Kurata H, Arai T, Yokota T, Arai K
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Japan.
J Allergy Clin Immunol. 1995 Dec;96(6 Pt 2):1083-99. doi: 10.1016/s0091-6749(95)70194-x.
Cytokines transduce their signals through specific receptors. Receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5 share the common signal transducing subunit (beta c), whereas the alpha subunits function as specific ligand binding components. In this study we prepared specific mouse monoclonal antibodies against human GM-CSF receptor-alpha subunit (hGMR alpha) by immunizing mice with Ba/F3 cells transfected with hGMR alpha complementary DNA. Using these anti-hGMR alpha antibodies in combination with antibodies against IL-3 receptor-alpha (IL-3R alpha), beta c subunits, and c-kit, we examined expression patterns and modulation of these receptor subunits on several human hematopoietic cells, including CD34+ cells and leukemic cell lines. GMR alpha and IL-3R alpha were expressed on GM-CSF- and IL-3-responsive cell lines, such as TF-1 and UT-7, whereas the expression levels were much lower on UT-7E, a GM-CSF- and IL-3-unresponsive subline of UT-7. The GMR alpha subunit was expressed only on mature granulocytes and monocytes, and IL-3R alpha was expressed on monocytes but not on mature granulocytes, and none of these subunits were expressed on lymphocytes. For CD34+ cells, GMR alpha was expressed more abundantly on CD34+ CD33high cells than on CD34+ CD33low cells, whereas IL-3R alpha was expressed more abundantly on CD34+ CD33low cells than on CD34+ CD33high and CD34+ CD33neg cells. Slight but significant expression of the beta c subunit was detected on CD34+ cells. Expression of not only GMR alpha and IL-3R alpha subunits but also c-kit was specifically downregulated by 48-hour incubation with their respective ligands. Receptor transmodulation between GM-CSF, IL-3, and stem cell factor (or kit ligand) was not detected on CD34+ cells in 48-hour cultures. We also detected upregulation of these alpha subunits by IL-1 alpha and interferon-gamma on leukemic cell lines. Our study showed expression levels for each receptor subunit--including GMR, IL-3R, and c-kit on human bone marrow and peripheral blood cells and leukemic cell lines--and revealed differential regulation of the expression of the receptor subunits.
细胞因子通过特定受体传导信号。粒细胞-巨噬细胞集落刺激因子（GM-CSF）、白细胞介素-3（IL-3）和白细胞介素-5的受体共享共同的信号转导亚基（βc），而α亚基则作为特异性配体结合成分发挥作用。在本研究中，我们通过用转染了人GM-CSF受体α亚基（hGMRα）互补DNA的Ba/F3细胞免疫小鼠，制备了针对人GM-CSF受体α亚基的特异性小鼠单克隆抗体。使用这些抗hGMRα抗体与抗IL-3受体α（IL-3Rα）、βc亚基和c-kit的抗体相结合，我们检测了包括CD34+细胞和白血病细胞系在内的几种人造血细胞上这些受体亚基的表达模式和调节情况。GMRα和IL-3Rα在GM-CSF和IL-3反应性细胞系（如TF-1和UT-7）上表达，而在UT-7的GM-CSF和IL-3无反应性子系UT-7E上表达水平要低得多。GMRα亚基仅在成熟粒细胞和单核细胞上表达，IL-3Rα在单核细胞上表达但不在成熟粒细胞上表达，并且这些亚基在淋巴细胞上均不表达。对于CD34+细胞，GMRα在CD34+ CD33high细胞上的表达比在CD34+ CD33low细胞上更丰富，而IL-3Rα在CD34+ CD33low细胞上的表达比在CD34+ CD33high和CD34+ CD33neg细胞上更丰富。在CD34+细胞上检测到βc亚基有轻微但显著的表达。用各自的配体孵育48小时后，不仅GMRα和IL-3Rα亚基的表达，而且c-kit的表达均被特异性下调。在48小时培养的CD34+细胞上未检测到GM-CSF、IL-3和干细胞因子（或kit配体）之间的受体转调节。我们还在白血病细胞系上检测到IL-1α和干扰素-γ对这些α亚基的上调作用。我们的研究显示了每个受体亚基在人骨髓、外周血细胞和白血病细胞系上的表达水平，包括GMR、IL-3R和c-kit，并揭示了受体亚基表达的差异调节。
