Hecker Andreas, Mikulski Zbigniew, Lips Katrin S, Pfeil Uwe, Zakrzewicz Anna, Wilker Sigrid, Hartmann Petra, Padberg Winfried, Wessler Ignaz, Kummer Wolfgang, Grau Veronika
Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Giessen, Germany.
J Leukoc Biol. 2009 Jul;86(1):13-22. doi: 10.1189/jlb.1107722. Epub 2009 Apr 30.
During acute rejection, large numbers of leukocytes accumulate in the blood vessels of experimental renal allografts. About 70% of them are activated, cytotoxic monocytes that appear to be involved in allograft destruction. ACh exerts anti-inflammatory effects upon monocytes/macrophages and has been proposed to be a key player in neuroimmunological interactions. Its short half-life, however, makes it unlikely that neuronal ACh affects blood leukocytes. Renal transplantation was performed in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested after 4 days, and the expression of CHT1, cChAT, pChAT, and nAChR subunits was investigated by RT-PCR, immunoblotting, and immunohistochemistry. Monocytes were identified by double-labeling with ED1-antibody, directed to a CD68-like antigen. ACh content was measured by HPLC. Ca(2+) was monitored by Fura-2. Intravascular graft leukocytes express CHT1 and cChAT mRNA and protein and pChAT protein. Their expression is strongly up-regulated in vivo during acute allograft rejection. Immunohistochemistry revealed CHT1, cChAT, and pChAT protein in ED1-positive monocytes. The ACh content of allograft intravascular leukocytes was sixfold higher than that of isografts. Intravascular leukocytes express nAChR subunits, and an ATP-induced increase in Ca(2+) was augmented in vitro by a nAChR inhibitor in allograft but not isograft leukocytes. Intravascular graft leukocytes, among them monocytes, up-regulate non-neuronal ACh synthesis and develop auto-/paracrine cholinergic attenuation of ATP signaling during acute allograft rejection.
在急性排斥反应期间，大量白细胞积聚在实验性肾移植的血管中。其中约70%是活化的细胞毒性单核细胞，似乎参与了移植肾的破坏。乙酰胆碱（ACh）对单核细胞/巨噬细胞具有抗炎作用，并且被认为是神经免疫相互作用中的关键因素。然而，其半衰期较短，使得神经元ACh不太可能影响血液中的白细胞。在异基因DA到LEW以及同基因LEW到LEW大鼠品系组合中进行肾移植。4天后采集血管内白细胞，通过逆转录聚合酶链反应（RT-PCR）、免疫印迹和免疫组织化学研究胆碱转运体1（CHT1）、胞质型胆碱乙酰转移酶（cChAT）、磷酸化胆碱乙酰转移酶（pChAT）和烟碱型乙酰胆碱受体（nAChR）亚基的表达。通过用针对CD68样抗原的ED1抗体进行双重标记来鉴定单核细胞。通过高效液相色谱法（HPLC）测量ACh含量。用Fura-2监测细胞内钙离子浓度（Ca(2+)）。血管内移植白细胞表达CHT1、cChAT mRNA和蛋白质以及pChAT蛋白质。在急性移植排斥反应期间，它们的表达在体内强烈上调。免疫组织化学显示在ED1阳性单核细胞中有CHT1、cChAT和pChAT蛋白质。移植肾血管内白细胞的ACh含量比同基因移植肾高六倍。血管内白细胞表达nAChR亚基，并且在体外，nAChR抑制剂增强了移植肾而非同基因移植肾白细胞中ATP诱导的Ca(2+)增加。在急性移植排斥反应期间，血管内移植白细胞（包括单核细胞）上调非神经元ACh合成，并产生ATP信号的自分泌/旁分泌胆碱能衰减。
