Evidence implicating utilization of different T cell receptor-associated signaling pathways by TH1 and TH2 clones

We have reported recently that high concentrations of anti-CD3 mAb inhibited IL-2-dependent proliferation of TH1 but not TH2 clones. The selective inhibitory effect on TH1 clones suggested that the two helper T lymphocyte subsets might utilize different TCR-associated signal transduction mechanisms. In the present study, we demonstrate that this distinction was not due to a gross difference in the level of TCR expression by TH1 and TH2 clones. Inhibition of TH1 proliferation by anti-CD3 mAb appeared to depend on calcium for maximal effect, suggesting that a substantial elevation of intracellular free calcium concentration ([Ca2+]i) might not occur after ligation of the TCR complex of TH2 clones. Calcium ionophore inhibited IL-2-dependent proliferation of both subsets, suggesting that receptor/ligand systems which stimulate elevated [Ca2+]i would be expected to inhibit proliferation. Although elevated [Ca2+]i and generation of inositol phosphates were readily detected in TH1 clones, these second messengers were not detected following stimulation of TH2 clones via the TCR complex. In addition, lymphokine production by TH1 clones was more sensitive to inhibition by cholera toxin, 8-bromoadenosine 3':5'-cyclic monophosphate, and cyclosporin A than was lymphokine production by TH2 clones. Collectively, these results suggest that TH1 and TH2 clones utilize distinct TCR-associated signal transduction mechanisms for lymphokine gene expression. The difference in signaling mechanisms suggests a potential pharmacologic target for intervention in situations where inappropriate activation of TH1 or TH2 cells occurs in vivo.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

The cGMP/protein kinase G pathway contributes to dihydropyridine-sensitive calcium response and cytokine production in TH2 lymphocytes

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.     

Role of intracellular calcium concentration and protein kinase C activation in IFN-gamma stimulation of U937 cells

IFN-gamma enhances many monocyte functions, including oxidative metabolism and Ag presentation. IFN-gamma has been reported to increase the intracellular concentration of calcium ([Ca2+]i) and modulate protein kinase C activity in murine macrophages, but the signal transduction pathways induced by IFN-gamma in human cells and their functional significance are poorly understood. Our study examined the hypothesis that an increases in [Ca2+]i and protein kinase C activation are required for functional responses to IFN-gamma. The U937 cell line was used as a model of an IFN-gamma responsive cell. IFN-gamma caused a rapid and concentration-dependent increase in [Ca2+]i, which was partly inhibited by calcium-free medium, diltiazem, and TMB-8. IFN-gamma induced a fourfold increase in the concentration of inositol 1,4,5-trisphosphate. Induction of HLA-DR, Fc gamma R, CR3, and Mo3e Ag expression by IFN-gamma was blocked by concentrations of TMB-8 that inhibited an increase in [Ca2+]i, but not by protein kinase C inhibition by H-7 or inhibition of calmodulin with W-7. Ionomycin did not enhance Ag expression and PMA induced the expression of only the Mo3e Ag. We conclude that IFN-gamma induces antigenic expression on human U937 cells by a mechanism dependent on, but not limited to, an increase in intracellular calcium, which is likely due to inositol 1,4,5-trisphosphate generation.     

Insulin-like growth factor I stimulates inositol phosphate accumulation, a rise in cytoplasmic free calcium, and proliferation in cultured porcine thyroid cells

Insulin-like growth factor (IGF-I) stimulates thyroid cell proliferation. Using primary cultured porcine thyroid cells, we studied the intracellular pathways that mediate the action of IGF-I on thyroid cell proliferation. IGF-I stimulates inositol phosphate accumulation, a rise in cytoplasmic free calcium [( Ca2+]i), and cell proliferation. Exposure to IGF-I results in a time- and dose-dependent accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. IGF-I also increases [Ca2+]i, measured using fura-2, a fluorescent Ca2+ indicator; the IGF-I-induced [Ca2+]i response occurs immediately, reaches a maximum within 1 min, and then slowly declines. IGF-I stimulates thyroid cell proliferation, stimulates thymidine incorporation, and increases cell numbers. The IGF-I-induced inositol phosphate accumulation and [Ca2+]i response parallel thyroid cell proliferation in a dose-dependent manner; the maximal response is observed at a concentration of 100 ng/ml IGF-I, with half-maximal stimulation at approximately 10 ng/ml. Inositol phosphate accumulation and [Ca2+]i response after IGF-I stimulation may function as intracellular messengers for thyroid cell proliferation. This report may constitute the first demonstration of IGF-I-stimulated inositol phosphate accumulation and [Ca2+]i response in the cells.     

Phorbol myristate acetate inhibits increases in membrane fluidity induced by anti-IgM in B cells

Anti-IgM or anti-IgD stimulates B cells to induce increases in inositol phospholipid metabolism and intracellular free calcium concentration [( Ca2+]i). Anti-IgM also causes increases in membrane fluidity that occur more promptly than those in [Ca2+]i in resting B cells as well as BAL17 B lymphoma cells. However, other B cell activators such as LPS or PMA did not induce the membrane fluidity changes. Furthermore, sodium fluoride, which is considered to be an activator of the guanine nucleotide-binding protein, caused increases in membrane fluidity as well as increased [Ca2+]i or inositol phospholipid metabolism. Anti-IgM- or sodium fluoride-induced increases in membrane fluidity were inhibited by 20-min pretreatment of cells with PMA, but not by 24-h pretreatment. These results indicate that membrane fluidity changes are closely associated with increased [Ca2+]i after cross-linkage of membrane Ig and are regulated by protein kinase C in B cells.     

Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells.

Intercellular communication of epithelial cells was examined by measuring changes in intracellular calcium concentration ([Ca2+]i). Mechanical stimulation of respiratory tract ciliated cells in culture induced a wave of increasing Ca2+ that spread, cell by cell, from the stimulated cell to neighboring cells. The communication of these Ca2+ waves between cells was restricted or blocked by halothane, an anesthetic known to uncouple cells. In the absence of extracellular Ca2+, the mechanically stimulated cell showed no change or a decrease in [Ca2+]i, whereas [Ca2+]i increased in neighboring cells. Iontophoretic injection of inositol 1,4,5-trisphosphate (IP3) evoked a communicated Ca2+ response that was similar to that produced by mechanical stimulation. These results support the hypothesis that IP3 acts as a cellular messenger that mediates communication through gap junctions between ciliated epithelial cells.     

The rise in concentration of free Ca2+ and of pH provides sequential, synergistic signals for secretion in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Ag stimulation of rat basophilic leukemia (RBL-2H3) cells results in hydrolysis of inositol phospholipids, a transient increase in concentration of cytosol Ca2+ [( Ca2+]i), a gradual increase in cytosolic pH (pHi) and the activation of protein kinase C. To determine whether all these changes serve as signals for secretion, studies were conducted with cells permeabilized with streptolysin O in which pHi and [Ca2+]i could be varied independently of each other and enzyme activities could be manipulated. At resting pHi (approximately 7.0) and [Ca2+]i (0.1 microM), the permeabilized cells showed little secretory response to Ag. At resting pHi, elevated levels of Ca2+ (0.33 microM) were required for maximal secretory response to Ag. At a pHi of 7.4, however, 0.1 microM [Ca2+]i was sufficient to sustain near maximal responses to Ag. Therefore, a small increase of [Ca2+]i to 0.33 microM was required to initiate secretion, but once the pHi was elevated secretion could be sustained at near basal levels of [Ca2+]i. Since elevating the [Ca2+]i and pHi, by themselves promoted little secretion, another potentiating signal must have been generated by antigen stimulation. This signal was possibly transduced via hydrolysis of inositol phospholipids and protein kinase C. Even with an elevated [Ca2+]i (0.33 microM) the hydrolysis of the phospholipids and secretion stimulated by Ag were inhibited by guanosine 5'(2-O-thio)diphosphate and neomycin. Furthermore, both protein-kinase C and the secretory response to Ag were lost after permeabilized cells were washed but both were retained if cells were exposed to PMA before permeabilization.     

Modulation of cytoplasmic calcium in human platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine

The calcium-sensitive, fluorescent dye Quin 2 was used to quantitate changes in free intracellular calcium [( Ca2+]i) induced in platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC). The Ca2+]i of unstimulated platelets was 91 +/- 18 nM (mean +/- SD, n = 8), and treatment with 1 to 16 nM AGEPC increased [Ca2+]i in a dose-related manner, with 16 nM AGEPC increasing [Ca2+]i by 102 +/- 20 nM. [Ca2+]i was not increased by analogs of AGEPC which do not activate platelets including the lysophospholipid precursor of AGEPC, the optical isomer, and a C-2 benzoyl analog. The capacity of AGEPC to increase [Ca2+]i exceeded that required to induce maximal platelet aggregation. In four experiments, 100% platelet aggregation was induced by 4.5 +/- 2.4 nM AGEPC (mean +/- SD) and was associated with a submaximal increase in [Ca2+]i of 56 +/- 22 nM. Pretreatment of platelets with AGEPC rendered the platelets specifically unresponsive to repeat stimulation with AGEPC in terms of both platelet aggregation and increased [Ca2+]i, whereas the platelet response to thrombin was undiminished by pretreatment with AGEPC. In contrast, the platelet response to 0.5 microM calcium ionophore A23187 was undiminished by pretreatment with the same concentration of ionophore, suggesting that AGEPC does not activate platelets by an ionophore-like mechanism. IgG aggregates and AGEPC in combination activate platelets synergistically, as shown by the observation that a 1-min exposure of platelets to 60 micrograms/ml of IgG aggregates increased the platelet aggregation response to 2 nM AGEPC from 44 to 100%. In contrast, sequential exposure of platelets to IgG aggregates and AGEPC increased [Ca2+]i additively, suggesting that increased [Ca2+]i contributes to but does not fully mediate synergistic platelet activation by IgG aggregates and AGEPC. Quantitation of free intracellular calcium with the fluorescent dye Quin 2 is a highly sensitive technique for delineating the role of calcium in mediating platelet activation.     

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.     

Signal transduction events and Fc gamma R engagement in human neutrophils stimulated with immune complexes.

Signal transduction events have been evaluated in human neutrophils stimulated with immune complexes consisting of polyclonal rabbit antibody complexed with BSA. Immune complexes induced dose-related O2- responses, but very small increases in intracellular calcium ([Ca2+]i) levels were observed, in contrast to FMLP-stimulated cells. Measurements employing [45Ca2+] demonstrated that calcium influx and efflux in cells stimulated with immune complexes was substantially less than fluxes found in FMLP-stimulated cells. With respect to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation under conditions in which the O2- responses to immune complexes or FMLP were similar, the Ins(1,4,5)P3 response to immune complexes was much smaller (by 65%) as compared to that induced by FMLP. Although pertussis toxin-treated cells showed a greatly diminished O2- response (by 89%) to FMLP, the response to immune complexes was largely resistant (only 26% reduction) to the inhibitory effects of this toxin. Antibodies to Fc gamma R indicated that engagement of Fc gamma RII and Fc gamma RIII, but not Fc gamma RI, receptors was related to the O2- response of neutrophils to immune complexes. O2- formation occurred in neutrophils incubated with Staphylococcus aureus cell walls bearing antibodies to Fc gamma RII or Fc gamma RIII. These data indicate that, in human neutrophils stimulated with immune complexes, signal transduction events involve engagement of Fc gamma RII and Fc gamma RIII. The O2- response is largely pertussis-toxin insensitive, is not associated with a significant increase in levels of [Ca2+]i, and is associated with relatively little formation of Ins(1,4,5)P3. This is in contrast to cells stimulated with FMLP in which O2- responses are largely pertussis toxin-sensitive and associated with large increases in [Ca2+]i as well as formation of Ins(1,4,5)P3. Signal transduction events involving Fc gamma R appear to be quite different from those events related to engagement of FMLP receptors.     

Protein kinase C activation blocks anti-IgM-mediated signaling BAL17 B lymphoma cells

Short term pretreatment of the B lymphoma, BAL17, with phorbol 12-myristate, 13-acetate (PMA) blocks elevation in inositol trisphosphate (InsP3) and increases in intracellular free calcium concentration ([Ca2+]i) in response to anti-IgM. The inhibition of enhanced InsP3 level is detected at 30 sec after the addition of anti-IgM, the earliest point measured, and is reversed by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C (PKC). The blockade of increased [Ca2+]i by PMA is also observed at the earliest time examined (15 sec), is reversed by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride, and is mimicked by dioctanoylglycerol, a physiologic activator of PKC. The enhanced production of inositol phosphates in response to NaF is also blocked in BAL17 cells pretreated with PMA. Extended treatment of BAL17 cells with PMA depletes cellular PKC. Such pretreatment with PMA enhances rather than inhibits increased InsP3 levels in response to anti-IgM and leads to more sustained elevations in [Ca2+]i than in normal BAL17 cells. These results lead us to conclude that PMA-blockade of the response of B cells to anti-IgM represents a disruption of the transmembrane signaling process (desensitization of the signaling pathway) as a result of a PKC-mediated phosphorylation event.     

Interactions between inositol phosphates and cytosolic free calcium following bradykinin stimulation in cultured human skin fibroblasts

The inositol triphosphate (IP3) that results from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is generally accepted to be responsible for the mobilization of intracellular calcium. However, some studies suggest that low concentrations of agonists elevate cytosolic free calcium concentration ([Ca2+]i) without IP3 formation. Thus, in the present studies, a comparison of the temporal response of inositol phosphates (IP3, IP2 and IP) and [Ca2+]i to a wide range of bradykinin concentrations was used to examine the relation of these two signal transduction events in cultured human skin fibroblasts (GM3652). In addition, the effects of alterations in internal or external calcium on the response of these second messengers to bradykinin were determined. Bradykinin stimulated accumulation of inositol phosphates and a rise of [Ca2+]i in a time- and dose-dependent manner. Decreasing the bradykinin concentration from 1 microM to 0.1 microM increased the time until the IP3 peak, and when the bradykinin concentration was reduced to 0.01 microM IP3 was not detected. [Ca2+]i was examined under parallel conditions. As the bradykinin concentration was reduced from 1 microM to 0.01 microM, the time to reach the peak of [Ca2+]i increased progressively, but the magnitude of the peak was unaltered. These two second messengers were variably dependent on external calcium. Although the bradykinin-stimulated initial spike of [Ca2+]i did not depend on extracellular calcium, the subsequent sustained levels of [Ca2+]i were abolished in calcium free medium. The bradykinin-stimulated inositol phosphate formation was not dependent on the extracellular calcium nor on the elevation of [Ca2+]i that was produced with Br-A23187. These results demonstrate that bradykinin-induced IP3 formation can be independent of [Ca2+]i and of external calcium, whereas changes in [Ca2+]i are partially dependent on external calcium.     

T cell receptor aggregation, but not dimerization, induces increased cytosolic calcium concentrations and reveals a lack of stable association between CD4 and the T cell receptor.

Exposure of T94, a CD4+ V beta 8-expressing murine Th cell clone, or immediately ex vivo CD4+ T cells to deaggregated, bivalent antibodies specific for either the TCR or CD3 failed to induce an increase in [Ca2+]i, or activation of phosphatidylinositol hydrolysis unless cross-linked with a secondary anti-Ig antibody. In contrast, we show that a combination of two mAb directed against different components of the TCR/CD3 complex (145.2C11, anti-CD3 epsilon and F23.1, anti-V beta 8) successfully induce second messenger formation, that is, without any requirement for a secondary antibody. This requirement for either a secondary antibody or two independent bivalent antibodies to activate second messenger production in T cells suggested that the signal transduction apparatus may be activated by multiple TCR/CD3 complexes being brought together on the T cell surface. This was supported by the observation that conditions inducing increased T cell [Ca2+]i through the TCR/CD3 complex also resulted in aggregation of the TCR/CD3 complex on the T cell surface. Conversely, binding of anti-TCR/CD3 antibodies to the T cell under conditions that did not induce increased [Ca2+]i also failed to induce surface TCR/CD3 redistribution. Cross-linking of the CD4 accessory molecule on T94 also resulted in increased [Ca2+]i, with kinetics similar to those observed after TCR/CD3 oligomerization. CD4 is involved in the recognition of invariant regions of MHC class II during Ag presentation and has been proposed to be associated with TCR/CD3 in the absence of Ag. Aggregation of TCR/CD3 and subsequent second messenger formation was achieved by combinations of mAb to distinct determinants within the complex due to the stable association of these determinants within the T cell membrane. We therefore assessed the functional association of CD4 with the TCR/CD3 complex by examining whether a combination of mAb directed against CD4 and CD3 or TCR induced second messenger formation. We found that anti-CD4 in combination with F23.1 or with 145.2C11 failed to induce increases in [Ca2+]i. Furthermore, mAb to CD4 failed to inhibit the increase in [Ca2+]i observed with the combination of 145.2C11 and F23.1. We therefore conclude that CD4 is not stably associated with TCR or CD3 in the absence of Ag/MHC class II composites.     

A Na(+)-dependent Ca2+ exchanger generates the sustained increase in intracellular Ca2+ required for T cell activation.

Movement of extracellular Ca2+ is required for the sustained increase in [Ca2+]i necessary for T cell activation. However, the mechanisms mediating mitogen-stimulated Ca2+ movement into T cells have not been completely delineated. To explore the possibility that a Na(+)-dependent Ca2+ (Na+/Ca2+) exchanger might play a role in the mitogen-induced increases in [Ca2+]i required for T cell activation, the effects of inhibitors of this exchanger were examined. Inhibitors of Na+/Ca2+ exchange suppressed the sustained increase in [Ca2+]i stimulated by ligation of the CD3-TCR complex, but did not affect mobilization of intracellular Ca2+ stores. Consistent with the importance of this prolonged increase in [Ca2+]i in T cell activation, Na+/Ca2+ exchange inhibitors, but not inhibitors of the Na+/H+ antiporter, inhibited DNA synthesis stimulated by immobilized anti-CD3 mAb. Inhibition only occurred when the agents were present during the first hours after stimulation. These agents also inhibited IL-2 production, but not expression of the IL-2R or of an early activation Ag, 4F2. Inhibition of IL-2 production did not account for the inhibition of T cell proliferation as addition of exogenous IL-2 or phorbol ester (PDB) did not overcome the inhibition. In contrast, activation pathways that are not thought to require an increase in [Ca2+]i such as IL-1 + PDB or engagement of CD28 in the presence of PDB were less sensitive to the suppressive effects of inhibitors of Na+/Ca2+ exchange. Thus, proliferation induced by these stimuli was not suppressed by low concentrations of these inhibitors and IL-2 production induced by mAb to CD28 + PDB was not inhibited by any concentration of inhibitors of Na+/Ca2+ exchange. These results suggest that stimulation of a Ca2+ transporter with the same spectrum of inhibition as the Na+/Ca2+ exchanger in other tissues mediates the sustained increase in [Ca2+]i required for T cell activation after CD3 ligation.     

Possible Existence of Two Subsets of Platelet-Activating Factor Receptor to Mediate Polyphosphoinositide Breakdown and Calcium Influx in Neuroblastoma × Glioma Hybrid NG 108–15 Cells

Platelet-activating factor (PAF) initiated polyphosphoinositide (polyPI) breakdown and a rise of intracellular calcium concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG 108-15 cells. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate was evident within 15 s after PAF stimulation, peaked at 1 min, and then gradually decayed. The increase in [3H]inositol monophosphate level was observed at 30 s, plateaued in 5 min, and was sustained up to 10 min in the presence of 10 mM LiCl. On the other hand, the rise of [Ca2+]i evoked by PAF reached a peak within 8-12 s and returned to basal levels within 1 min as measured in fura 2-loaded cells. When cells were suspended in Ca(2+)-depleted medium, the PAF-induced [Ca2+]i rise was reduced by 80%, indicating that the increase of [Ca2+]i was predominantly due to the Ca2+ influx from an extracellular source. Both PAF-induced accumulation of 3H-labeled inositol phosphates and [Ca2+]i elevation were concentration dependent with EC50 values of approximately 1 x 10(-10) and 5 x 10(-8) M, respectively. The PAF analogs 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-O-methyl-rac-glycerol-3-phosphocholine were much poorer agonists at eliciting the same responses in these cells. Pretreatment of cells with pertussis toxin caused a substantial inhibition of PAF-induced accumulation of 3H-inositol phosphates. In contrast, the rise in [Ca2+]i was not significantly affected by toxin treatment at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of the dissociation between IL-2 production and proliferation in a response of a T cell clone to the antigen presented by B cells

Using B cells as APC, antigen specific responses of two murine T cell clones, 34-7F and 35-8H, were analyzed. 34-7F cells produced IL-2 but failed to proliferate, whereas 35-8H cells both produced IL-2 and proliferate. The antigenic stimulation increased intracellular free Ca2+ concentration in both clones, but enhanced inositol phospholipid metabolism only in 35-8H cells. The treatment of 34-7F cells with PMA, an activator of protein kinase C, synergized with the antigenic stimulation to induce the proliferation of the T cells. Thus, the failure of 34-7F cells to proliferate in the Ag response appears to result from the absence of an increase in inositol phospholipid metabolism. The absence is likely due to the defect in B cells as APC, inasmuch as the antigenic stimulation of 34-7F cells with whole spleen cells induced increases in inositol phospholipid metabolism and proliferation. The PMA treatment synergized with the Ag on B cells to enhance IL-2R expression, which was not inhibited by the addition of nifedipine, a calcium channel blocker. The agent inhibited the IL-2 production. Taken together, the results in the present experiments suggest the association of IL-2 production with increases in intracellular free Ca2+ concentration but not in inositol phospholipid metabolism, and that of IL-2R expression with increases in the metabolism but not in intracellular free Ca2+ concentration.     

Antigen-induced Ca2+ signaling and desensitization in B cells

Cross-linking of B cell surface Ig (sIg) by anti-Ig results in transmembrane signaling. However, the capacity of a thymus-dependent (TD) Ag to mediate B cell signal transduction has been less well documented. Therefore, we examined Ag-induced intracellular free calcium concentration [( Ca2+]) in B cells by using TD Ag that would be expected to either cross-link or not cross-link sIgM and/or induce the coupling of sIgM to FcR. Stimulation of mouse TA3 hybridoma B cell transfectants that express the SP6 anti-TNP specific sIgM with either TNP-OVA or anti-IgM antibodies resulted in a maximal fourfold increase in [Ca2+]i. The net increase in [Ca2+]i in response to TNP-OVA was dependent upon both the Ag dose and the TNP:OVA molar ratio. Because occupancy of several cell-surface receptor types leads to a loss of response to subsequent stimulation by ligand (homologous desensitization), we examined the ability of Ag to induce homologous desensitization of sIgM in these B cells. TNP1-OVA at all concentrations tested (up to 500 micrograms/ml) did not lead to any change in [Ca2+]i or desensitization. Cross-linking of TNP1-OVA (10 micrograms/ml) with F(ab')2 of anti-OVA antibody induced both a rise in [Ca2+]i and homologous desensitization of sIg, suggesting that cross-linking of sIgM by Ag is sufficient to induce both these processes. TNP6-OVA at a concentration of 10 micrograms/ml induced changes in [Ca2+]i and partially desensitized TNP-specific B cells to stimulation by anti-IgM. Interestingly, a high dose (180 micrograms/ml) of TNP6-OVA stimulated minimal changes in [Ca2+]i yet did not lead to desensitization. However, cross-linking of TNP6-OVA at this high dose with F(ab')2 of rabbit anti-OVA elevated [Ca2+]i and elicited partial desensitization. Complete desensitization of sIgM by Ag was achieved when intact (Fc-containing) anti-OVA antibody was used, suggesting that the FcR can play a role in desensitization. Ag- and antibody-mediated desensitization was not caused by steric hindrance of sIg. Thus, we have observed two forms of Ag-induced desensitization of sIgM, both of which involve sIg cross-linking and one of which is mediated by the physiologic coupling of sIg to FcR.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Early biochemical events after MHC class II-mediated signaling on human B lymphocytes

These studies examined the role of the MHC class II Ag in signal transduction using human B lymphocytes. Early events in signal transduction were considered including the intracellular calcium [Ca2+)i) flux, the activation of phospholipase C, and induction of protein phosphorylation. The (Ca2+)i was enhanced after incubation of B lymphocytes with several mAb anti-HLA class II and cross-linking with rabbit anti-mouse-F(ab')2. We have also demonstrated an enhancement of the (Ca2+)i in response to a suboptimal concentration of a monoclonal anti-IgM either in the presence of or after preincubation with a mAb anti-HLA class II. The activation of phospholipase C was assessed by measuring the generation of inositol phosphates in permeabilized B lymphocytes. mAb anti-HLA-class II of two different epitopes were used to demonstrate both the (Ca2+)i flux and the generation of inositol phosphates. Two-dimensional gel electrophoresis was used to investigate the phosphorylation pattern of resting B lymphocytes and the changes in the pattern after stimulation with soluble mAb anti-HLA-DR, immobilized mAb anti-HLA-DR, and PMA. In addition to the augmentation of phosphorylation observed with regard to phosphoproteins already present in resting B lymphocytes, new phosphorylations were observed after stimulation by any one of the reagents. Furthermore, stimulation by PMA did not result in an identical pattern to that observed after stimulation by mAb anti-HLA class II. An inhibition of the proliferative response to PMA was demonstrated after prestimulation of cells with immobilized mAb anti-HLA-DR, supporting the notion of a shared pathway of activation. In summary, these data demonstrate signal transduction via MHC class II Ag as assessed by three different measures of early events in human B lymphocyte activation and suggest that a protein kinase C pathway is at least partly involved.