The B-chain of mistletoe lectin I efficiently stimulates calcium signaling in human Jurkat T-cells

Mistletoe lectin I (ML I), a heterodimeric disulfide-linked type II ribosome inactivating protein, exhibits immunomodulatory potency in stimulating the cytokine release in vitro and in vivo. However, data concerning early activation events in T-cells induced by ML I and its A and B chain preceding cytokine secretion and the receptors involved are of limited availability. Here we show by flow cytometric measurements that human T-lymphoblastoid Jurkat cells express surface glycoprotein receptors for ML I. One of which is shown to be the CD2 antigen involved in a variety of T-cell signaling events. The lectin induces in Jurkat T-cells an increase of the cytosolic calcium concentration ([Ca2+](i)) consisting of both, the transient release of Ca2+ from internal stores and a sustained influx of extracellular Ca2+. Studies with isolated A- and B-chains provided evidence that the lectin-induced increase in [Ca2+](i) is mediated by ML IB. The ML I and ML IB stimulated cellular calcium responses are inhibited by saccharidic competitors. In transiently transfected E6.1 cells ML IB stimulated the expression of the luciferase reporter construct pNFAT-TA-Luc that is activated through the nuclear factor of activated T-cells (NFAT). The ML IB stimulated expression of the reporter luciferase (Luc) is completely inhibited by cyclosporin A (0.2 muM) and by FK 506 at 0.05 muM. Pretreatment of Jurkat E6.1 cells with 1-deoxymannojirimycin (dMJ), an inhibitor of cis-Golgi alpha -mannosidase 1, strongly reduced cell binding of ML IB-FITC and the ML IB induced calcium response. Benzyl-alpha -GalNAc, an inhibitor of O-linked glycosylation, has slightly decreasing effects in ML IB-FITC binding and was without effects on the lectin stimulated increase in [Ca2+](i). Inhibition of the lectin induced calcium responses by cholera toxin and by inhibitors of protein kinases as well as the absence of calcium responses in CD3(-) and CD45(-) Jurkat T-cell clones suggest that ML IB has the potency to induce early T-cell activation events. (C) 2001 Elsevier Science B.V. All rights reserved.