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PubMed2006

Triggering receptor expressed in myeloid cells 2 (TREM2) trafficking in microglial cells: continuous shuttling to and from the plasma membrane regulated by cell stimulation.

Cell biology of triggering receptor expressed in myeloid cells 2, a receptor expressed in brain cells (microglia and possibly neurons and oligodendrocytes) which is responsible for a neurological and psychiatric genetic disease, polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy otherwise called the Nasu-Hakola disease, is still largely unknown. Using immortalized mouse N9 microglial cells we demonstrate that triggering receptor expressed in myeloid cells 2 is mostly distributed intracellularly in two pools: a deposit in the Golgi complex and a population of exocytic vesicles, distinct from endosomes and lysosomes, which is continuously translocated to, and recycled from the cell surface. Results with ionomycin and gamma-interferon, showing rapid and slow increases, respectively, of triggering receptor expressed in myeloid cells 2 surface density, documented that the exocytosis of the receptor-rich vesicles is regulated. Pulse labeling in the cold of surface triggering receptor expressed in myeloid cells 2 with its antibody (or Fab fragment) followed by chase at 37 degrees C showed internalization, with recovery of the antibody in endosomes and lysosomes. However, part of the receptor/antibody complex, internalized for up to 30 min chase, was recycled to the cell surface within 2 min of ionomycin stimulation, together with a fraction of the total biotinylated surface protein chased in parallel. The internalized receptor appears therefore to get access to exocytic organelles distinct from lysosomes which may resemble the exocytic vesicles of resting cells. These results document that, in microglial cells, the surface density of the triggering receptor expressed in myeloid cells 2 and thus, presumably, the response to its activation, is continuously adapted and can be greatly increased, even at rapid rate, as a function of cell activation.

Animals↗
PubMed2005

Immunomodulatory effects of the fungicide Mancozeb in agricultural workers.

Available data suggest that ethylenebisdithiocarbamates (EBDCs) may have immunomodulatory effects. This study aimed to investigate the immunological profile of farmers exposed to Mancozeb, an EBDC fungicide, through the determination of several serum, cellular, and functional immune parameters. Twenty-six healthy subjects entered the study, 13 vineyards exposed to Mancozeb and 13 unexposed controls. Exposure was assessed through the determination of ethylentiourea (ETU) in urine. Complete and differential blood count, serum immunoglobulins, complement fractions, autoantibodies, lymphocyte subpopulations, proliferative response to mitogens, natural killer (NK) activity, and cytokine production were measured. Post-exposure samples showed ETU urine concentration significantly higher than pre-exposure and control groups. A significant increase in CD19+ cells, both percentage and absolute number, and a significant decrease in the percentage of CD25+ cells were found in post-exposure samples compared to controls. A statistically significant increase in the proliferative response to phorbol myristate acetate plus ionomycin (PMA + ionomycin) was observed in the post-exposure group compared to controls and baseline, while a significant reduction in LPS-induced TNF-alpha release in post-exposure samples was observed. Overall, our results suggest that low-level exposure to Mancozeb has slight immunomodulatory effects, and point out a method adequate to reveal immune-modifications in workers occupationally exposed to potential immunotoxic compounds, based on a whole blood assay.

Adult↗
PubMed1998

Functional expression of sperm angiotensin II type I receptor in Xenopus oocyte: modulation of a sperm Ca2+-activated K+ channel.

In addition to Ca2+ and K+ fluxes, angiotensin II (Ang II) has been shown to influence sperm motility. The present study investigated the involvement of angiotensin II type 1 receptor (AT1) in mediating the modulatory effect of Ang II on a sperm Ca2+-activated K+ channel expressed in Xenopus oocytes injected with RNAs of spermatogenic cells. Ang II at a concentration of 1 microM was found to potentiate the ionomycin-induced current, previously demonstrated to be mediated by a 'Maxi' Ca2+-activated K+ channel. However, at higher concentration, 20 microM, Ang II was found to suppress the ionomycin-induced current. Both potentiating and inhibitory effects of Ang II were blocked by losartan, a specific antagonist of AT1 receptors. Immunohistochemical studies further confirmed the presence of AT1 receptors in spermatogenic cells while expression of AT1 receptor mRNA was demonstrated by RT-PCR. These results suggest that Ang II may influence sperm motility as well as other sperm function by acting on AT1 receptors, and exerting potentiating and inhibitory effects on the Ca2+-activated K+ channels.

Angiotensin II↗
PubMed1990

Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells.

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.

Animals↗
PubMed1997

Influence of the protein kinase C inhibitor 3-[1-[3-(amidinothio) propyl]-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl) maleimide methane sulfonate (Ro-318220) on surfactant secretion in type II pneumocytes.

We have investigated the influence of 3-[1-[3-(amidinothio)propyl]-1H-indoyl-3-yl]-3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate (Ro-318220), a potent and selective inhibitor of protein kinase C, on phosphatidylcholine secretion in response to surfactant secretagogues in rat type II cells. Freshly isolated cells were cultured overnight with [3H]choline to label the phosphatidylcholine pool and were preincubated for 30 min in fresh medium with or without Ro-318220. Secretagogues were then added, and the incubation was continued for 90 min after which [3H]phosphatidylcholine secretion was measured. Ro-318220 (10 microM) almost completely abolished the stimulatory effects of 36 microM terbutaline, 10 microM ATP, and 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) and significantly antagonized the effects of 10 microM 5'-(N-ethylcarboxyamido) adenosine (NECA), 100 microM dioctanoylglycerol, and 0.05 microM ionomycin. The effect of Ro-318220 was dependent on concentration. The IC50 values for Ro-318220 inhibition of the effects of terbutaline, NECA, TPA, and ionomycin were not significantly different. The IC50 value for Ro-318220 inhibition of the effect of TPA in the type II cell (0.05 microM) was similar to that reported for inhibition of protein kinase C in vitro (0.08 microM). We conclude that Ro-318220 antagonizes the effects of the different surfactant secretagogues by antagonizing a step common to the different signaling pathways. It remains to be established if it is protein kinase C or another step that is inhibited.

Adenosine↗
PubMed2003

Down-regulation of cell surface CXCR6 expression during T cell activation is predominantly mediated by calcineurin.

CXCR6, the receptor for the membrane-anchored chemokine, CXCL16, is expressed on a subset of CCR5-bearing memory T cells, and may play a role in recruiting these cells to sites of inflammation. Here, we set out to determine the effect of T cell activation on CXCR6 expression. Highly purified human peripheral blood T cells were cultured for 7-8 days in presence of IL-2 (400 U/ml) to enhance CXCR6 expression. Overnight stimulation with anti-CD3 mAb+anti-CD28 mAb, which resulted in CD69 induction and cytokine (IL-2 and IFN-gamma) production, reduced cell surface expression of CXCR6 by 85% and that of CCR5 by 76%. The Ca(2+) ionophore, ionomycin (125-500 ng/ml), also markedly diminished CXCR6 expression (85%), but without inducing CD69 expression or cytokine production, and reduced CCR5 expression by only 40%. In contrast, the phorbol esters, PdBu or PMA had little effect on CXCR6 expression (23% reduction) but induced CD69 expression and caused a profound down-regulation (92%) of CCR5 expression. Moreover, CCR7, whose expression was low on CXCR6(+) T cells, was little affected by any of these modes of activation. The down-regulation of CXCR6 expression induced by CD3/CD28 activation was blocked by the broad kinase inhibitor, staurosporine, and by the src kinase inhibitor, PP2, but not by the MEK1 inhibitor, U0106. Most interestingly, the calcineurin inhibitor, FK506, consistently inhibited CD3/CD28-induced CXCR6 down-regulation. FK506 also blocked the decrease of CXCR6 expression caused by ionomycin, whereas staurosporine or PP2 had no effect on this decrease. Altogether, these data indicate that CXCR6 expression is down-regulated, independent of CCR5 or CD69 expression and of cytokine induction, by T cell activation signals that involve predominantly the Ca(2+)-dependent calcineurin pathway.

Antigens, CD↗
PubMed1997

Studies on endothelium-dependent vasorelaxation by hydralazine in porcine coronary artery.

Hydralazine relaxed porcine coronary artery strips in a concentration-dependent manner by distinct endothelium-dependent and endothelium-independent actions. With lower doses (< or = 10(-6) M), the hydralazine-induced relaxation appears to be completely endothelium-dependent and we designed the present study to elucidate the mechanisms of this endothelium-dependent relaxation. Hydralazine (10(-6) M)-induced endothelium-dependent relaxation was not affected by the presence of 10(-5) M indomethacin, 10(-3) M L-NG-nitro-arginine (L-NOARG) or 10(-5) M haemoglobin, and was accompanied by accumulation of cGMP but not of cAMP in artery strips. There was a close time-dependent parallel relationship between endothelium-dependent relaxation and accumulation of cGMP induced by hydralazine (10(-6) M). The endothelium-dependent relaxation and accumulation of cGMP induced by hydralazine showed much slower kinetics than those induced by ionomycin (10(-7) M). Pretreatment of the strips with actinomycin D (10 micrograms/ml) significantly inhibited not the endothelium-dependent relaxation and accumulation of cGMP induced by ionomycin (10(-7) M) but those induced by hydralazine (10(-6) M). These results suggest that hydralazine induces endothelium-dependent vasorelaxation via the slow accumulation of cGMP in the strips. This does not occur through the release of nitric oxide or prostaglandin I2 but through immediate transcription and probably expression of a molecule in the endothelium.

Animals↗
PubMed1997

Determination of intracellular cytokines by flow-cytometry following whole-blood culture.

Various methods have been reported for measuring intracellular cytokines in peripheral blood mononuclear cells isolated by density-gradient centrifugation. In this report, we describe a whole-blood method for the determination of intracellular cytokines (IFN-gamma, TNF-alpha and IL-2) that uses small-volume (500 microl) blood samples. Directly conjugated anti-cytokine antibodies and commercial cell membrane fixation and permeabilisation reagents were used. Blood was cultured in a 1:3 dilution with a combination of PMA and ionomycin to reveal the cytokine synthetic potential of each cell, together with monensin to increase the sensitivity by retaining cytokines within the cell to detectable levels. The optimum concentrations of PMA (10 ng/ml (16.2 nmol/l)), ionomycin (2 micromol/l) and monensin (3 micromol/l) were determined. Kinetic studies showed maximal cytokine expression after 2 h of culture for TNF-alpha and IFN-gamma and 4 h for IL-2. Assessment of TNF-alpha and IFN-gamma production within the CD4 and CD8 lymphocytes from 10 normal volunteers showed that considerably more CD8 + than CD4 + cells produced IFN-gamma. This technique could be used by routine immunology laboratories and will be of use in studies to determine whether cytokine assays are of value in the investigation of immune disorders.

Adult↗
PubMed1999

Detection of intracellular cytokines in citrated whole blood or marrow samples by flow cytometry.

We have used three-color flow cytometric analysis for the detection of intracellular cytokines (IFN-gamma and IL-4) in CD3(+) cells, after stimulation for 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin. We report in the present paper a validation study for analysing IFN-gamma and IL-4 production by bone marrow (BM)-derived T cells and peripheral blood T cells after BM transplantation. Using citrate as anticoagulant for blood and marrow sampling interfered with PMA+ionomycin-based cell stimulation. Indeed, removing this anticoagulant by two washes with 10% pooled human AB serum-supplemented RPMI 1640 before cell stimulation improved the percentages of IL-4(+) (0.02+/-0.01% to 0. 47+/-0.17% without and with washes, respectively; p<0.01) and IFN-gamma(+) (6.8+/-2.75% to 39.33+/-4.6%; p<0.01) cells to levels similar to those observed in heparin-based whole blood cultures (0.38+/-0.17% IL-4(+) and 34.27+/-4.96% IFN-gamma(+) cells; p>0.05). Delaying the cell cultures for 24 h did not significantly modify the detection of IFN-gamma in washed whole blood, but significantly altered IFN-gamma secretion in culture supernatants, as assessed by ELISA. Moreover, the percentage of IFN-gamma-producing cells within the CD3(+) lymphocyte population was stable, since similar results were obtained in two or three different independent experiments performed with the same healthy donors. This method was shown to be applicable for different kinds of citrated samples, such as blood or BM-derived cells. Overall, our data suggest that in addition to allowing for the identification of cytokine-producing cell phenotype, intracellular cytokines staining using flow cytometry is more reliable than ELISA for the biological follow-up of clinical samples.

Animals↗
PubMed1997

Activation of mitogen-activated protein kinase in freshly isolated rat hepatocytes by both a calcium- and a protein kinase C-dependent pathway.

In the present study, we investigated the role of calcium and protein kinase C (PKC) in the activation of mitogen-activated protein kinase (MAPK) in isolated rat hepatocytes. We found that the glycogenolytic hormone norepinephrine (NE), acting through the alpha1-adrenergic receptor and the G protein Gq, was able to induce a dose- and time-dependent activation of MAPK in hepatocytes. Vasopressin, which acts through a different receptor but also through stimulation of the Gq-dependent pathway, also caused a twofold activation of MAPK. Activation of MAPK by both agonists required the presence of free extracellular calcium and was blocked by the specific PKC inhibitor, Ro 31-8220. MAPK activation was also induced by phorbol myristate acetate (PMA), confirming that a PKC-dependent pathway exists for MAPK activation in liver. Furthermore, calcium-mobilizing agents such as thapsigargin and ionomycin were able to induce an activation of MAPK by a PKC-independent pathway that was totally abolished by preincubation of cells with EGTA. A second pathway for MAPK activation that relies solely on calcium may therefore exist. Ro 31-8220 did not affect phosphorylase activation by NE, vasopressin, thapsigargin, and ionomycin, indicating that PKC inhibition did not interfere with the signaling pathway leading to inositol-1,4,5-triphosphate (IP3)-induced calcium mobilization or with changes in calcium fluxes. The role of MAPK activation by NE and vasopressin in the regulation of hepatic carbohydrate metabolism is discussed.

Animals↗
PubMed1997

Calcium-induced activation of the rat vascular myocyte Na+/H+ exchanger isoform-1.

An established intermediate phenotype of human hypertension and diabetic nephropathy is an elevation of Na+/H+ exchanger (NHE) activity, but the mechanism for this is unclear. This phenotype is maintained in vascular myocytes from the spontaneously hypertensive rat (SHR) compared with the normotensive Wistar Kyoto rat (WKY). Since intracellular calcium levels ([Ca2+]i) following agonist stimulation were elevated in cells from both hypertensive humans and SHR, we have examined the role of calcium-calmodulin (CaM) in the mechanism of increased NHE activity in vascular myocytes of SHR by determining the activity and phosphorylation state of NHE isoform-1 (NHE-1) in cells from SHR and WKY when [Ca2+]i was elevated by the ionophores A23187 or ionomycin. NHE activity was measured using fluorometry and NHE-1 phosphorylation by immunoprecipitating the exchanger from 32P-orthophosphate-labeled cells with a polyclonal NHE-1-specific antibody. The ionophore A23187 increased [Ca2+]i in both cell types to approximately 700 to 800 nmol x L(-1), and led to stimulation of NHE-1 activity only in WKY myocytes, with no effect on SHR cells. An inhibitor of CaM kinase II (KN-62) failed to abolish stimulation of NHE-1 by A23187 in WKY cells, and had no effect on unstimulated NHE-1 activity in both cell types. Ionomycin also elevated [Ca2+]i in both cell types to approximately 1,000 nmol x L(-1) and activated NHE-1 activity in only WKY cells. Activation of NHE-1 in WKY cells by an increased [Ca2+]i was not mediated by an increase in NHE-1 phosphorylation, whether in the presence or absence of KN-62. The elevated NHE-1 phosphorylation in SHR cells was not affected by elevated [Ca2+]i or KN-62. Calmodulin-agarose beads bound NHE-1 extracted from SHR cells to a lesser extent than that from WKY cells. We conclude that calcium-induced NHE-1 activation in WKY cells was not mediated by CaM kinase II. The elevated NHE-1 activity and phosphorylation of SHR cells was not further modulated by increased [Ca2+]i, and was also independent of CaM kinase II. Non-phosphorylation-dependent mechanisms of activation of NHE-1 may therefore be responsible for alterations of NHE-1 activity in these cells, such as the direct binding of CaM to NHE-1. This direct binding of CaM to NHE-1 may be impaired in SHR compared with WKY cells.

Animals↗
PubMedDate not supplied

Modulation by calcium/calmodulin-dependent protein kinase II of functional response of delta opioid receptor in neuroblastoma x glioma hybrid (NG108-15) cells.

The potential modulation of opioid receptor signaling by calcium/calmodulin-dependent protein kinase II (CaMKII) has been investigated in NG108-15 cells. Both CaMKII specific inhibitors used, KN62 and KN93, time- and dose-dependently blocked inhibition of cAMP accumulation by [D-Pen2, D-Pen5]enkephalin (DPDPE), with an IC50 of about 1.2 microM and 0.8 microM, respectively. In the presence of 1 microM KN62 or KN93, the DPDPE dose-response curve shifted to the right (IC50 from 0.7 to 20 nM for KN62 and from 0.65 to 10 nM for KN93, respectively), and the maximal response was also significantly reduced. KN92, an inactive analogue of KN93, showed no significant impact, while ionomycin, an activator of CaMKII, greatly potentiated the opioid receptor response, suggesting that the effects of KN62, KN93 and ionomycin were likely mediated through CaMKII. In addition, KN62 did not affect ligand binding, receptor/Gi coupling, or basal and forskolin-stimulated adenylyl cyclase activity, suggesting its possible interference in the Gi/adenylyl cyclase interaction. Furthermore, a CaMKII inhibitor potently blocked the functional responses of other Gi-coupled receptors (m4 muscarinic and alpha2 adrenergic receptors) tested, but not that of Gs-coupled receptors (prostaglandin E1 and adenosine receptors). Our results clearly demonstrate that CaMKII modulates the signaling of opioid receptor and other Gi-coupled receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1999

Calcium signal abnormalities in murine T lymphocytes that express the multidrug transporter P-glycoprotein.

Previous work has established that aging in mice leads to an accumulation of T cells that express high levels of P-glycoprotein, a plasma membrane pump that mediates multiple drug resistance in tumor cells but whose function in normal T cells is still obscure. Pgp+ cells seem to be functionally defective: isolated from the CD4 memory population of young mice, they are unresponsive to T cell receptor-dependent stimuli in tests for proliferation and cytokine production. The proliferative defect can, however, be overcome by exposure to PMA plus the calcium ionophore ionomycin, suggesting that the Pgp+ cells may have a specific defect in calcium signal generation. We show here that Pgp+ T cells, from young or old mice, do indeed show smaller changes in intracellular calcium ion concentration than Pgp- cells, when activated either by Con A, anti-CD3 antibodies, or ionomycin. The difference between Pgp+ and Pgp- cells is apparent even in experiments on isolated CD4 memory T cells from young mice and thus is not simply a consequence of the age-dependent increase in memory cell numbers. Although the molecular basis for the abnormality in calcium signal generation by Pgp+ cells is still uncertain, our data suggest that the effect could be due to inter-subset differences in levels of sorcin, a 22 kDa cytoplasmic protein that is co-expressed with P-glycoprotein in many tumor cells and which binds free calcium ion with high affinity. Sorcin levels are higher in Pgp+ CD4 cells than in Pgp- CD4 cells of young mice and increase with age in CD4 cells, consistent with the hypothesis that sorcin interferes with calcium signals in the age-sensitive Pgp+ T cell subset.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets.

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.

Adenosine Diphosphate↗
PubMed1995

Antigen presenting cell-independent cytokine and spontaneous in vitro IgE production in patients with atopic dermatitis: increased interferon-gamma production and lack of effects of in vivo low-dose interferon-gamma treatment.

Atopic dermatitis is characterized by elevated serum IgE concentrations and dysregulation of T-lymphocyte function. To examine the pattern of cytokine production associated with elevated IgE levels, phorbol ester plus ionomycin-stimulated production of interleukin (IL)-4, IL-5, and interferon-gamma (IFN-gamma) by blood mononuclear cells from 16 patients with atopic dermatitis was compared with that of 18 healthy subjects. Spontaneous in vitro IgE production was also studied longitudinally in patients receiving placebo or daily treatment with 0.05 mg/m2 IFN-gamma. Spontaneous in vitro IgE production and mitogen-driven IL-4 and IFN-gamma synthesis did not differ when patients were receiving interferon treatment compared with no treatment. Furthermore, ionomycin plus phorbol ester-stimulated mononuclear cells from patients with atopic dermatitis produced less IL-4 and more IFN-gamma than did cells from healthy subjects. IL-5 production by cells from patients with atopic dermatitis did not differ from that of cells from healthy subjects. The ratio of IL-4 to IFN-gamma produced in vitro was significantly lower (p = 0.04) in the cells of patients with atopic dermatitis (0.9) as compared with those of healthy subjects (2.7). The findings suggest that when circulating T cells are stimulated under antigen presenting cell-independent conditions, atopic dermatitis is not characterized by the shift in the reciprocal relationship between IL-4 and IFN-gamma production, which has been postulated to explain the pathogenesis of IgE elevation and the therapeutic action of IFN-gamma in patients with atopic dermatitis.

Adult↗
PubMed1995

Decreased frequency of interferon-gamma- and interleukin-2-producing cells in patients with atopic diseases measured at the single cell level.

BACKGROUND: Recently, diminished interferon-gamma (IFN-gamma) and increased interleukin (IL-4 production in peripheral blood mononuclear cells (PBMCs) from atopic patients have been described by several groups, measured as total cytokine content in culture supernatants. These studies suggested a predominance of TH2-like cells producing large amounts of IL-4 in atopic patients. It is not clear whether the reported cytokine imbalances are the result of an alteration in the distribution of specific T-cell subsets or whether intrinsic dysregulation in cytokine production is a characteristic of atopic individuals. OBJECTIVE: This study examined the production of IFN-gamma, and IL-2 in PBMCs from atopic patients at the single cell level with the use of freshly isolated lymphocytes. METHODS: We recently described a flow cytometric assay in which three-color analysis was used to study the production of a cytokine of interest in a T-cell subpopulation defined by two cell surface markers. PBMCs from 23 atopic patients and 14 control subjects were stimulated with phorbol ester and ionomycin for 5 hours. PBMCs from seven patients and seven control subjects were also cultured with immobilized anti-CD3 antibodies for 24 hours. Cells were fixed, made permeable, and stained for intracellular cytokines in combination with cell surface markers CD3, CD8, and CD45RO. Cytokine-producing cells were analyzed by gating on T-cell subsets. RESULTS: IFN-gamma-producing cells were significantly decreased (p < 0.05) in CD4+ T cells but not in CD8+ T cells of atopic patients. CD45R0+ and CD45R0-T cells showed a decreased proportion of IFN-gamma-producing cells (p < 0.05 and p < 0.01, respectively). IL-2 production was diminished in all T-cell subsets (p < 0.01). The number of IL-4-producing cells was not elevated, and such cells were exclusively found in the CD45RO+ T cells. Analysis of culture supernatants of sorted CD45RO+ T cells for IL-4 and IFN-gamma production confirmed these results. CONCLUSION: Our findings provide evidence that a reduced IFN-gamma production in atopic patients is due to an intrinsic defect selectively found in the CD4+ T cells. Because IL-2 production was markedly decreased but IL-4 production was unchanged, our data demonstrate a deficiency in the ability of atopic T cells to produce TH1-like cytokines on stimulation with phorbol ester, ionomycin, or anti-CD3 monoclonal antibodies.

Adolescent↗
PubMed1999

Frequency and characterization of antigen-specific IL-4- and IL-13- producing basophils and T cells in peripheral blood of healthy and asthmatic subjects.

BACKGROUND: Both basophils and T cells are known to secrete IL-4 and IL-13 after activation with either nonspecific stimuli or specific antigen, but the relative contribution of these 2 cell types to overall cytokine production is unclear. OBJECTIVES: To further characterize basophil cytokine production and compare it with that of T cells, we examined the frequency of IL-4- and IL-13-producing basophils and T cells in human PBMCs by means of flow cytometry after activation in allergic asthmatic and normal subjects. METHODS: PBMCs obtained from whole blood after Percoll gradient were activated with specific antigen or ionomycin and fixed. PBMCs were made permeable; stained with antibodies to IgE, CD3, and either IL-4 or IL-13; and analyzed by means of flow cytometry. RESULTS: Preformed cytokines were not detected in unactivated basophils. After ionomycin activation, 60% to 90% of basophils from both control and allergic asthmatic subjects expressed IL-4 and IL-13. Specific antigen induced cytokine expression by 10% to 20% of basophils from the asthmatic group only. After specific antigen activation, basophils accounted for 4 times more IL-4-producing cells than did T cells. IL-4 and IL-13 production at 2 hours was exclusively from basophils. After allergen activation, CD40 ligand was upregulated on a subset of peripheral blood basophils. CONCLUSIONS: These data demonstrate that basophils are the predominant peripheral blood cells that express IL-4 and IL-13 in the first 6 hours after antigen activation and strengthen the putative role of basophils both in IgE production and in the generation of allergic inflammation.

Adult↗
PubMed1996

Injection of rat hepatocyte poly(A)+ RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels.

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP3F) and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulated Ca2+ and Mn2+ inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin- and InsP3F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inhibition at less thari 5 microM Gd3+ for InsP3F-stimulated Ca2+ inflow). GTP gamma S-stimulated Ca2+ inflow was insensitive to 50 microM Gd3+ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca2+ channels can be detected in Xenopus oocytes using Ca(2+)-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A)+ RNA exhibited greater rates of Ca2+ and Mn2+ inflow in the basal (no agonist) state, greater rates of Ca2+ inflow in the presence of vasopressin or InsP3F and greater rates of Ba2+ inflow in the presence of InsP3F, when compared with "mock"-injected oocytes. In poly(A)+ RNA-injected oocytes, vasopressin- and InsP3F-stimulated Ca2+ inflow, but not basal Ca2+ inflow, was inhibited by Gd3+. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn2+ as well as Ca2+ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.

Animals↗