Role of Eosinophil in the Onset and Chronicity of Ulcerative Colitis

背景：溃疡性结肠炎中，结肠粘膜表现出明显的活化嗜酸性粒细胞浸润，伴有嗜酸性粒细胞颗粒蛋白之一的嗜酸性阳离子蛋白（ECP）的分泌形式。虽然有证据表明ECP与溃疡性结肠炎的炎症过程密切相关，但ECP对肠道的直接影响尚不清楚。为了阐明ECP对粘膜的损害，我们从正常人嗜酸性粒细胞中纯化ECP，然后观察ECP对猪小肠眼膜平滑肌收缩的影响。方法：1；ECP的纯化：ECF是从健康人的褐皮嗜酸性粒细胞颗粒中纯化得到的。采用Peterson et al.（1988）的方法进行纯化，步骤包括：1)预处理：(1)用葡聚糖处理从大约21个正常人的褐皮中分离白细胞，（2)用匀浆分离颗粒，(3)用超滤分离颗粒浓度为20倍的白细胞，以及2）舱层析：(1)Superdex 75凝胶过滤，(2)SOURCE离子交换层析。(3)锌螯合Sepharose的螯合色谱。采用RIA （Pharmacla ECP RIA试剂盒）测定ECP。2. 收缩检查：将豚鼠小肠条（2cm长，从离盲肠连接处近端10cm处取下）置于克雷布斯-林格溶液存在的器官浴中。施加约600毫克静息张力并保持恒定。将ecp10杯/l加入器官液中，通过等距传感器记录机械活动。同时观察阿托品是否抑制ECP收缩作用。结果：1；ECP的纯化：分离最后阶段的ECP活性在电泳分子量为16 ~ 21.4kD的47号组分（ECP浓度为4292马克杯/l）和48号组分（ECP浓度为3805马克杯/l）存在明显的I)COk，认为ECP具有较高的纯化度。2. 收缩检查：以正常血清的切断浓度10杯/升ECP加入器官浴后，观察短暂性收缩。ECP诱导的收缩宽度约为10^-^6 M乙酰胆碱诱导的收缩宽度的13%。此外，在初步实验中，阿托品对豚鼠平滑肌的收缩作用有部分抑制作用。结论：首次证实了ECP对肠道的作用。ECP与桂皮猪肠道的光滑接触，可能与溃疡性结肠炎的病理生理有关。少

Background : In ulcerative colitis, the colonic mucosa exhibited the marked infiltration of activated eoslnophils with the secretoly form of eosinophil cationic protein(ECP), one of the eosinophll granule proteins. Although it Is strongly suggested that ECP is closely associated with the inflammatory process of ulcerative colitis, the direct effect of ECP to the intestine remains obscure.To elucidate the mucosal damage caused by ECP, purification of ECP from normal human eosinophils was performed, and then, to examine the effect of ECP on the contraction of the smooth muscle of the glnea pig small intestine.Methods : 1. Purification of ECP : ECF was purified from the granules of human buffy coat eosinophils obtained from healthy Individuals. Method of Peterson et al. (1988) was followed for purification The procedure included 1) pretreatment : (1) separation of leukocytes by Dextran treatment from approximately 2l buffy coats of normal subjects, (2) separation of granules by homogenizi … More ng of leukocytes, and (3) 20-fold concentration of the granules by ultra-filtration, and 2) cabin chromatography : (1) gel filtration on Superdex 75, (2) ion exchange chromatography on SOURCE.and (3) chelating chromatography on zinc-chelating Sepharose. ECP was measured by RIA (Pharmacla ECP RIA kit). 2. Examinatlon of the contraction : The strips of the guinea pig small intestine (2cm long were removed from a region 10cm proximal from the lleo-cecal Junction) were placed in a oragan bath In the presence of Krebs-Ringer solution. ApproxImately 600 mg of resting tension was applied and was kept constant. ECP1O mug/l was added to the oragan bath, and mechanical activity was recorded by means of an isometric transducer. In addition, examination whether atropine inhibits the contraction effect of ECP was performed.Results : 1. Purification of ECP : The ECP activity at the final stage of separating process showed a definite I)COk at fractions No. 47(the concentration of ECP was 4292 mug/l) and No. 48 (that was 3805 mug/l) ECP was existent in the area of molecular weight l6kD to 21.4kD in electrophoresis, and it was considered that a high degree of ECP purification was available. 2. Examination of the contraction : After addition of 10 mug/l ECP, which was the cut off concentration of normal serum, to the organ bath, the transient contraction was observed. The width of the contraction Inducted by ECP was approximately 13% of that of inducted by 10^-^6 M acetylcholine. Furthermore, the ECP contraction effect to the smooth muscle of guinea-pig were inhibited partially by atropine at preliminary experiment.Conclusion : This is the first demonstration of the effect of ECP to the intestine. ECP conrtacts the smooth of the guipan pig intestene and may contribute the pathopbysiology of ulcerative colitis. Less