Clark B D, Ikejima T, Mancilla J, Orencole S F, Sirko S P, Ishii N, Okuda K, Dinarello C A
Department of Medicine, Tufts University School of Medicine, Boston, MA, USA.
J Interferon Cytokine Res. 1996 Dec;16(12):1079-88. doi: 10.1089/jir.1996.16.1079.
On the basis of their relative hydropathy and alpha-helical structure, we prepared antibodies to four synthetic peptides with amino acid sequences homolgous to four hydrophilic, extracellular regions of the murine 80 kDa type I interleukin-1 receptor (IL-1RI). Antibodies to each of the four peptides recognized their specific immunogen. Human [125I]-IL-1 alpha or -beta was crosslinked to murine EL4 and D10S cells. Antiserum to peptide 150-166 precipitated the IL-1/IL-1R complex, whereas antibodies to peptide 66-84, 190-200, or 266-285 did not. Antibody to peptide 150-166 did not precipitate the type II IL-1R. Anti-IL-1RI150-166 blocked 71% of the binding of radiolabeled human IL-1 beta to EL4 cells and 50% of the binding to D10S cells. Using affinity-purified anti-IL-1RI150-166, we compared the ability of this antibody to inhibit the binding of murine or human IL-1 alpha to that of murine or human IL-1 beta. At a concentration of 20 ng/ml, affinity-purified anti-IL-1RI150-166 blocked 50% binding of murine IL-1 beta. At 1 microgram/ml, 90% blockage was observed. In contrast, no significant blockade of IL-1 alpha binding was observed at concentrations as high as 3 micrograms/ml of anti-IL-1RI150-166. The selective blockade of IL-1 beta forms was not due to differences in the affinities of these ligands for receptors on these cells. The antibody also blocked the binding of human IL-1 beta but not human IL-1 alpha to EL4 cells. The biologic activity of murine IL-1 beta but not IL-1 alpha on EL4 cells was also inhibited by this antibody. These data suggest (1) that antibody to a specific epitope on the extracellular domain interferes with the binding of IL-1 beta but not IL-1 alpha, (2) the differential inhibition of binding of IL-1 beta but not IL-1 alpha by anti-IL-1RI150-166 also blocks biologic activity, and (3) IL-1 alpha and IL-1 beta may transduce different signals by binding to separate loci on the IL-1RI.
基于它们相对的亲水性和α-螺旋结构，我们制备了针对四种合成肽的抗体，这些肽的氨基酸序列与小鼠80 kDa I型白细胞介素-1受体（IL-1RI）的四个亲水性细胞外区域同源。针对这四种肽中每一种的抗体都识别其特异性免疫原。人[125I]-IL-1α或-β与小鼠EL4和D10S细胞交联。针对肽150-166的抗血清沉淀了IL-1/IL-1R复合物，而针对肽66-84、190-200或266-285的抗体则没有。针对肽150-166的抗体没有沉淀II型IL-1R。抗IL-1RI150-166阻断了放射性标记的人IL-1β与EL4细胞结合的71%以及与D10S细胞结合的50%。使用亲和纯化的抗IL-1RI150-166，我们比较了该抗体抑制小鼠或人IL-1α与小鼠或人IL-1β结合的能力。在浓度为20 ng/ml时，亲和纯化的抗IL-1RI150-166阻断了小鼠IL-1β 50%的结合。在1μg/ml时，观察到90%的阻断。相比之下，在抗IL-1RI150-166浓度高达3μg/ml时，未观察到对IL-1α结合的显著阻断。对IL-1β形式的选择性阻断并非由于这些配体与这些细胞上受体的亲和力存在差异。该抗体也阻断了人IL-1β与EL4细胞的结合，但未阻断人IL-1α与EL4细胞的结合。该抗体还抑制了小鼠IL-1β而非IL-1α对EL4细胞的生物学活性。这些数据表明：（1）针对细胞外结构域上特定表位的抗体干扰IL-1β而非IL-1α的结合；（2）抗IL-1RI150-166对IL-1β而非IL-1α结合的差异抑制也阻断了生物学活性；（3）IL-1α和IL-1β可能通过与IL-1RI上不同位点结合来转导不同信号。
