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PubMed1990

Second messenger modulation of epidermal growth factor receptor function does not occur at the level of receptor dimerization.

Epidermal growth factor (EGF)-induced receptor dimerization may provide a mechanism for activation of the receptor protein tyrosine kinase and for initiation of post-receptor signalling pathways. We have examined whether second messengers and agents that modulate EGF receptor function act at the level of receptor dimerization. Both the Ca2+ ionophore ionomycin and the tumour promotor tetradecanoylphorbol acetate (TPA), added shortly before EGF, inhibit EGF receptor protein tyrosine kinase activity in intact cells. In permeabilized cells, elevation of Ca2+ similarly inhibits EGF receptor function. The inhibitory effect of Ca2+, unlike that of TPA, appears not to be dependent on protein kinase C activity. Neither ionomycin nor phorbol ester affects EGF-induced receptor dimerization, as shown by cross-linking and immunoblotting techniques, although the phosphotyrosine content of both monomeric and dimeric receptors is strongly decreased. Furthermore, we show that EGF receptor dimerization is not affected by increases in cyclic AMP or intracellular pH, nor by changes in transmembrane potential, medium osmolarity or the glycosylation state of the receptor. These result suggest that modulation of EGF receptor function occurs at a step other than receptor dimerization.

Animals↗
PubMed1992

Ca2+ homeostasis and fast-type sarcoplasmic reticulum Ca(2+)-ATPase expression in L6 muscle cells. Role of thyroid hormone.

The effect of thyroid hormone (L-tri-iodothyronine; T3) on the cytosolic free Ca2+ concentration ([Ca2+]i) in L6 myotubes was studied at rest and during activation to explore the possible mediating role of [Ca2+]i in the T3-induced net synthesis of fast-type sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The mean [Ca2+]i at rest was approx. 115 nM in myoblasts, control myotubes and T3-treated myotubes. Therefore it is unlikely that the T3-induced elevation of Ca(2+)-ATPase levels is mediated by [Ca2+]i changes. To investigate the influence of the 4-fold higher Ca(2+)-ATPase levels in T3-treated myotubes (compared with controls) on [Ca2+]i, interventions with caffeine (10 mM) and a high extracellular K+ concentration ([K+]o) (30 mM) were applied which initially mobilize Ca2+ predominantly from the SR. The results showed a lower (caffeine) or not significantly different (high [K+]o) increase in [Ca2+]i in T3-treated myotubes compared with controls. No rise in [Ca2+]i was found in myoblasts with caffeine or high [K+]o. The role of [Ca2+]i in the regulation of Ca(2+)-ATPase levels was investigated by varying [Ca2+]i through exposure of cells to different concentrations of extracellular Ca2+ (0.2-1.8 mM) and ionomycin (0.1-0.25 microM). At subnormal [Ca2+]i (55 nM) the T3-induced net synthesis of Ca(2+)-ATPase was virtually abolished, and at supranormal [Ca2+]i (195 nM) it was greatly depressed. Intermediate stimulation of net Ca(2+)-ATPase synthesis was found at [Ca2+]i of 95 and 165 nM, with an optimum at approx. 125 nM. Similar but less pronounced effects were found for the basal Ca(2+)-ATPase levels. In contracting primary rat myotubes, Ca(2+)-ATPase levels were significantly lower than in tetrodotoxin-arrested myotubes. The same results were obtained in the presence of T3. Since the mean [Ca2+]i in contracting cells is higher than in resting cells, these data agree with those obtained in the L6 cells with ionomycin. A major conclusion of this study is the existence of a [Ca2+]i optimum, near resting levels, for the expression of the fast-type Ca(2+)-ATPase in the L6 muscle cell line.

Animals↗
PubMed1992

Quantification of contributions of phospholipid precursors to diradylglycerols in stimulated mononuclear phagocytes.

Phosphatidylcholine (PC) hydrolysis has been shown to occur in hormone-stimulated cells and represents a potential metabolic source, in addition to phosphoinositides, for the generation of diradylglycerols (DG). We performed studies in order to quantify the importance of this pathway in DG formation. We incubated murine peritoneal macrophages with platelet-activating factor (PAF), ionomycin, phorbol myristate acetate (PMA) or no stimulus in a series of timed incubations ranging from 15 s to 20 min. We quantified the profiles of the molecular species in the accumulated DG after extraction, specific radiolabelling to give [32P]phosphatidic acid by DG kinase, and conversion to the dimethyl derivative. We used two independent methods for molecular species analysis: (1) reversed-phase h.p.l.c. separation with in-line beta-radiation detection of peaks, and (2) an argentation-t.l.c. separation with scintillation counting of bands. Our results showed a clearly biphasic sequence in the composition of accumulated DG. The molecular species composition of early DG (up to 1 min stimulation time) was very similar to that of unstimulated DG, whereas the proportions of the species present in later DG were substantially altered. In the same experiments, we extracted native phospholipids from unstimulated macrophages, separated phosphatidylinositol (PI), PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), converted them to the corresponding DGs by using phospholipase C, and determined their molecular species compositions as above. In comparison with the diradyl compositions of stimulated DG, the diradyl composition of PI closely matched that of early DG, the differences between the PC and PI compositions matched the differences between early and late DG very closely, and the compositions of PE and PS were unique and unrelated. We quantified these relationships more precisely by multilinear regression analysis to calculate the theoretical best mix of five molecular species compositions (PI, PC, PE, PS and unstimulated DG) that would most closely replicate the early and late accumulated DG compositions. We found that by both h.p.l.c. and t.l.c. analyses, 15-30% (PAF) or 25-50% (ionomycin and PMA) of the later DG could be accounted for by PC hydrolysis. These results represent quantifications of phospholipid class contributions to stimulated DG formation, and demonstrate the potential importance of PC hydrolysis in phagocytic leucocytes.

Animals↗
PubMed1992

Agonist-induced inhibition of phosphatidylserine synthesis is secondary to the emptying of intracellular Ca2+ stores in Jurkat T-cells.

The biosynthesis of phosphatidylserine (PtdSer) by the serine base-exchange enzyme system, in Jurkat T-lymphocytes, was inhibited in intact cells maintained in low-Ca(2+)-containing buffer (< 10 microM-Ca2+) by using Ca2+ ionophores (A23187 or ionomycin). The rise in cytosolic Ca2+ concentration under these experimental conditions was only due to the release of Ca2+ from intracellular compartments, suggesting that the inhibition of PtdSer synthesis was correlated with the emptying of intracellular Ca2+ pools. This was further studied in saponin-permeabilized cells, in which PtdSer synthesis was found to be inhibited by EGTA, Ca2+ ionophores (A23187 or ionomycin) and Ca(2+)-ATPase inhibitors [thapsigargin or 2,5-di-(t-butyl)-benzohydroquinone]. Since Ca(2+)-ATPase inhibitors impaired refilling of the Ca2+ stores with Ca2+, and since in CD3-activated Jurkat T-cells the Ca2+ stores remained empty after 1 h of treatment with anti-CD3 monoclonal antibodies, we suggest that PtdSer synthesis is mainly regulated by the level of Ca2+ in the intracellular compartments and that the Ca(2+)-dependent serine base-exchange system responsible for PtdSer synthesis is probably located within or close to a Ca(2+)-storage organelle.

Antibodies, Monoclonal↗
PubMed1993

Changes in free cytoplasmic magnesium following activation of human lymphocytes.

Activation of lymphocytes with 10 microM ionomycin leads to a rapid increase in the concentration of free cytoplasmic calcium ([Ca2+]i) and, at a slower rate, also to an increase in the cytoplasmic free magnesium concentration ([Mg2+]i). The ionomycin-induced Mg(2+)-mobilization response is dependent on the influx of extracellular Ca2+. After receptor-mediated lymphocyte activation, induced by mitogens or anti-receptor antibodies, a Mg(2+)-mobilization response does occur in a small fraction of the cells. Simultaneous measurement of [Ca2+]i and [Mg2+]i in individual cells showed that the receptor-triggered Mg(2+)-mobilization response is restricted to cells that have a high [Ca2+]i. It can therefore be concluded that a high [Ca2+]i induces the release into the cytoplasm of Mg2+ from intracellular stores.

Burkitt Lymphoma↗
PubMed1993

A steady-state mechanism can account for the properties of inositol 2,4,5-trisphosphate-stimulated Ca2+ release from permeabilized L1210 cells.

We have investigated the effects of sub-maximal Ins(2,4,5)P3 concentrations on the Ca2+ permeability of the residual undischarged Ca2+ stores in electroporated or digitonin-permeabilized L1210 cells by measuring Ca(2+)-efflux rate after addition of the ATPase inhibitor thapsigargin. Low concentrations of Ins(2,4,5)P3, causing rapid discharge of a small proportion of the releasable Ca2+, result in a substantial stimulation of Ca2+ efflux after thapsigargin addition. This indicates firstly that in the absence of thapsigargin there must have been a substantial, counterbalancing, increase in rate of Ca2+ pumping, and secondly that the increased Ca2+ permeability is more consistent with a steady state than with a quantal model of Ca2+ release. Similar increases in passive Ca2+ permeability are produced by addition of concentrations of ionomycin which produce equivalent changes in Ca2+ loading to those produced by Ins(2,4,5)P3, although the time course and initial rate of Ca2+ release are very much slower. In the presence of a Ca(2+)-buffering system, the time course of Ca2+ release by Ins(2,4,5)P3 becomes superimposable on that of ionomycin, indicating that the initial rapid phase of Ins(2,4,5)P3-stimulated Ca2+ is at least partially due to positive feedback from extravesicular Ca2+.

Aniline Compounds↗
PubMed1994

Hydrogen peroxide activates agonist-sensitive Ca(2+)-flux pathways in canine venous endothelial cells.

The effect of the biological oxidant H2O2 on purinergic-receptor-stimulated Ca2+ signalling was determined in canine venous endothelial cells. H2O2 increased cytosolic free [Ca2+] ([Ca2+]i), the rate of rise of which was dose-dependently related to H2O2 concentration. The response of [Ca2+]i to H2O2 resulted in part from release of Ca2+ from internal stores. The H2O2-sensitive intracellular Ca2+ pool was characterized in cells suspended in Ca(2+)-free/EGTA buffer and stimulated in sequence with H2O2 and ionomycin or ATP. Under this condition, the rank order of apparent compartment size sensitive to each compound was ionomycin > H2O2 > ATP. Stimulation of cells with H2O2 eliminated any response of [Ca2+]i to subsequent addition of ATP. To test more directly whether H2O2 accesses the inositol trisphosphate-sensitive Ca2+ store, cells were pretreated with thapsigargin, a selective inhibitor of that store's Ca2+ pump. Release of Ca2+ from internal Ca2+ stores by H2O2 declined as the interval after thapsigargin addition increased, a finding that supports the contention that H2O2 accesses the inositol trisphosphate-sensitive Ca2+ store. H2O2-stimulated Ca2+ influx across the cell membrane was sensitive to Ni2+, La3+, and 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole HCl (SKF-96365), a selective inhibitor of the agonist-stimulated Ca(2+)-influx pathway. Ca2+ entry triggered by H2O2 appears to occur via the agonist-sensitive Ca2+ influx pathway. Together, these results suggest that H2O2, which is normally secreted by activated neutrophils and monocytes, may act as an intercellular messenger and stimulate Ca2+ signalling in target endothelial cells.

Adenosine Triphosphate↗
PubMed1994

Phenylarsine oxide and phorbol myristate acetate inhibit the CD3-induced rise of cytosolic Ca2+ in Jurkat cells by refilling internal Ca2+ stores.

Phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, has been found to inhibit the early elevation in cytosolic Ca2+ concentration ([Ca2+]i), related to the CD3 activation pathway in Jurkat T cells. This inhibition was dose-dependent, consistent with previously reported effects of PAO on tyrosine phosphatases, and reversed by dimercaptopropanol. By contrast, okadaic acid, an inhibitor of serine/threonine phosphatases, had no effect on CD3-induced Ca2+ flux. PAO was compared with phorbol 12-myristate 13-acetate (PMA), which caused a similar, although less potent, inhibition as previously described. The two reagents produced additive inhibition of the CD3-induced [Ca2+]i rise, but did not affect thapsigargin- or ionomycin-driven Ca2+ flux in Jurkat cells. PAO and PMA prevented cells from complete depletion of intracellular Ca2+ stores by an anti-CD3 monoclonal antibody (mAb) and restored, at least partially, the ionomycin-sensitive pool, when added after anti-CD3 mAb. Moreover, the CD3-induced inhibition of phosphatidylserine synthesis, due to depletion of internal Ca2+ stores, is reversed by PAO and PMA. Anti-phosphotyrosine immunoblot analysis show that these effects cannot be accounted for by an inhibition of CD3-induced tyrosine phosphorylations. We propose that PAO and, to a lesser extent, PMA allow the refilling of internal compartments by Ca2+, which consequently abrogates a capacitative entry of external Ca2+.

Arsenicals↗
PubMed1994

Intracellular Ca2+, inositol 1,4,5-trisphosphate and additional signalling in the stimulation by platelet-activating factor of prostaglandin E2 formation in P388D1 macrophage-like cells.

In the P388D1 macrophage-like cell line, phospholipase A2 activity and prostaglandin production are stimulated by platelet-activating factor (PAF) and bacterial lipopolysaccharide (LPS). We have investigated the role of Ins(1,4,5)P3 and Ca2+ in signal transduction of PAF-induced prostaglandin E2 (PGE2) formation in these cells. The role of Ca2+ in the activation mechanism was studied by fluorescence imaging of intracellular Ca2+ in individual adherent cells and by determining the PGE2 production in the same population of cells. This new approach enabled us to correlate directly events on the single-cell level with a physiologically relevant response of the cell population. Priming the cells with LPS was required for PAF to stimulate PGE2 formation, yet LPS affected neither the intracellular free Ca2+ concentration ([Ca2+]i) nor the PAF-induced rise in [Ca2+]i. In addition, basal and PAF-stimulated Ins(1,4,5)P3 levels were not affected by LPS priming. However, the Ca2+ transient, the release of Ins(1,4,5)P3 and the formation of PGE2 induced by PAF were inhibited in cells pretreated with pertussis toxin. Buffering the [Ca2+]i with intracellular BAPTA [bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid] blocked the PAF-stimulated rise in [Ca2+]i and PGE2 formation. Removal of extracellular Ca2+ during PAF stimulation prevented the influx of Ca2+, but did not affect the initial [Ca2+]i transient, nor did it inhibit PGE2 formation. Under the same conditions, ionomycin stimulated an identical [Ca2+]i transient, but, in contrast with PAF stimulation, no PGE2 formation was observed. PGE2 production could be rescued by prompt subsequent addition of PAF, which caused no further [Ca2+]i change on its own. These results show that the transient initial rise in [Ca2+]i, produced either by PAF via the formation of Ins(1,4,5)P3 or directly by ionomycin, is necessary, but not sufficient for the formation of PGE2 in LPS-primed P388D1 cells. Furthermore, we have demonstrated for the first time that PAF triggers a second signal that is not mediated by a change in [Ca2+]i. However, both signals are required to induce PGE2 formation.

Calcium↗
PubMed1995

Two classes of agonist-sensitive Ca2+ stores in platelets, as identified by their differential sensitivity to 2,5-di-(tert-butyl)-1,4-benzohydroquinone and thapsigargin.

In the absence of extracellular Ca2+, extensive Ca2+ release from the platelet intracellular stores [monitored as an increase of intracellular Ca2+ concentration ([Ca2+]i)] is produced by the combined action of the endomembrane Ca(2+)-ATPase inhibitor thapsigargin and 2 nM ionomycin. The titration of Ca2+ unloading with thapsigargin (plus ionomycin) shows that a substantial fraction of the store-associated Ca2+ is released by 8-10 nM thapsigargin, but that 100-200 nM thapsigargin is required for the complete release. The store depletion obtained in similar conditions with a different endomembrane Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBHQ), is always incomplete. It is completed by thrombin or by 10 nM thapsigargin. We conclude that two different types of Ca2+ pumps exist in platelets, one sensitive to TBHQ and to high thapsigargin, the other insensitive to TBHQ and sensitive to low thapsigargin. They are distributed separately in discrete subpopulations of the agonist-sensitive stores. The influx of external Ca2+ is maximal when both types of stores are Ca(2+)-depleted, either by high thapsigargin or by the combined action of low thapsigargin and TBHQ.

Antioxidants↗
PubMed1995

Bradykinin-stimulated phosphatidylcholine hydrolysis in airway smooth muscle: the role of Ca2+ and protein kinase C.

The regulation of phosphatidylcholine (PtdCho) hydrolysis by Ca2+ and protein kinase C (PKC) was measured in [3H]palmitate-labelled cultured guinea-pig airway smooth-muscle cells as phosphatidylbutanol ([3H]PtdBut) and phosphatidate ([3H]PtdOH) formation in the presence of butanol. The former is a direct measure of phospholipase D (PLD) activity, whereas the latter, in airway smooth muscle, is indicative of net PtdCho-specific phospholipase C (PLC)-like/diacylglycerol (DG) kinase activity. Bradykinin-stimulated responses exhibited a requirement for extracellular Ca2+ influx, since they were inhibited in the presence of EGTA. This influx was independent of voltage-operated channels, since the L-type channel blocker nifedipine (up to 10 microM) was without effect on bradykinin-stimulated responses. In support of this, membrane depolarization with KCl (30 mM) failed to elicit either response. However, bradykinin-stimulated formation of both [3H]PtdBut and [3H]PtdOH was partially inhibited by 100 microM SKF96365. Ionomycin, a Ca2+ ionophore, induced PtdCho hydrolysis to a greater extent than bradykinin, also in an extracellular-Ca(2+)-dependent manner. Thapsigargin-induced emptying of intracellular Ca2+ pools elicited the formation of both [3H]PtdBut and [3H]PtdOH and displayed a requirement for extracellular Ca2+. Bradykinin-stimulated PtdCho-specific PLC-like/DG kinase pathway and PLD responses were unaffected by thapsigargin pretreatment, thereby questioning the role of Ins(1,4,5)P3/Ins(1,3,4,5)P4-dependent Ca2+ stores in the receptor stimulation of these activities in airway smooth-muscle cells. In this regard, we have previously demonstrated that the bradykinin-stimulated PtdCho-specific PLD and PLC-like activities can occur under conditions of apparent complete blockade of bradykinin-stimulated Ins(1,4,5)P3 formation by receptor antagonist in guinea-pig airway smooth muscle. The PKC inhibitor, Ro31-8220, selectively blocked both bradykinin- and ionomycin-stimulated PLD activity in a concentration-dependent manner (IC50 approx. 1 microM), but was without effect on bradykinin-stimulated PtdCho-PLC-like/DG kinase-derived PtdOH formation. In contrast, an inhibitor of PtdCho-PLC, D609, selectively blocked the formation of [3H]PtdOH in the presence of butanol (PtdCho-PLC-like/DG kinase activity), but not [3H]PtdBut formation. In conclusion, PtdCho hydrolysis appears to occur via two distinguishable routes which both require extracellular Ca2+, whereas only the PLD route is regulated by PKC.

Animals↗
PubMed1996

Induction of calreticulin expression in HeLa cells by depletion of the endoplasmic reticulum Ca2+ store and inhibition of N-linked glycosylation.

Calreticulin is now considered to be a multifunctional Ca(2+)-binding protein. Its primary role is as a Ca2+ storage protein within the lumen of the endoplasmic reticulum (ER), where it also seems to assist in the correct folding and assembly of proteins. We have investigated whether agents that affect these processes can alter calreticulin expression in HeLa cells. Perturbation of intracellular Ca2+ levels by prolonged exposure to either thapsigargin or ionomycin induced calreticulin mRNA, both in the presence and absence of extracellular Ca2+, consistent with the proposal that sustained depletion of the ER Ca2+ store can trigger these increases. The mechanism underlying the induction seems to be transcriptional up-regulation as both agents increased calreticulin promoter-driven firefly luciferase expression in transfected cells to the same degree as the observed increases in calreticulin mRNA. Experiments with a truncated promoter construct showed that the sequences that confer this inducibility reside within the 225 bp immediately upstream of the putative major transcriptional start site. We also examined the effect of tunicamycin, which inhibits N-linked glycosylation in the ER thereby interfering with protein processing. This caused increases in calreticulin mRNA greater than those with either thapsigargin or ionomycin, but failed to transactivate the calreticulin promoter. Thus either additional cis sequences that reside outside our promoter region are necessary for transcriptional activation by tunicamycin, or the increases in calreticulin mRNA occur post-transcriptionally. This suggests that there are probably different mechanisms by which calreticulin expression can be induced in response to agents that affect normal ER functioning.

Base Sequence↗
PubMed1996

Elevated intracellular calcium concentration increases secretory processing of the amyloid precursor protein by a tyrosine phosphorylation-dependent mechanism.

Secretory cleavage of the amyloid precursor protein (APP), a process that releases soluble APP derivatives (APPs) into the extracellular space, is stimulated by the activation of muscarinic receptors coupled to phosphoinositide hydrolysis. The signalling pathways involved in the release process exhibit both protein kinase C- and protein tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337-8344]. The possibility that elevations in intracellular Ca2+ concentration initiate the tyrosine phosphorylation-dependent release of APPs was examined in human embryonic kidney cells expressing muscarinic m3 receptors. Inhibition of protein kinase C with the bisindolylmaleimide GF 109203X decreased the carbachol-evoked release of APPs by approx. 30%, as shown previously. The residual response was further decreased, in an additive manner, by the Ca2+ chelator EGTA, or by the tyrosine kinase inhibitor tyrphostin A25. The Ca2+ ionophore, ionomycin, like carbachol, stimulated both the release of APPs and the tyrosine phosphorylation of several proteins, one of which was identified as paxillin, a component of focal adhesions. The effects of ionomycin on APPs release and on protein tyrosine phosphorylation were concentration-dependent, and occurred over similar concentration ranges; both effects were inhibited only partly by GF 109203X, but were abolished by EGTA or by tyrosine kinase inhibitors. The results demonstrate for the first time that ionophore-induced elevations in intracellular Ca2+ levels elicit APPs release via increased tyrosine phosphorylation. Part of the increase in APPs release evoked by muscarinic receptor activation might be attributable to a similar mechanism.

Amyloid beta-Protein Precursor↗
PubMed2001

Nerve growth factor in combination with second messenger analogues causes neuronal differentiation of PC12 cells expressing a dominant inhibitory Ras protein without inducing activation of extracellular signal-regulated kinases.

In the present work, nerve growth factor (NGF) was used in combination with the calcium ionophore, ionomycin or dibutyryl cyclic AMP (dbcAMP), to study the connection between neuronal differentiation and extracellular signal-regulated kinase (ERK) activation of PC12 rat pheochromocytoma cells expressing a dominant negative, Ha-Ras Asn17 protein. Due to the block of endogenous Ras activity, neurite outgrowth in response to NGF is completely inhibited in these cells. However, this blockade can be bypassed by combined treatment with NGF plus ionomycin or NGF plus dbcAMP. The mitogen-activated protein kinase (MAPK) /ERK kinase inhibitor, PD98059, proved to be insufficient in inhibiting the neurite outgrowth under these conditions. Moreover, although both long-term ERK activation and nuclear translocation of ERKs are believed to be key events in neuronal differentiation, neither detectable ERK phosphorylation, nor nuclear translocation of these enzymes, occurred upon combination treatments in our experimental system. However, the neuritogenesis induced by either the combination of NGF/ionomycin or NGF/dbcAMP was inhibited by the Trk inhibitor, K252a. Ras-independent pathways, originating from the NGF receptor, can thus synergize with second messenger analogues bypassing the ERK cascade but leading to the same biological result--neurite formation.

Active Transport, Cell Nucleus↗
PubMed2001

In vitro generation of human CD86+ dendritic cells from CD34+ haematopoietic progenitors by PMA and in serum-free medium.

The cytokine requirements to differentiate CD34+ progenitor cells from different origins either cord blood (CB) or peripheral blood (PB) into dendritic cells (DC) are known to be different. In addition to DC, macrophages and neutrophils are generated. On the other hand, phorbol esters such as PMA induce primary human CD34+ bone marrow (BM) progenitor cells to differentiate into functional DC and no other lineages are generated. In addition, FCS is used as culture supplement in most of the protocols described which contains additional foreign antigens potentially skewing the resulting immune response. Therefore, we evaluated the ability to differentiate CB- and PB-CD34+ progenitor cells into DC with PMA and under serum-free conditions. In this study, we delineate the maturation of cultured human blood DC by analysis of expression co-stimulatory molecule B7-2 (CD86). Human mature DC with typical morphology and surface antigen phenotype (CD1a-, CD83+ and CD86+) were obtained from CB- and PB-CD34+ progenitor cells after 1 week of culture in serum-free medium upon stimulation with PMA alone. The same result was obtained from ex vivo-expanded BM-CD34+ cells. CD86+ yield was increased by PMA compared to cytokine cocktails (28.0% +/- 7.0 versus 15.3% +/- 5.6 for CB and 44.6% +/- 7.5 versus 28.1% +/- 7.5 for PB, respectively). CD86 was most up-regulated in the presence of the calcium ionophore ionomycin. However, the number of viable cells after differentiation was decreased by PMA plus ionomycin (P < 0.05) or plus TNF-alpha (P > 0.05) as compared with that in PMA alone. We conclude that PMA is a potent activator to differentiate human CD34+ cells into mature DC in serum-free medium. This may be used for in vitro studies of primed or genetically modified DC against infectious and tumour-associated antigens.

Antigen Presentation↗
PubMed2001

In vitro cytokine production and growth inhibition of lymphoblastoid cell lines by CD4+ T cells from Epstein-Barr virus (EBV) seropositive donors.

In vitro stimulation of peripheral blood lymphocytes (PBL) from healthy Epstein-Barr Virus (EBV) seropositive individuals with autologous lymphoblastoid cell lines (LCL) gives rise to CD4+ and CD8+ T cells both of which are cytotoxic for autologous lymphoblastoid cells. Activated EBV-specific CD4+ T cells are cytotoxic towards autologous LCL but, paradoxically, CD4+ T cells have also been shown to enhance tumour formation in SCID/Hu mice. Here, we show that despite being cytotoxic, CD4+ T-cell lines from different donors show considerable variation in their ability to inhibit the long-term growth of autologous LCLs in vitro. Following re-stimulation in vitro with PMA and ionomycin, CD4+ T cells produced IFNgamma, TNFalpha, TNFbeta, IL-2, IL-4, IL-10 and IL-13. TNFalpha, TNFbeta and IL-10 production were also detected in LCL. IL-6 was only detected in trace amounts in either cell type. The ratio of IFNgamma to IL-4 production varied between the CD4+ T-cell lines, indicating differences in the Th1/Th2 balance of the response. When CD4+ T cells were re-stimulated using autologous LCL as antigen-presenting cells, they produced more IL-4 and less IFNgamma or IL-13 when compared with cells re-stimulated by phorbol myristate acetate (PMA) and ionomycin. Using two colour cytokine staining, we showed that many individual CD4+ T cells produced IFNgamma along with either IL-4 or IL-13. Purified CD4+ T cells completely inhibited the outgrowth of autologous LCL in five out of nine cases, and partially inhibited outgrowth in the remaining four. There was no correlation between the pattern of CD4+ T-cell cytokine production and the capacity to inhibit outgrowth of autologous LCL. The killing of LCLs was contact-dependant and not mediated by soluble factors. We conclude that the ability of CD4+ T cells to inhibit autologous LCL growth is not directly related to T-helper cell cytokine production, but may depend on cytoxicity through surface ligands such as CD95L (FasL) and TNFalpha-related apoptosis-inducing ligand (TRAIL).

Antibodies, Viral↗
PubMed2001

Polymeric IgA binding to the human pIgR elicits intracellular signalling, but fails to stimulate pIgR-transcytosis.

The intracellular pathway of polymeric immunoglobulin receptor (pIgR) is governed by multiple signals that lead to constitutive transcytosis. In addition, in transfected polarized MDCK cells, polymeric immunoglobulin A (pIgA) binding stimulates rabbit pIgR-transcytosis, owing to phospholipase-C gamma 1 activation and increase of intracellular calcium. Transcytosis of rat pIgR across hepatocytes is similarly accelerated by pIgA injection. In contrast we show here that human Madrin-Darby Canine Kidney (pIgR)-transcytosis, in human Calu-3 and human pIgR-transfected MDCK cells, is not promoted by pIgA, as monitored by a continuous apical release of its secreted ectodomain. However, the incubation of cells expressing human or rabbit pIgR with pIgA induces a comparable IP3 production, and pIgR-transcytosis of either species is accelerated by the protein kinase C (PKC)-activator phorbol myristate acetate. Without pIgA, mimicking phospholipase-C activation by combining low concentrations of phorbol myristate acetate with ionomycin, or high concentrations of ionomycin alone, stimulates the rabbit, but not the human, pIgR transcytosis. These data suggest that the species difference in pIgA-induced pIgR-transcytosis does not stem from the defective production of second messengers, but from a different sensitivity of pIgR to intracellular calcium. Our results outline the danger of extrapolating to humans the abundant data obtained from mucosal vaccination of laboratory animals.

Adenocarcinoma↗
PubMed1999

Cyclosporin A inhibits Ca2+-mediated upregulation of the DNA repair enzyme DNA polymerase beta in human peripheral blood mononuclear cells.

Alterations in gene expression may represent an underlying cause of undesired side-effects mediated by the immunosuppressant cyclosporin A (CsA). We employed the method of differential display PCR to identify new genes whose expression is modulated by CsA. Human peripheral blood mononuclear cells (PBMCs), or subpopulations thereof, were simultaneously stimulated with the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. We identify the gene encoding the DNA repair enzyme DNA polymerase beta (Pol beta) as a novel CsA-sensitive transcription unit. Our data show that transcription of pol beta mRNA is induced by Ca2+ and that CsA significantly inhibits PMA/ionomycin- and ionomycin-mediated upregulation of both pol beta mRNA and Pol beta protein. The CsA-mediated inhibition of pol beta upregulation is maintained for at least 21 h after gene activation and is exerted via the phosphatase calcineurin. FK506, another immunosuppressant that targets calcineurin, also inhibits pol beta upregulation, while rapamycin competes with FK506 action. This work identifies Ca2+ as an inducer of pol beta gene activity in primary blood cells. The demonstrated CsA sensitivity of this process suggests a novel molecular mechanism that may contribute to the increased tumor incidence in patients receiving CsA treatment.

Animals↗