Juliana Echevarria-Lima

Monocytes from HTLV-1–infected patients are unable to fully mature into dendritic cells

Human T-cell lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1-associated myelopathy/tropical spastic paraparesis is a chronic inflammatory disease characterized by loss of motor movement in response to spinal marrow cell destruction by T lymphocytes. To perform their cellular function, T cells need to be activated by antigen-presenting cells, such as dendritic cells (DCs). The aim of this work was to analyze DC differentiation and activation from monocytes of HTLV-1-infected individuals. We demonstrated that monocytes from HTLV-1-infected patients who had been stimulated to differentiate had an impaired loss of CD14 expression, expressed low levels of CD1a, and maintained secretion of tumor necrosis factor-α compared with monocytes from noninfected donors. We further evaluated DC activation by tumor necrosis factor-α. We observed that in response to activation, DCs that were derived from noninfected donors had an increase in the percentage of CD83(+), CD86(+), and human leukocyte antigen-DR(+) cells, whereas in DCs derived from HTLV-1-infected patients, the percentage of CD83(+), CD86(+), and human leukocyte antigen-DR(+) cells remained similar to that of nonactivated cells. Moreover, these cells had an impaired capacity to stimulate allogeneic T lymphocytes. We demonstrated that DC maturation was altered in HTLV-1-infected patients, which could contribute to the development of HTLV-1-associated diseases.

CD69 expression induced by thapsigargin, phorbol ester and ouabain on thymocytes is dependent on external Ca2+ entry

In the present work murine thymocytes exposed to Thapsigargin (TG 10, 20 and 50 nM), Phorbol-12,13,20-triacetate (TPA16 nM) and Ouabain (OUA100 nM) exhibited an increased expression of CD69, a molecule related to cellular activation and associated to Ca(++) influx in other systems. The kinetics of CD69 appearance depended on the stimuli and dose used. TG 50 nM induced an increased expression by 6 h whereas with lower doses (10 and 20 nM) an increase was detected at 18 h. TPA maximal increase was evident at 6 h. OUA lead to an observable increase at 18 h. However, in the case of TPA or TG the presence of the stimuli was only necessary for the first 2 h of culture, whereas OUA needed to be present during the whole assay. It was also demonstrated that Ca(++) influx was an essential feature, as EGTA diminished or abolished CD69 increased expression. Nevertheless, EGTA was only capable of this effect when present at the time of the stimuli. No correlation of CD69 expression with thymocyte death was observed. Similarly, the agents under study did not promote the maturation from double-positive into single-positive thymocytes. TPA and Thapsigargin were capable of decreasing the level of CD4 molecules on the cell surface, probably due to the loss of these molecules. OUA, on the other hand, did not modify CD4/CD8 expression on these cells.

ABCC transporter inhibition reduces zymosan-induced peritonitis.

Inflammatory mediators are released from injured tissues being responsible for the first steps of inflammatory processes. Multidrug efflux transporters, members of the ATP‐binding cassette (ABC) family, are ubiquitously expressed. ABCC molecules transport several endogenous substances, including leukotriene C4 (LTC4) and PGE2, which are involved in zymosan‐induced inflammation. The present study investigated the role played by ABCC transporters on zymosan‐induced peritonitis in mice. Most of the resident peritoneal cells were macrophages, based on their morphology and membrane‐activated complex 3 expression. RT‐PCR demonstrated that these cells expressed ABCC, and ABCC activity was analyzed in vivo via the s.c. injection of ABCC inhibitors [probenecid (PROB) 200 mg/kg or MK571 20 mg/kg], followed by an i.v. injection of carboxyfluorescein diacetate (CFDA), an ABCC fluorescent substrate. Both inhibitors increased CFDA accumulation, suggesting ABCC impairment. Moreover, ABCC reversors decreased zymosan‐induced plasma exudation by 86.6 ± 7.4 and 97.6 ± 2.3%, a feature related to a diminished secretion of LTC4 (65.1±11 and 47.8±9.9%) and PGE2 (under basal levels). Cell migration was inhibited similarly. Furthermore, PROB and MK571 inhibited IL‐1ß by 83.4 ± 13 and 71.2 ± 13.4% and TNF‐α content by 47 ± 4.5 and 28.9 ± 0.8%, respectively. NO metabolites and reactive oxygen species production were also reduced. The present results suggest that ABCC molecules have a relevant role in the acute inflammatory response produced by zymosan in mice.

Ca2+ Mobilization Induced by Ouabain in Thymocytes Involves Intracellular and Extracellular Ca2+ Pools

Abstract— Immune dysfunction has been reported in hypertensive rats, and circulating levels of ouabain are increased in some experimental models of hypertension. Ouabain is an inhibitor of the Na+/K+‐ATPase capable of diverse effects on cells of the immune system, but its mode of action on these cells is still unknown. The levels of cytoplasmic calcium ions play an important role in cell signaling, and ouabain may induce an increase in intracellular calcium indirectly through the Na+/Ca2+ exchanger. The current work examined the possibility that this drug could be exerting its effects on thymocytes through calcium mobilization and an increase in the cytosolic calcium concentration. Intracellular calcium was evaluated by using Balb‐c mouse thymocytes loaded with FURA‐2. Both intracellular and extracellular calcium pools were mobilized by ouabain (3 to 1000 nmol). The influx of extracellular calcium depended on the Na+/Ca2+ exchanger and on voltage‐dependent calcium channels, as it was inhibited by amiloride and benzamil, consistent with the inhibition of the Na+/K+ pump. In addition, the increase of calcium from intracellular stores was extremely rapid. Furthermore, an increase in cytosolic calcium levels was obtained with the combination of ouabain and thapsigargin, which was greater than that seen with either drug alone. Our data suggest that low concentrations of ouabain may be acting on thymocytes through a mechanism different from the traditional inhibition of the Na+/K+‐ATPase, as the cytosolic calcium rise was partly dependent on the release from intracellular stores.

Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

Multidrug resistance proteins [MRPs and P‐glycoprotein (Pgp)] are members of the family of ATP‐binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti‐cancer agents in tumour cells. These proteins act as ATP‐dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy‐2′‐7′‐dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration‐dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double‐positive cells and CD8 single‐positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation.

Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition

PurposeGlioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells.MethodsThe cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of β3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry.ResultsWe found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of β3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling.ConclusionFrom our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

Expression of c-kit and Sca-1 and their relationship with multidrug resistance protein 1 in mouse bone marrow mononuclear cells

P‐glycoprotein (Pgp) and multidrug resistance protein 1 (MRP1) are members of the ATP‐binding cassette (ABC) family of transporter proteins. Both molecules are membrane‐associated, energy‐dependent efflux pumps with different substrate selectivity and they may play a role in the activation, differentiation and function of haematopoietic cells. Mouse haematopoietic cells are characterized by the expression of the cell surface molecules c‐kit and Sca‐1. Herein, the presence and activities of Pgp and MRP1 in mouse bone marrow mononuclear cells (BMMC) and their relationship with the proteins c‐kit and Sca‐1 were evaluated. Pgp and MRP activities were measured based on the extrusion of rhodamine 123 (for Pgp) and Fluo‐3 (for MRP). Cell populations were assessed by cytometry using anti‐c‐kit and anti‐Sca1 antibodies. Pgp activity was present in 5% of BMMC while 50% of BMMC cells showed MRP activity. These findings agreed with the proportion of cells expressing the MRP1 surface molecule (51·3 ± 4·17%). About 14% of BMMC were positive for c‐kit and/or Sca‐1 (9·3% c‐kit– Sca‐1+, 4·2% c‐kit+ Sca‐1– and 0·9% c‐kit+ Sca‐1+). Among these subpopulations only c‐kit– Sca‐1+ cells presented Pgp activity (21·36%). On the other hand, MRP activity was present in all three subpopulations. Most cells (82·5%) of the c‐kit+ Sca‐1– subpopulation presented MRP1 activity compared to only 54·1% of c‐kit+ Sca‐1+ and 38·8% of c‐kit– Sca‐1+. This study demonstrates the expression and activity of MRP1 in BMMC. While only a small proportion of precursor cells had Pgp activity, MRP1 activity was present among different subpopulations of precursor cells. Further studies are necessary to establish the role of these transporters in haematopoietic cells.

ABCB1 (P-glycoprotein) but not ABCC1 (MRP1) is downregulated in peripheral blood mononuclear cells of spontaneously hypertensive rats

Although the kidney is a major target in hypertension, several studies have correlated important immune alterations with the development of hypertension in spontaneously hypertensive rats (SHR), like increased secretion of pro-inflammatory cytokines, inflammatory infiltration in kidneys and thymic atrophy. Because adenosine-triphosphate-binding cassette sub-family B member 1 (ABCB1; P-glycoprotein) and adenosine-triphosphate-binding cassette sub-family C member 1 (ABCC1; multidrug resistance protein 1), two proteins first described in multidrug resistant tumors, physiologically transport several immune mediators and are required for the adequate functioning of the immune system, we aimed to measure the expression and activity of these proteins in peripheral blood mononuclear cells (PBMC), thymocytes, and also kidneys of normotensive Wistar Kyoto rats and SHR. Our results showed that ABCB1, but not ABCC1, activity was diminished (nearly 50%) in PBMC. Moreover, Abcb1b gene was downregulated in PBMC and kidney of SHR and this was not counterbalanced by an upregulation of its homolog Abcb1a, suggesting that the diminished activity is due to downregulation of the gene. No alteration was detected in ABCB1 activity in SHR thymocytes, indicating that this downregulation occurs after lymphocytes leave the primary lymphoid organs. Even though it is not known at present which parameter(s) is(are) responsible for this downregulation, it may contribute for the altered immune response observed in hypertension and to possible altered drug disposition in hypertensive individuals, resulting in greater drug interaction and increased drug toxicity.

Independent Regulation of ABCB1 and ABCC Activities in Thymocytes and Bone Marrow Mononuclear Cells during Aging

Aging modifies a number of functional and phenotypic parameters of cells from the immune system. In this study, the activities of two members of the superfamily of ATP‐binding cassette (ABC) transport proteins, ABCB1 and ABCC (measured by rhodamine 123 efflux and Fluo‐3 efflux respectively), were compared in murine bone marrow cells and thymocytes of young (3–4 weeks old), adult (2–3 months old) and old (18 months old) mice. ABCB1 activity was shown to be age regulated in murine bone marrow mononuclear cells and thymocytes. In the bone marrow, the increased amount of cells with ABCB1 activity observed in old mice was restricted to the c‐kit−Sca‐1+ and c‐kit+Sca‐1+ subpopulations. Only a small percentage of c‐kit+ cells in the thymus had ABCB1 activity, and this subpopulation increased with age. In the thymus, old age augmented this activity in the CD4− CD8− double‐negative cells and in the CD4+ and CD8+ single‐positive populations. The activity of another ABC transporter, the ABCC‐related activity, was also modified by age in the bone marrow. However, the age‐related increase was observed in the subpopulations were ABCB1 was not modified, namely the non‐progenitor population (c‐kit−Sca‐1−cells) and c‐kit+Sca‐1− cells. Nearly, all thymocytes expressed the ABCC1 molecule in an active form and aging did not affect this pattern. This study demonstrates an independent upregulation of ABCB1 and ABCC activities during the aging process. The increases were observed in different subsets of cells but followed a developmentally regulated pattern. The functions played by these transporters and alterations in aging are discussed.

Resistance to thapsigargin-induced intracellular calcium mobilization in a multidrug resistant tumour cell line

A multidrug resistant (MDR) cell line, derived from the human leukaemic cell K562 and selected for its resistance to Vincristine, was shown to be resistant to Thapsigargin (TG). A concentration of 50 nM TG was toxic to K562 cells whereas the MDR cell line, known as Lucena I cells, survived unaffected for up to seven days in culture. Similarly, no intracellular Ca2+ mobilization was observed in the MDR cell line treated with TG. This effect was not a result of TG extrusion by P glycoprotein (Pgp), as no mobilization was observed even in the presence of the Pgp inhibitors Verapamil (5 μM) and Cyclosporin A (0.16 μM). In the present study, both cell lines expressed comparable levels of Bcl-2 making it unlikely that Bcl-2 was involved in this process. Similarly, no overexpression of the endoplasmic reticulum Ca2+ ATPase (SERCA) could be detected in the MDR cell line and Ca2+ uptake by vesicles of the two cell types were equally sensitive to TG. These results confirm that MDR cells do not mobilize Ca2+ in the presence of TG but go against the possibility that this might be due to TG extrusion or to the overexpression of a resistant SERCA isoform.