Di Sabatino A, Ciccocioppo R, Cinque B, Millimaggi D, Morera R, Ricevuti L, Cifone M G, Corazza G R
Gastroenterology Unit, IRCCS Policlinico S Matteo, University of Pavia, Pavia, Italy.
Gut. 2004 Jan;53(1):70-7. doi: 10.1136/gut.53.1.70.
To verify whether targeting defective mucosal T cell death underlies the sustained therapeutic benefit of infliximab in Crohn's disease, we explored its in vivo proapoptotic effect after 10 weeks of treatment, and its in vitro killing activity on lamina propria T cells (LPT) and peripheral blood T cells (PBT), both isolated from Crohn's disease patients.
Endoscopic intestinal biopsies were collected from 10 Crohn's disease patients (six steroid refractory and four fistulising) before and after three consecutive infusions of infliximab, administered at week 0, 2, and 6 in a single intravenous dose (5 mg/kg), and from 10 subjects who proved to have functional diarrhoea. Apoptosis was determined in vivo by TUNEL assay, and in vitro by fluorescein isothiocyanate-annexin V/propidium iodide staining on LPT and PBT from Crohn's disease patients cultured with infliximab. The effect of the broad caspase inhibitor Z-VAD-FMK and the neutralising anti-Fas antibody ZB4 was tested in vitro on LPT and PBT treated with infliximab. Caspase-3 activity was determined by immunoblotting.
In Crohn's disease patients, infliximab treatment induced a sustained LPT apoptosis, still evident four weeks after the last infusion. In vitro infliximab induced death of LPT from Crohn's disease patients occurred via apoptosis rather than necrosis. LPT showed a higher susceptibility to infliximab induced apoptosis than PBT in Crohn's disease patients. The signalling pathway underlying the restoration of infliximab induced LPT apoptosis occurred via the caspase pathway but not Fas-Fas ligand interaction in Crohn's disease.
These findings demonstrate that apoptosis is the major mechanism by which infliximab exerts its killing activity on LPT in Crohn's disease. The sustained LPT proapoptotic action of infliximab, which extends far beyond its circulating half life, may be responsible for the sustained remission induced in Crohn's disease patients by infliximab retreatment.
为验证靶向缺陷性黏膜T细胞死亡是否为英夫利昔单抗对克罗恩病持续治疗益处的基础，我们探究了治疗10周后其体内促凋亡作用，以及对从克罗恩病患者分离的固有层T细胞（LPT）和外周血T细胞（PBT）的体外杀伤活性。
从10例克罗恩病患者（6例激素难治性和4例瘘管形成性）在第0、2和6周单次静脉注射（5mg/kg）连续三次输注英夫利昔单抗之前和之后采集内镜下肠道活检组织，并从10例经证实有功能性腹泻的受试者采集活检组织。通过TUNEL法在体内测定凋亡，通过异硫氰酸荧光素-膜联蛋白V/碘化丙啶染色在与英夫利昔单抗共培养的克罗恩病患者的LPT和PBT上在体外测定凋亡。在体外测试广谱半胱天冬酶抑制剂Z-VAD-FMK和中和性抗Fas抗体ZB4对用英夫利昔单抗处理的LPT和PBT的作用。通过免疫印迹法测定半胱天冬酶-3活性。
在克罗恩病患者中，英夫利昔单抗治疗诱导LPT持续凋亡，在最后一次输注后四周仍很明显。体外，英夫利昔单抗诱导克罗恩病患者LPT死亡是通过凋亡而非坏死。在克罗恩病患者中，LPT比PBT对英夫利昔单抗诱导的凋亡更敏感。英夫利昔单抗诱导LPT凋亡恢复的信号通路是通过半胱天冬酶途径而非克罗恩病中的Fas-Fas配体相互作用。
这些发现表明凋亡是英夫利昔单抗对克罗恩病LPT发挥杀伤活性的主要机制。英夫利昔单抗对LPT的持续促凋亡作用远远超出其循环半衰期，可能是英夫利昔单抗再治疗诱导克罗恩病患者持续缓解的原因。
