30% Percoll to remove the most of epithelial cells and then centrifuged in a discontinuous 40%-70% Percoll gradient. The time required for the SI-IEL isolation using two-step Percoll centrifugation was-2 h, while less half an hour for one-step Percoll centrifugation. From a large series of SI-IEL isolates (n=8 each group), there was a significant increase in the purity of SI-IEL after two-step purification (93.2%) compared to the purity following one-step purification (88.8%), thus representing an overall increase in purity of 4.4%. The contaminating cells were mainly epithelial cells. There was no significant difference in the yields between two groups. The recovery of SI-IEL remained both high, with 0.92-1.34×107 cells (mean value of 1.123×107) after two-step Percoll and 0.81-1.48×107 cells (mean value of 1.141×107) after one-step Percoll centrifugation procedure. Percentages of CD3+, TCRγδ+, CD8α+ and CD8a+TCRγδ+T cells in SI-IEL from BALB/c mice were determined by three color fluorescence flow cytometry. SI-IEL contained mostly T lymphocytes and majority of these cells were CD8a+. TCRγδ+T cells on CD3+gating were abundant, comprising up to 30-50% of the total cell population. Approximately 90%of TCRγδ+T cells were CD8a+. The phenotypic composition of the cell populations was also compared between the two SI-IEL purification protocols. Results showed no significant difference in the proportional distribution of SI-IEL subsets from the two purification protocols
SI-IEL isolated following two-step Percoll purification procedure was used for separation of TCRγδ+T cells. During the process, the amount of antibody and microbeads needed to be optimized to get sufficient purity and recovery. Optimal purity and recovery were achieved when 1.5×10 cells were stained with 3μg of PE-conjugated anti-TCRδ-chain antibody and sorted with 45μl of anti-PE microbeads. SI-IEL could produce much more IFN-y and TGF-β1 than sortedγδT cells upon stimulation with plate-bound anti-CD3εmAb. y8 T cells secrete moderate amounts of TGF-β1 and minimal amounts of IFN-γ.
In summary, the SI-IEL isolation protocol described here is a reliable and simple method with high purity and high yields. One-step Percoll purification by discontinuous 40%-70% Percoll gradients is less laborious yet it is sufficient to get exact profiles for use in phenotypic analysis of cell subsets. SI-IEL isolated after two-step Percoll centrifugation by 10 ml of 30% Percoll and then by discontinuous 40%-70% Percoll gradients are fit to purify y8 T cells by MACS. Purification of y8 T cells using an appropriate method is helpful in investigating the phenotype and function of intestinal mucosalγδT cells.
PartⅢProtective role of small intestinal intraepithelial lymphocytes in murine colitis induced by dextran sulfate sodium
To research the roles of SI-IEL and y8 T cells in DSS-induced colitis, SI-IEL (3×106) or SI-y8 IEL (9×105) from untreated mice, colitis mice, CEP-fed colitis mice and BSA-fed colitis mice were injected into naive mice (n= 8 each group) via tail vein in 150μl of PBS. Then mice were given 4% DSS for 7 days. Results showed that transfer of SI-IELs or sorted y8 T cells from untreated and colitis mice alleviated experimental colitis. Transfer of SI-IELs or sortedγδT cells from CEP-fed and BSA-fed colitis mice also alleviated experimental colitis. The protective effects of SI-IEL from CEP-fed colitis mice were stronger than SI-IEL from BSA-fed colitis mice. The DAI and histological scores of colitis mice upon transfer of SI-IEL were less than those of colitis mice upon transfer of SI-γδIEL on day 7, but differences were not statistically significant in this experiment.
SI-IEL and sorted y8 T cells from CEP-fed colitis mice produced more TGF-β1 than SI-IEL and sortedγδT cells from BSA-fed colitis mice upon stimulation with plate-bound anti-CD3εmAb. No difference in IFN-y production was found in supernatants of SI-IELs or sortedγδT cells between CEP-fed and BSA-fed colitis mice, or untreated and colitis mice. Serum TGF-β1 and IFN-y levels were measured in cell-transferred colitis mice by ELISA. Transfer of cells, including SI-IEL and sortedγδT cells from untreated, colitis, CEP-fed colitis and BSA-fed colitis mice, induced a two-to three-fold increase in serum TGF-β1 levels of the recipient mice. The production of serum TGF-β1 was negatively related to weight loss and histological scores of colitis (r=-0.762 and r=-0.770, p= 0.002 and p= 0.002). There were higher levels in colitis mice following transfer of SI-IEL from CEP-fed colitis mice than from BSA-fed colitis mice (p= 0.04). No significant difference was detected in serum IFN-y levels among cell-transferred mice. In vitro culture showed SI-IEL responded poorly to anti-CD3s antibody and ConA.
In summary, SI-IEL and sortedγδT cells play protective roles in DSS-induced colitis in B ALB/c mice. Oral tolerance strengthens the suppressive effects of regulatory subsets in SI-IEL, includingγδT cells. TGF-β1 seems to be the best candidate for mediating these suppressive effects. Future studies should explore the therapeutic potential of oral immune regulation, regulatory cells and immunosuppressive cytokines in human IBD.
γδT细胞倾向性分布于上皮组织。日益增多的证据表明γδT细胞能发挥多种免疫调节功能。它们诱导对感染或转化细胞的溶细胞效应,支持粘膜IgA生成,维持上皮的自稳。γδT细胞亦在口服耐受中发挥关键的调节作用。然而,关于γδT细胞在炎症过程中有利及有害的作用均有报道,在IBD患者及动物模型中的作用多年来存在争议,而且关于肠粘膜γδT细胞在口服耐受干预结肠炎过程中所起的作用尚未明确。CD8+T细胞在肠粘膜中相当丰富,尤其是上皮层。已有报道CD8+调节性T细胞对IBD的抑制效应。口服耐受能诱导产生脾CD8+调节性T细胞。但对肠粘膜CD8+T细胞在口服耐受中的作用研究甚少。
因此,本课题以多次低剂量口服CEP对葡聚糖硫酸钠(DSS)诱导BALB/c小鼠结肠炎的预防模型为基础,利用流式细胞术检测T细胞亚群CD8α+和TCRγδ+T细胞在肠相关淋巴组织(GALT)及脾中的改变,探讨T细胞亚群在DSS诱导结肠炎中的作用以及T细胞亚群在口服耐受干预结肠炎中的作用,同时过继转移来自未处理小鼠或口服耐受处理小鼠的小肠上皮内淋巴细胞(SI-IEL)及经磁性活化细胞分选技术(MACS)分选的SI-γδIEL,观察受体鼠结肠炎的症状及组织学病变,并初步探索细胞作用的机制,为IBD的发病机制及临床治疗提供新的依据。
第一部分小鼠结肠炎的改善与结肠粘膜中CD8α+和TCRγδ+T细胞增多相关
为建立BALB/c小鼠结肠炎,予7周龄雄性小鼠自由饮用4%DSS,每日监测疾病活动指数(DAI),7日后取脾曲至肛门的肠段用于组织学评分。结果表明结肠炎小鼠的第7日疾病活动指数和组织学评分均较未处理对照鼠显著升高。
取结肠炎诱导7日后小鼠的结肠标本,制备结肠炎提取蛋白(CEP)。以每只200μl 1.25 mg/ml CEP灌胃6周龄小鼠,隔日1次,共5次。以等浓度等量牛血清白蛋白(BSA)灌胃为对照。于末次灌胃后休息3天开始用4%DSS诱导结肠炎,评估两组小鼠(每组12只)的疾病活动指数及病理学改变。从结肠炎诱导第4日至第7日,口服CEP结肠炎小鼠的DAI显著低于口服BSA结肠炎小鼠。两组的组织学评分具有显著性差异(33.6±2.8 vs 25.4±5.0,p<0.01),表明口服五次低剂量CEP能缓解DSS诱导的结肠炎。Spearman分析显示组织学评分与第7日疾病活动指数相关(r=0.78,p<0.01)。
收集未处理小鼠、结肠炎小鼠、口服CEP结肠炎小鼠、口服BSA结肠炎小鼠的血清,ELISA法检测血清中TGF-β1和IFN-y的浓度。结果示结肠炎小鼠血清中的TGF-β1浓度低于未处理鼠,与口服BSA对照鼠相比,口服CEP结肠炎小鼠血清中的TGF-β1有2至3倍的增加。然而,血清IFN-y浓度在各组小鼠之间未见明显差异。
然后,我们重建结肠炎模型、口服CEP模型及对照组(每组8只小鼠),提取小肠(SI)和大肠(LI)的上皮内淋巴细胞(IEL)、固有层淋巴细胞(LPL)、脾细胞、派氏集合淋巴结(PP)及肠系膜淋巴结(MLN)的淋巴细胞,利用流式细胞术检测各组小鼠中CD3+、TCRγδ+, CD8a+和CD8a+TCRγδ+T细胞的改变。
与未处理组相比,结肠炎小鼠SI-IEL和SI-LPL的TCRγδ5+和CD8a+TCRγδ+T细胞的比例增加,相应的TCRγδ-T细胞和CD8a+TCRγδ-T细胞的比例降低。与口服BSA结肠炎对照组相比,口服CEP结肠炎小鼠LI-IEL和LI-LPL的TCRγδ+T细胞、CD8α+TCRγδ+T细胞、CD8α+T细胞和CD8α+TCRγδ-T细胞的比例均增加,相应的TCRγδ- T细胞和CD8αTCRγδ-T细胞的比例降低。第7日的DAI与大肠粘膜中CD8α+T细胞或TCRγδ+T细胞的百分比之间呈负相关(p均小于0.05)。
口服CEP结肠炎小鼠的结果表明结肠炎的改善伴随大肠粘膜淋巴细胞中CD8α+T细胞和TCRγδ+T细胞的增多,因此我们检测DSS处理终止5日的修复期小鼠的肠粘膜淋巴细胞。与未处理对照组相比,结肠炎修复期小鼠LI-IEL和LI-LPL的TCRγδ+T细胞、CD8α+TCRγδ+T细胞、CD8α+T细胞和CD8α+TCRγδ-T细胞的比例均增加,相应的TCRγδ- T细胞和CD8α-TCRγδ-T细胞的比例降低。
口服CEP结肠炎小鼠脾淋巴细胞中CD8α+T细胞的比例高于口服BSA结肠炎小鼠。PP的CD3+T细胞在结肠炎小鼠中的比例高于未处理鼠,CD8α+T细胞的比例亦增多,口服CEP结肠炎小鼠的PP中CD3+T细胞和CD8α+T细胞的比例均低于口服BSA结肠炎小鼠。与PP中细胞的变化模式相反,MLN的CD3+T细胞在结肠炎小鼠中的比例低于未处理鼠,CD8α+T细胞的比例亦减少,口服CEP结肠炎小鼠的肠系膜淋巴结中CD3+T细胞和CD8α+T细胞的比例均高于口服BSA结肠炎小鼠。
总之,口服多次低剂量CEP能缓解DSS诱导的结肠炎,口服抗原致结肠炎的改善与大肠粘膜淋巴细胞中CD8α+T细胞和TCRγδ+T细胞的比例增多相关,修复期粘膜的修复也伴随大肠粘膜淋巴细胞中CD8α+T细胞和TCRγδ+T细胞比例的增多,提示肠粘膜CD8α+T细胞和TCRγδ+T细胞在DSS诱导结肠炎中具有保护性调节作用。口服耐受诱导脾淋巴细胞中CD8α+T细胞增多。PP与MLN中的T细胞在DSS诱导结肠炎及口服耐受中发挥不同的作用。
第二部分小肠上皮内γδT细胞的提纯
γδT细胞在外周血及大多数淋巴器官中比例较少,但富集于上皮组织如肠上皮内,该部分主要建立BALB/c小鼠小肠上皮内γδT细胞的提纯过程。
取BALB/c小鼠的小肠,外翻并振荡,以离散上皮组织。在一部分实验中,细胞悬液直接用40%／70%不连续Percoll离心,在另一部分实验中,离散上皮首先用10ml的30%Percoll离心,去除大部分上皮细胞,然后用40%／70%不连续Percoll离心。利用两步Percoll离心获取SI-IEL的总过程耗时约2小时,而一步提纯法则缩短半小时左右时间。对每组8份提取细胞的分析显示：一步40%／70%Percoll离心后纯度约88.8%,两步30%/40%/70% Percoll离心后纯度约93.2%,两组之间存在显著性差异,污染的细胞主要为上皮细胞。然而在细胞量方面未见明显差异,一步Percoll离心后细胞产量为0.81-1.48×107不等,平均1.141×107细胞,两步Percoll离心后细胞产量为0.92-1.34×107不等,平均1.123×107细胞。利用三色流式细胞术检测SI-IEL中CD3+、TCRγδ+、CD8a+和CD8a+TCRγδ+T细胞的比例。SI-IEL主要由T细胞构成,而且绝大多数是CD8a+T细胞。CD3+T细胞中,TCRyγδ+T细胞占了较高的比例,大约占30-50%, TCRγδ+T细胞中大约90%是CD8a+的。两种Percoll提纯的细胞也被用于流式检测,结果表明两组之间细胞亚群的构成无显著差异。
利用MACS分选两步Percoll提纯的SI-IEL中的γδT细胞。在这个过程中,抗体和微珠的量需要经过优化处理,以获得足够的纯度和产量。实验结果显示1.5×107细胞用3μg PE-conjugated anti-TCRδ-chain抗体染色,45μl anti-PE微珠分选能获得95%以上的纯度。经包被的anti-CD3ε抗体刺激,SI-IEL比分选的γδT细胞分泌更多的IFN-y和TGF-β1,γδT细胞产生中等量的TGF-β1和极少量的IFN-γ。
总之,我们建立的SI-IEL分离程序是可依赖的简单的方法,获得的细胞纯度高及细胞量多。利用一步40%/70% Percoll提纯法可以少费力,足以用于细胞亚群的表型分析。先用10 ml的30% Percoll离心再用40%/70% Percoll提纯法获得的细胞适合用MACS技术分选出γδT细胞,从而为表型及功能研究奠定基础。
第三部分小肠上皮内淋巴细胞对葡聚糖硫酸钠诱导小鼠结肠炎的保护作用
为进一步研究SI-IEL及γδT细胞对DSS诱导小鼠结肠炎的作用,来自未处理鼠、结肠炎鼠、口服CEP结肠炎鼠及口服BSA结肠炎鼠的SI-IEL 3×106或SI-γδIEL 9×105重悬在150μl PBS中,通过尾静脉注射至受体鼠(每组8只),以注射PBS作为空白对照组,然后受体鼠自由饮用4%DSS 7日以诱导结肠炎,评估受体鼠的DAI和组织学评分。结果显示来自未处理小鼠及结肠炎小鼠的SI-IEL或分选的γδT细胞的转移均能缓解受体鼠的结肠炎,来自口服CEP及BSA结肠炎小鼠的小肠上皮内淋巴细胞或分选的γδT细胞的转移均能缓解受体鼠的结肠炎,来自口服CEP结肠炎鼠的SI-IEL的保护效果强于来自口服BSA结肠炎小鼠的SI-IEL.转移各种SI-IEL的受体结肠炎鼠的DAI及组织学评分在第7日与转移SI-γδIEL的受体结肠炎鼠相比均有所减少,但未达到统计学意义。
经包被的anti-CD3ε抗体刺激,来自口服CEP结肠炎鼠的SI-IEL和分选的γδT细胞比来自口服BSA结肠炎鼠的细胞分泌更多的TGF-β1。来自未处理鼠与结肠炎鼠之间、来自口服CEP与口服BSA结肠炎鼠之间的SI-IEL和γδT细胞分泌的IFN-γ无显著差异。细胞转移的受体鼠用于检测血清中TGF-β1和IFN-y的水平。与PBS对照组相比,细胞的转移包括来自未处理鼠、结肠炎鼠、口服CEP结肠炎鼠、口服BSA结肠炎鼠的SI-IEL或分选的γδT细胞,均引起受体鼠血清中TGF-β1水平提高2-3倍。血清TGF-β1的浓度与结肠炎体重下降百分比或组织学评分呈负相关(r=-0.762和r=-0.770,p=0.002和p=0.002)。转移口服CEP结肠炎鼠SI-IEL的受体鼠比转移口服BSA结肠炎鼠SI-IEL的受体鼠拥有更高浓度的TGF-β1 (p=0.04).血清IFN-γ在细胞转移受体鼠与PBS对照组之间均无明显差异。体外培养显示SI-IEL对anti-CD3s抗体和ConA刺激均无明显增殖。
总之,SI-IEL及分选的γδT细胞对DSS诱导的BALB/c小鼠结肠炎具有保护作用,口服耐受加强了SI-IEL中调节细胞亚群的抑制效应,包括γδT细胞。TGF-β1最可能介导这一抑制效应。口服免疫耐受、调节性T细胞与免疫抑制因子对人类IBD的治疗潜能是下一步研究的重点。
Inflammatory bowel diseases (IBD) include ulcerative colitis (UC) and Crohn's disease (CD), which are the major chronic inflammatory diseases of the gastrointestinal tract in humans. Although the causes of these disorders remain unknown, various features strongly suggest the involvement of immune responses, particularly autoimmune reactions, in the pathogenesis of IBD. Tolerance to normal flora seems to be disrupted in IBD patients, suggesting an active immune response against luminal antigens. IBD patients have elevated serum antibodies against dietary antigens. Therefore, the study of oral tolerance may have a significant clinical impact on IBD management. Several low doses of antigens induce tolerance through the negative immunoregulatory cytokines secreted by regulatory T cells. It was reported that oral colitis-extracted proteins (CEP) or normal colon-extracted proteins (NCEP) with five low doses can prevent experimental colitis induced by 2,4, 6-trinitrobenesulfonic acid (TNBS) in rodent animals. Tolerance induction was mediated by immunosuppressive cytokines such as TGF-β1, IL-4, IL-10 and so on.
γδT cells are preferentially localized in the epithelial tissues. Multiple functions have been ascribed toγδT cells. They induce cytolysis of infected or transformed intestinal epithelial cells, support mucosal IgA production and maintain homeostasis of the intestinal epithelium through an intranet withαβT and epithelial cells.γδT cells may also play an important regulatory role in oral tolerance induction. However, both the beneficial and detrimental roles ofγδT cells in the inflammatory process are evident. The role ofγδT cells in IBD patients and animal models is still controversial. The relative contributions of intestinal mucosal y8 T cells in the protective effect of oral tolerance on IBD also remain unclear. The CD8+T cells are abundant in the intestinal mucosa, especially in the intraepithelial compartment. It was reported that CD8+ regulatory T cells could play suppressive effects on IBD. Splenic CD8+Treg are induced upon oral antigen feeding. Despite of much recent interest in the intestinal mucosal CD8+T cells, the immunological function of these cells in oral tolerance is less understood.
Therefore, we evaluated the changes of CD8+T cells and TCRγδ+T cells in gut-associated lymphoid tissues (GALT) and spleens in dextran sulfate sodium (DSS)-induced colitis mice and CEP-fed DSS-induced colitis mice by flow cytometry (FCM). Then we evaluated the roles of SI-IEL or SI-γδIEL sorted by magnetic activated cell sorting (MACS) technology in DSS-induced colitis by adoptive transfer of SI-IEL or SI-y8 IEL from untreated, colitis and CEP-fed colitis mice.
Part I Improvement of colitis is associated with increases of CD8α+and TCRγδ+T cells in large intestinal mucosa
Acute colitis was induced in BALB/c mice by treatment with 4% DSS in drinking water for 7 days. The daily disease activity index (DAI) was evaluated and the large intestine from the anus to the splenic flexure area was used to estimate histological scores. DAI and histological scores in DSS-treated mice were significantly higher than those in untreated mice.
The colons were removed from DSS-induced colitis mice and used for preparation of oral antigen. CEP was orally administered into mice using a feeding atraumatic needle,250μg/mouse in 200μl, every other day for five doses. Bovine serum albumin (BSA) was used in parallel with a protocol identical to that used with CEP. After three days of rest, mice were given 4% DSS for 7days. The daily monitored disease activity index was reduced in CEP-fed colitis mice (n=12) compared with that of BSA-fed colitis mice (n=12) from day 4 to 7. There is significant difference in histological scores between two groups (33.6±2.8 vs 25.4±5.0, p<0.01).The relationship between histological scores and DAI of the last day correlated well (r=0.78,p<0.01).
Serum TGF-β1 and IFN-y levels were measured in CEP-fed colitis mice, colitis mice, and untreated mice by enzyme-linked immunosorbent assay (ELISA). Compared to untreated mice, colitis mice demonstrated significantly lower levels of serum TGF-β1. The feeding of CEP induced a two-to three-fold increase in serum TGF-β1 levels, compared to the BSA-fed colitis mice. However, no significant change was found in serum IFN-y levels.
Then percentages of CD3+, TCRγδ+, CD8α+and CD8α+TCRγδ+T cells in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) of small intestine (SI) and large intestine (LI), Peyer's patches (PP), the spleens and mesenteric lymph nodes (MLN) from model mice (n=8 each group) were determined by flow cytometry.
The proportion of TCRγδ+T cells and CD8a+TCRγδ+T cells in SI-IEL and SI-LPL from colitis mice was significantly higher compared to untreated mice. Accordingly, TCRγδ- T cells and CD8a+TCRγδ- T cells were significantly reduced in SI-IEL and SI-LPL from colitis mice. The proportion of TCRγδ+T cells, CD8α+TCRyγδ+T cells, CD8α+T cells and CD8a+TCRγδ- T cells in LI-IEL and LI-LPL from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice. Accordingly, TCRγδ-T cells and CD8αTCRγδ-T cells were significantly reduced. The relationship between DAI of the last day and percentages of TCRγδ+T cells/CD8α+T cells in large intestinal mucosa was also negatively correlated well (p<0.05).
From the results of CEP-fed DSS mice, it seemed that improvement of colitis was accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. Hence, the percentages of these cells in repair-period mice five days after termination of DSS treatment were also investigated. The proportion of TCRγδ+T cells, CD8α+TCRγδ+T cells, CD8α+T cells and CD8α+TCRγδ- T cells in LI-IEL and LI-LPL from repair-period mice was significantly higher compared to untreated mice. Accordingly, TCRγδ- T cells and CD8α-TCRγδ- T cells were significantly reduced.
The proportion of CD8α+T cells in spleens from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice. The proportion of CD3+T cells in PP from colitis mice was significantly higher than that from untreated mice. The proportion of CD8α+T cells was also increased. The proportion of CD3+T cells and CD8α+T cells from CEP-fed colitis mice was significantly lower compared to BSA-fed colitis mice. The proportion of CD3+T cells in MLN from colitis mice was significantly lower than that from untreated mice. The proportion of CD8α+T cells was also reduced. The proportion of CD3+T cells and CD8α+T cells from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice.
In conclusion, oral administration of colitis-extracted proteins could alleviate experimental colitis. Improvement of colitis resulted from oral administration of antigen was accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. Mucosal repair in repair-period mice was also accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. These results support protective regulatory role of intestinal mucosal CD8α+T cells and TCRγδ+T cells in DSS-induced colitis. Oral tolerance induced an increase of CD8α+T cells in spleens. PP and MLN may play differential roles not only in DSS-induced colitis but also in oral immune regulation.
PartⅡIsolation of murine small intestinal intraepithelialγδT cells
γδT cells represent a small leukocyte subpopulation in the peripheral blood and most lymphoid organs. However, they are enriched at epithelial barriers such as the gastrointestinal mucosa. The experiments presented here describe a purification procedure for small intestinal intraepithelialγδT lymphocytes in BALB/c mice.
Each small intestine was removed from BALB/c mice, everted and shaken to individualize epithelial content. In some experiments, the discrete epithelium was directly centrifuged using discontinuous 40%-70% Percoll gradients. In other experiments, the discrete epithelium was first centrifuged in 10 ml of 30% Percoll to remove the most of epithelial cells and then centrifuged in a discontinuous 40%-70% Percoll gradient. The time required for the SI-IEL isolation using two-step Percoll centrifugation was-2 h, while less half an hour for one-step Percoll centrifugation. From a large series of SI-IEL isolates (n=8 each group), there was a significant increase in the purity of SI-IEL after two-step purification (93.2%) compared to the purity following one-step purification (88.8%), thus representing an overall increase in purity of 4.4%. The contaminating cells were mainly epithelial cells. There was no significant difference in the yields between two groups. The recovery of SI-IEL remained both high, with 0.92-1.34×107 cells (mean value of 1.123×107) after two-step Percoll and 0.81-1.48×107 cells (mean value of 1.141×107) after one-step Percoll centrifugation procedure. Percentages of CD3+, TCRγδ+, CD8α+ and CD8a+TCRγδ+T cells in SI-IEL from BALB/c mice were determined by three color fluorescence flow cytometry. SI-IEL contained mostly T lymphocytes and majority of these cells were CD8a+. TCRγδ+T cells on CD3+gating were abundant, comprising up to 30-50% of the total cell population. Approximately 90%of TCRγδ+T cells were CD8a+. The phenotypic composition of the cell populations was also compared between the two SI-IEL purification protocols. Results showed no significant difference in the proportional distribution of SI-IEL subsets from the two purification protocols
SI-IEL isolated following two-step Percoll purification procedure was used for separation of TCRγδ+T cells. During the process, the amount of antibody and microbeads needed to be optimized to get sufficient purity and recovery. Optimal purity and recovery were achieved when 1.5×10 cells were stained with 3μg of PE-conjugated anti-TCRδ-chain antibody and sorted with 45μl of anti-PE microbeads. SI-IEL could produce much more IFN-y and TGF-β1 than sortedγδT cells upon stimulation with plate-bound anti-CD3εmAb. y8 T cells secrete moderate amounts of TGF-β1 and minimal amounts of IFN-γ.
In summary, the SI-IEL isolation protocol described here is a reliable and simple method with high purity and high yields. One-step Percoll purification by discontinuous 40%-70% Percoll gradients is less laborious yet it is sufficient to get exact profiles for use in phenotypic analysis of cell subsets. SI-IEL isolated after two-step Percoll centrifugation by 10 ml of 30% Percoll and then by discontinuous 40%-70% Percoll gradients are fit to purify y8 T cells by MACS. Purification of y8 T cells using an appropriate method is helpful in investigating the phenotype and function of intestinal mucosalγδT cells.
PartⅢProtective role of small intestinal intraepithelial lymphocytes in murine colitis induced by dextran sulfate sodium
To research the roles of SI-IEL and y8 T cells in DSS-induced colitis, SI-IEL (3×106) or SI-y8 IEL (9×105) from untreated mice, colitis mice, CEP-fed colitis mice and BSA-fed colitis mice were injected into naive mice (n= 8 each group) via tail vein in 150μl of PBS. Then mice were given 4% DSS for 7 days. Results showed that transfer of SI-IELs or sorted y8 T cells from untreated and colitis mice alleviated experimental colitis. Transfer of SI-IELs or sortedγδT cells from CEP-fed and BSA-fed colitis mice also alleviated experimental colitis. The protective effects of SI-IEL from CEP-fed colitis mice were stronger than SI-IEL from BSA-fed colitis mice. The DAI and histological scores of colitis mice upon transfer of SI-IEL were less than those of colitis mice upon transfer of SI-γδIEL on day 7, but differences were not statistically significant in this experiment.
SI-IEL and sorted y8 T cells from CEP-fed colitis mice produced more TGF-β1 than SI-IEL and sortedγδT cells from BSA-fed colitis mice upon stimulation with plate-bound anti-CD3εmAb. No difference in IFN-y production was found in supernatants of SI-IELs or sortedγδT cells between CEP-fed and BSA-fed colitis mice, or untreated and colitis mice. Serum TGF-β1 and IFN-y levels were measured in cell-transferred colitis mice by ELISA. Transfer of cells, including SI-IEL and sortedγδT cells from untreated, colitis, CEP-fed colitis and BSA-fed colitis mice, induced a two-to three-fold increase in serum TGF-β1 levels of the recipient mice. The production of serum TGF-β1 was negatively related to weight loss and histological scores of colitis (r=-0.762 and r=-0.770, p= 0.002 and p= 0.002). There were higher levels in colitis mice following transfer of SI-IEL from CEP-fed colitis mice than from BSA-fed colitis mice (p= 0.04). No significant difference was detected in serum IFN-y levels among cell-transferred mice. In vitro culture showed SI-IEL responded poorly to anti-CD3s antibody and ConA.
In summary, SI-IEL and sortedγδT cells play protective roles in DSS-induced colitis in B ALB/c mice. Oral tolerance strengthens the suppressive effects of regulatory subsets in SI-IEL, includingγδT cells. TGF-β1 seems to be the best candidate for mediating these suppressive effects. Future studies should explore the therapeutic potential of oral immune regulation, regulatory cells and immunosuppressive cytokines in human IBD.
引文
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