Effect of temperature on raft-dependent endocytic cluster formation during activation of Jurkat T cells by concanavalin A.

Temperature plays an important role in the immune response. Acclimatization occurs when there are changes in ambient temperature over a long period. In this study, we used the human leukemic Jurkat T cell line to study the effect of temperature on the immune system using concanavalin A (ConA), a plant-derived immunostimulant, as a trigger for T-cell activation. Previously, we have reported endocytic intracellular cluster formation during T-cell activation by ConA with the aid of rafts and polymerization of the cytoskeleton (actin and microtubules). Here, we investigated the effect of temperature on cluster formation (with the aid of three-dimensional images of the cells) and on the stability of rafts, actin, and microtubules. When the temperature was changed between 23°C and 37°C (physiological temperature), clusters could be observed throughout this temperature range. Raft structure was stabilized at lower temperatures but destabilized at higher temperatures. Actin was stable when the temperature was higher than 27°C. When actin was depolymerized, clustering was not observed at 37°C but could be observed at 23°C. There were no changes in microtubules within this temperature range. Thus, raft clustering may be associated with raft stability at lower temperatures (<27°C) and with actin at higher temperatures (≥27°C). Hence, we provided insight into the associations between temperature, rafts, actin, and microtubules in the immune response.

Raft-dependent endocytic movement and intracellular cluster formation during T cell activation triggered by concanavalin A.

Certain food ingredients can stimulate the human immune system. A lectin, concanavalin A (ConA), from Canavalia ensiformis (jack bean) is one of the most well-known food-derived immunostimulants and mediates activation of cell-mediated immunity through T cell proliferation. Generally, T cell activation is known to be triggered by the interaction between T cells and antigen-presenting cells (APCs) via a juxtacrine (contact-dependent) signaling pathway. The mechanism has been well characterized and is referred to as formation of the immunological synapse (IS). We were interested in the mechanism behind the T cell activation by food-derived ConA which might be different from that of T cell activation by APCs. The purpose of this study was to characterize T cell activation by ConA with regard to (i) movement of raft domain, (ii) endocytic vesicular transport, (iii) the cytoskeleton (actin and microtubules), and (iv) cholesterol composition. We found that raft-dependent endocytic movement was important for T cell activation by ConA and this movement was dependent on actin, microtubules, and cholesterol. The T cell signaling mechanism triggered by ConA can be defined as endocrine signaling which is distinct from the activation process triggered by interaction between T cells and APCs by juxtacrine signaling. Therefore, we hypothesized that T cell activation by ConA includes both two-dimensional superficial raft movement on the membrane surface along actin filaments and three-dimensional endocytic movement toward the inside of the cell along microtubules. These findings are important for developing new methods for immune stimulation and cancer therapy based on the function of ConA.

Glycosyl chains and 25-hydroxycholesterol contribute to the intracellular transport of amyloid beta (Aß-42) in Jurkat T cells.

Amyloid beta (Aß) is a peptide responsible for the development of Alzheimer's disease (AD). Misfolding and accumulation of endogenous Aß can lead to neural cell apoptosis through endoplasmic reticulum (ER) stress. Added exogenous Aß can also result in ER stress, leading to neurotoxicity and apoptosis, which is identical to that caused by the endogenous peptide. We have speculated that the endocytic transport of Aß causes ER stress and have previously shown that the oxysterol, in particular, 7-ketocholesterol (7-keto) induces more surface interaction between Aß-42 and Jurkat cells than cholesterol. However, the interaction was not enough to induce intracellular transfer of the peptide. In this study, we investigated the effect of another oxysterol, 25-hydroxycholesterol (25-OH) on the membrane raft-dependent transport of Aß-42 in Jurkat cells. Interestingly, intracellular transfer of Aß-42 was observed in the presence of 25-OH only after the inclusion of cholera toxin B subunit (CT-B), a marker used to detect the raft domain. We speculated that 25-OH can induce intracellular movement of Aß peptides. Furthermore, CT-B together with GM1 provided negative curvature, which resulted in the intracellular transport of Aß-42. Notably, we used a protofibrillar species of Aß-42 in this study. We have shown that the transport was microtubule-dependent since it could not be observed in depolymerized microtubules. These results demonstrate that oxysterols and glycosyl chains are important factors affecting intracellular transport. These compounds are also associated with aging and advanced glycation are risk factors for AD. Thus, this study should further understanding of the pathology of AD.

Localization of amyloid beta (Aß1-42) protofibrils in membrane lateral compartments: effect of cholesterol and 7-Ketocholesterol.

Cholesterol plays an important role in the interaction of Alzheimer's amyloid beta (Aß) with cell membranes, an important event in Aß-induced cytotoxicity. However, it is not fully understood how cholesterol influences the association of Aß with membrane lateral compartments. We have shown that by modulating membrane fluidity, cholesterol decreased peptide localization in solid-ordered domains and increased that in liquid-ordered domains. It changed the amount of Aß associating with liquid-disordered (Ld) phase with different tendencies depending on the composition of heterogeneous membrane systems. 7-Ketocholesterol, an oxidized derivative of cholesterol, majorly enhanced the fluidity of and Aß interaction with Ld phase. These findings are useful for clarifying the impact of cholesterol and its oxidation in Aß-induced toxicity.