1,25-Dihydroxyvitamin D3 induces a myelomonocytic phenotype with enhanced effector cell function in the HL-60 promyelocytic leukemia cell line

Human promyelocytic leukemia (HL-60) cells were induced by 1,25-dihydroxyvitamin D3 (calcitriol) to differentiate and examined using a panel of monoclonal antibodies (MoAbs) and functional assays. Although morphologically and histochemically these cells appeared to be of the monocyte-macrophage phenotype, there was a decline in Fc receptors for IgGl and no induction of class II HLA antigens. There was, however, dramatic induction of the antigen detected by the myeloid-specific MoAb AML-2-23. These data suggest that the phenotypic changes induced by calcitriol in HL-60 cells are consistent with myelomonocytic differentiation in that the resultant cells possess characteristics of both monocytes (morphology, non-specific esterase staining, high levels of AML-2-23 reactivity) and granulocytes (PMN 29 binding, decreased Fc receptors for IgGl, absence of class II HLA antigens). Perhaps more important, the ability of calcitriol-treated cells to perform antibody-dependent cellular cytotoxicity and phagocytosis was markedly augmented. Lysis of antibody-coated erythrocytes by HL-60 cells increased from 5% in controls to 30-35% with calcitriol treatment for 4 days. This enhanced effector cell function was seen despite a decline in Fc receptors measured by cytofluorography. These data suggest that calcitriol may be involved in both differential and functional activation of myeloid cells.     

Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye

Accurate detection of apoptotic cells is important for the determination of cell viability. The aim of this study was to compare the sensitivity of the cell permeant SYTO 16 fluorescent dye for detecting early apoptotic mononuclear cells (MNCs) in normal donor blood with other apoptosis assays [i.e. Annexin-V, light scatter/7-amino-actinomycin-D (7-AAD) and chloromethyl-X-rosamine (CMXRos)] and to identify critical parameters for optimal SYTO 16 staining. Apoptosis was induced in normal human leukocytes from adult peripheral blood or cord blood, or the Jurkat T-lymphocytic cell line and assessed by fluorescence microscopy and flow cytometry. Dual labelling showed that SYTO 16 detected more apoptotic MNCs compared to Annexin-V. SYTO 16 staining intensity was consistent with the light scatter profiles expected of live, apoptotic and necrotic MNCs and was more objective than light scatter/7-AAD. CMXRos staining required considerable care and may not be a robust marker of apoptotic primary MNCs. For SYTO 16 flow cytometric analysis, the optimal conditions for staining 1x10(6) leukocytes were 4 nM SYTO 16 in the presence of 30 muM verapamil for 25-45 min at 37 degrees C in media containing calcium/magnesium supplemented with protein. A P-glycoprotein inhibitor, such as verapamil, and calcium/magnesium are essential for optimal loading of SYTO 16 into live MNCs and discrimination of apoptotic MNCs in normal blood samples. SYTO 16 is a sensitive, simple, inexpensive 'live cell' method for the discrimination of live, apoptotic and necrotic normal blood MNCs and is more sensitive for detecting apoptosis in these cells than Annexin-V or light scatter/7-AAD.     

The exposed carbohydrates of schistosomula of Schistosoma mansoni and their modification during maturation in vivo

Lectins labeled with 125I or conjugated with fluorescein were employed to study the carbohydrates on the surface of different stages of schistosomula of Schistosoma mansoni. Newly transformed schistosomula were shown to bind concanavalin A; the 60 000 and 120 000 dalton agglutinins from Ricinus communis; the fucose-binding protein from Lotus tetragonolobus; wheat germ agglutinin and peanut agglutinin. Soybean agglutinin, Ulex europaeus agglutinin and Dolichos biflorus agglutinin, on the other hand, failed to bind to the schistosomulum surface. The binding of peanut and soybean agglutinin was unaffected by pretreatment of the parasites with neuraminidase. Binding of concanavalin A, the 120 000 dalton agglutinin from Ricinus communis, wheat germ agglutinin and peanut agglutinin to the surface of 5-day schistosomula, recovered from the lungs of mice, was also demonstrated. In each case, however, the level of binding was approximately 70% less than that observed with newly transformed schistosomula and the binding of the fucose-binding protein from L. tetragonolobus practically disappeared. In contrast with newly transformed schistosomula, lung stage schistosomula, pretreated with neuraminidase, displayed a significant increase in the binding of peanut and soybean agglutinin. The results indicate that a significant alteration in the surface carbohydrates of S. mansoni occurs during in vivo maturation of the parasite. This change may contribute to the organism's ability to survive in the vertebrate host.     

Reshaping of T-lymphocyte compartment in adult prepubertaly ovariectomised rats: A putative role for progesterone deficiency

This study explores the role of ovarian hormones in the phenotypic shaping of peripheral T-cell pool over the reproductive lifespan of rats. For this purpose, 2-month-old prepubertally ovariectomised (Ox) rats, showing oestrogen and progesterone deficiency, and 11-month-old Ox rats, exhibiting only progesterone deficiency, were examined for thymus output, and cellularity and composition of major TCRαβ+ peripheral blood lymphocyte (PBL) and splenocyte subsets. Although ovariectomy increased thymic output in both 2- and 11-month-old rats, the count of both CD4+ and CD8+ PBLs and splenocytes increased only in the former. In the blood and spleen of 11-month-old Ox rats only the count of CD8+ cells increased. Although ovariectomy affected the total CD4+ count in none of the examined compartments from the 11-month-old rats, it increased CD4+FoxP3+ PBL and splenocyte relative proportions over those in the age-matched controls. The age-related differences in the cellularity and the major subset composition in Ox rats were linked to the differences in the ovarian steroid hormone levels registered in 2- and 11-month-old rats. The administration of progesterone to Ox rats during the seven days before the sacrificing confirmed contribution of this hormone deficiency to the ovariectomy-induced changes in the TCRαβ+ PBL and splenocyte pool from 11-month-old rats. The expansion of the CD8+ splenocyte subset in the 11-month-old Ox rats reflected increases in cellularity of memory and, particularly, naïve cells. This was due to greater thymic output of CD8+ cells and homeostatic proliferation than apoptosis in 11-month-old Ox rats when compared with age-matched sham-Ox control rats. The homeostatic changes within CD8+ splenocyte pool from 11-month-old Ox rats, most likely, reflected the enhanced splenic IL-7 and TGF-β mRNA expression. Overall, in adult female rats, circulating oestrogen and progesterone provide maintenance of T-cell counts, a diversity of T-cell repertoire, and the main T-cell subset composition in the periphery. Progesterone deficiency affects mainly the CD8+ lymphocyte compartment through increasing thymic CD8+ cell export and upsetting homeostatic regulation within the CD8+ splenocyte pool. These alterations were reversible through progesterone supplementation.     

Stimulation of Fas signaling down-regulates activity of neutrophils from major trauma patients with SIRS

Posttrauma apoptosis resistance of neutrophils (PMN) is related to overshooting immune responses, systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Recently, we have shown that the apoptosis resistance in circulating PMN from severely injured patients which is known to be mediated by high serum levels of pro-inflammatory cytokines can be overcome by the activation of Fas death receptor. Here, we aimed to study whether stimulation of surface Fas leads to the inactivation of hyperactivated PMN from critically ill patients with SIRS. PMN from 23 multiple trauma patients (mean injury severity score (ISS) 34 ± 1.9) were isolated at day 1 after admission to the trauma center. PMN from 17 volunteer blood donors served as controls. Neutrophil activity has been determined after ex vivo short (1 h) and long-term (4 h) stimulation of freshly isolated PMN with immobilized agonistic anti-Fas antibodies. We found neutrophil chemotactic migration in response to IL-8, phagocytosis and oxidative burst to be significantly inhibited in control cells already after short-term (1 h) Fas stimulation. In contrast, inactivation of trauma PMN by agonistic anti-Fas antibodies was found to be efficient only after long-term (4 h) incubation of cells with agonistic antibodies. Thus, in trauma PMN down-regulation of neutrophil activity seems to be delayed when compared to cells isolated from healthy controls, suggesting impaired susceptibility for Fas stimulation in these cells. Interestingly, whereas Fas-mediated inhibition of phagocytosis and oxidative burst could be prevented by the broad range caspase inhibitor t-butoxycarbonyl-aspartyl(O-methyl)-fluoromethyl ketone (BocD-fmk), the chemotactic activity in response to IL-8 was unaffected. In conclusion, we demonstrate that stimulation of neutrophil Fas does not only initiate apoptosis but also induces inhibition of neutrophil functions, partially by non-apoptotic signaling.     

Nef enhances c-Cbl phosphorylation in HIV-infected CD4+ T lymphocytes

The multifunctional HIV-1 protein Nef possesses several motifs that interact with signaling molecules in infected T cells. In order to determine whether Nef influences T cell activation, cells were infected with Nef-positive and Nef-negative clones of HIV. CD28 expression and changes in tyrosine phosphorylation were monitored. We observed no Nef-dependent changes in CD28 expression or function. However, infection with Nef-positive virus led to changes in tyrosine phosphorylation. This Nef-induced phosphorylation was observed in unstimulated cells, and c-Cbl was identified as one of the proteins whose phosphorylation was upregulated by Nef. Furthermore, Lck is required for Nef-mediated c-Cbl tyrosine phosphorylation. These results suggest that Nef modifies T cell signaling in the absence of T cell receptor engagement and co-stimulation.     

Chloride transport in NCL-SG3 sweat gland cells: channels involved

The aim of the study was to assess whether NCL-SG3, the only immortalized sweat gland cell line available, can be used as an in vitro model to study chloride ion transport in cultured sweat gland cells. Cl(-) efflux was measured using the MQAE dye fluorescence technique after stimulating the cells with different agonists. A significant stimulation of chloride efflux was achieved with the calcium ionophore A23187 resulting in an efflux rate of 0.9 mM/s. Both ATP and UTP activated chloride efflux in these cells, with the ATP response being larger. IBMX and forskolin stimulation did not induce a rate of chloride efflux above the basal level. Immunocytochemistry showed no detectable CFTR in NCL-SG3 cells. This finding was confirmed with flow cytometry analysis. Niflumic acid (20 and 100 microM NFA) and 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (100 ìM) decreased the rate of ATP-stimulated chloride efflux significantly (0.40 and 0.31 mM/s with NFA, 0.37 mM/s with H2DIDS). Gadolinium (20 ìM) had no effect on the chloride transport rate. In conclusion, the NCL-SG3 cells retain some of the aspects of human sweat gland epithelium, such as the ability to form cell-cell contacts. The CFTR protein is neither functional nor expressed in cultured NCL-SG3 sweat gland cells. Ca(2+)-activated chloride conductance is confirmed and the putative Ca(2+)-activated chloride channel (CaCC) is further characterized in term of its pharmacological sensitivity. The NCL-SG3 sweat gland cell line can be used to investigate the characteristics of the CaCC and to identify the channel.     

Eosinophil peroxidase induces expression of cholinergic genes via cell surface neural interactions

Eosinophils localize to and release their granule proteins in close association with nerves in patients with asthma and rhinitis. These conditions are associated with increased neural function. In this study the effect of the individual granule proteins on cholinergic neurotransmitter expression was investigated. Eosinophil peroxidase (EPO) upregulated choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) gene expression. Fluorescently labeled EPO was seen to bind to the IMR-32 cell surface. Both Poly-l-Glutamate (PLG) and Heparinase-1 reversed the up-regulatory effect of EPO on ChAT and VAChT expression and prevented EPO adhesion to the cell surface. Poly-l-arginine (PLA) had no effect on expression of either gene, suggesting that charge is necessary but insufficient to alter gene expression. EPO induced its effects via the activation of NF-κB. MEK inhibition led to reversal of all up-regulatory effects of EPO. These data indicate a preferential role of EPO signaling via a specific surface receptor that leads to neural plasticity.