concanavalin-a and diphenyleneiodonium

The lectin, concanavalin A (Con A), is the most extensively investigated member of the lectin family of plant proteins, but its effects on cortical neurons and astrocytes are poorly understood. In cultured cortical neurons and astrocytes, Con A exhibited dose-dependent neurotoxicity, but this was not observed in astrocytes. Similarly, in the cortical areas of rat brains, intracranial administration of Con A caused neuronal but no astrocyte damage. Methyl-α-D-mannopyranoside, a competitor of Con A, blocked Con A-induced cell death, whereas AMPA/KA receptor antagonists showed partial blocking effects. Furthermore, the mRNA levels of TNF-α, IL-1β, and IL-6 were elevated in astrocytes and cortical neurons treated with Con A. Intracellular reactive oxygen species (ROS) levels were increased in Con A-treated cortical neurons, and N-acetyl-cysteine (NAC, an antioxidant) and diphenyleneiodonium (DPI, a NADPH oxidase inhibitor) reduced intracellular ROS accumulation. Likewise, AG556 (a TNF-α inhibitor) and AG82 (a tyrosine kinase inhibitor) both reduced Con A-induced intracellular ROS accumulation. Furthermore, Con A-induced tyrosine phosphorylation was decreased by NAC and by AG556. Taken together, Con A-induced apoptosis in cortical neurons occurred as a sequel to Con A binding to neuronal glycoproteins and intracellular ROS accumulation. Interestingly, Con A-induced cellular damage was observed in cortical neurons but not in astrocytes or microglia.

Topics: Animals; Antioxidants; Apoptosis; Astrocytes; Cell Death; Cells, Cultured; Concanavalin A; Male; Microglia; NADPH Oxidases; Neurons; Onium Compounds; Phosphorylation; Rats, Sprague-Dawley; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Molecular events mediating the T-lymphocyte response to lectins are still incompletely understood, although much evidence suggests that both the mitogenic and the death-promoting effects of these agents involve the biochemical cascade initiated by the CD3/T-cell antigen receptor (TCR) complex. Reactive oxygen species (ROS) and in particular H(2)O(2) have been shown to have a role in cell response to cytokines and growth factors. Here we report that the proliferation of mouse thymocytes in response to the mitogenic lectin concanavalin A (ConA) is strongly and selectively inhibited by the intracellular ROS scavenger N-acetylcysteine (NAC) and by diphenyleneiodonium (DPI), a potent inhibitor of NADPH-dependent membrane oxidases activated by surface receptors. A rapid 'burst' of intracellular oxygen radicals was observed in mouse thymocytes stimulated by ConA, with kinetics that paralleled the appearance of tyrosine-phosphorylated proteins. This burst was abrogated by the pretreatment of cells with NAC or DPI. Only a modest increase in intracellular oxygen species was found in thymocytes stimulated by strong cross-linking of TCR together with CD4 or CD28. Pharmacological interference with ROS production in ConA-stimulated thymocytes resulted in a decreased tyrosine phosphorylation of multiple protein species, including a 38 kDa band able to recruit the adapter protein Grb2 and corresponding to the recently identified transducer LAT (linker for activation of T-cells), a molecule involved in linking activated TCR to the production of interleukin 2 and the proliferation of T-cells. Furthermore, ROS inhibition markedly attenuated the activation of stress-activated protein kinase/JNK-1 (c-Jun N-terminal kinase 1) in response to lectins. Taken together, these results identify ROS as important modulators of the signalling cascade initiated by mitogenic lectins in thymocytes and, by extension, as a novel class of mediators downstream of antigen receptors.

Topics: Acetylcysteine; Animals; Antioxidants; CD4 Antigens; CD8 Antigens; Cells, Cultured; Concanavalin A; Enzyme Inhibitors; Kinetics; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Onium Compounds; Phosphorylation; Phosphotyrosine; Reactive Oxygen Species; Receptor-CD3 Complex, Antigen, T-Cell; T-Lymphocytes; Tetradecanoylphorbol Acetate