Role of calcium and magnesium and of cytochalasin-sensitive processes in lectin-stimulated lymphocyte activation

We have investigated possible interactions of divalent-cation-requiring processes and cytochalasin-sensitive processes in the early events of lymphocyte activation. In lectin-stimulated responses, Ca and Mg were synergistic in support of protein and DNA synthesis, as expressed in the increased requirement for one ion under conditions of depletion of the other. Experiments evaluating the temporal relationships between cytochalasin sensitivity and requirements for Ca and Mg demonstrated that both cations were required before or during the early cytochalasin B-sensitive events. Furthermore, we observed that Ca and Mg and cytochalasin B-sensitive processes were required for lectin-dependent commitment to DNA synthesis. Additional studies comparing the relative potencies of cytochalasin E, D, and B suggested that the probable target for cytochalasins in the inhibition of commitment was a motility-related process. These data demonstrate an early period of activation which can be characterized by its requirement for divalent cations and its sensitivity to cytochalasins.     

Physiology of mixed leukocyte reaction suppressor factor. I. Role of cytoskeleton and protein synthesis in production and secretion.

The secretory physiology of the T cell-produced lymphokine, mixed leukocyte rection suppressor factor (MLR-TsF), was characterized with respect to its kinetics of secretion and its sensitivity ot a variety of metabolic blocking agents. It was found that spleen cells from alloantigen-immunized mice released active MLR-TsF after freeze-thaw lysis. Upon restimulation with the same priming alloantigen, MLR-TsF was secreted into culture supernatants, and the rate of secretion was determined to be nearly constant. Although colchicine and vinblastine, which bind to microtubules, are known inhibitors of lectin-induced proliferation, it was demonstrated that these drugs had no effect on the secretion of MLR-TsF. However, cytochalasin B, an inhibitor which also binds to some cytoskeletal and membrane-associated proteins, did inhibit the production of MLR-TsF. The above findings indicated that the activation-secretion mechanism of of MLR-TsF was much like that described for lymphotoxin and macrophage migration inhibition factors. The dissociation between DNA synthesis and lymphokine secretion was also demonstrated in the MLR-TsF system. DNA synthesis plays no role in the in vitro production of suppressor factor, as determined by resistance to treatment with mitomycin C and gamma-irradiation. However, new protein synthesis is required as indicated by the potent inhibitory effects of cycloheximide. Experiments utilizing timed addition and removal of cycloheximide defined a broad period of drug sensitivity, starting from the beginning of culture and lasting for 12 to 16 hr. In addition, experiments measuring the effect of cycloheximide on the MLR-TsF content of cell lysates demonstrated that the cell-associated lymphokine activity is lost when protein synthesis is interrupted. These experiments support the conclusion that MLR-TsF is synthesized de novo in culture. In addition, the secretory process itself may require protein synthesis.     

Suppressor T cell growth and differentiation: production of suppressor T cell differentiation factor by the murine thymoma BW5147

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

Cellular and antigenic requirements for production of mixed leukocyte reaction suppressor factor

When murine spleen cells, alloantigen-sensitized previously in vivo, are incubated with spleen cells bearing the sensitizing alloantigens, a supernatant factor is produced that inhibits 3H-thymidine incorporation by responding lymphocytes in the mixed leukocyte reaction. This study evaluates the cellular and antigenic requirements during restimulation for elaboration of this suppressor factor, MLR-TsF. BALB/c spleen cells, sensitized to C57BL/6 (B6) alloantigens in vivo, produced MLR-TsF when cultured with B6 spleen cells in vitro, despite depletion of Sephadex G-10-adherent cells from factor-producing cells, stimulator cells, or from both populations. T cells were not required within the stimulating population, but a requirement for viable stimulator cells was demonstrated when heat-killed or glutaraldehyde-fixed stimulator cells failed to induce MLR-TsF production. The alloantigenic requirements for MLR-TsF production were addressed by 2 approaches. Treatment of stimulator cells with appropriate anti-I region antisera and complement did not affect MLR-TsF production, demonstrating that an absolute requirement for cells expressing I region determinants did not exist. However, spleen cells primed against entire H-2 haplotype differences produced significant quantities of MLR-TsF when they were restimulated with spleen cells homologous to the priming cells in only the I region, in the K and D regions, or in the D region alone. The additive nature of subregion-specific restimulation suggests that distinct subpopulations of K, I, and D region-specific MLR-Ts comprise the MLR-Ts population primed to entire H-2 haplotype differences.     

Activation of pig oocytes by intracytoplasmic injection of strontium and barium

Ovulated mouse oocytes are activated by exposure to culture medium containing Sr2+ or Ba2+ or by intracytoplasmic injection of the divalent cations. It is known that in vitro matured pig oocytes are activated by the intracytoplasmic injection of Ca2+. In this study, we examined the effect of exposure and of intracytoplasmic injection of Sr2+ or Ba2+ on in vitro matured pig oocytes (MII-oocytes). When MII-oocytes were exposed to the medium containing divalent cations, no oocytes were activated. However, in the case of oocytes that were injected with Sr2+, Ba2+ and Ca2+, at 6 h after injection, 64%, 71% and 86% of the oocytes had been released from MII-arrest, and 51%, 67% and 84% formed female pronuclei, respectively. The initial transient in intracellular Ca2+ concentration ([Ca2+]i) was measured by the Ca2+ indicator dye fluo-4 dextran. Microinjection of Sr2+, Ba2+ or Ca2+ induced a rapid elevation of [Ca2+]i. The exocytosis of cortical granules was examined by staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin. After an injection of divalent cations, a release of cortical granules was observed in the oocytes. Maturation promoting factor (MPF) activity declined to a low level after 6 h in all the oocytes injected with divalent cations. To test their developmental ability, injected oocytes were treated with cytochalasin B and then cultured for 168 h in NCSU23 medium. After 168 h, 29% (Sr2+), 29% (Ba2+) and 51% (Ca2+) of the oocytes had developed to the blastocyst stage. These results indicate that intracytoplasmic injection of Sr2+ and Ba2+, like that of Ca2+, induces in vitro matured pig oocytes to be released from MII-arrest and leads them into a series of events related to oocyte activation.     

Changes in ionic movements across rabbit polymorphonuclear leukocyte membranes during lysosomal enzyme release. Possible ionic basis for lysosomal enzyme release.

Changes in the movements of Na+, K+, and Ca+2 across rabbit neutrophils under conditions of lysosomal enzyme release have been studied. We have found that in the presence of cytochalasin B, the chemotactic factor formyl methionyl leucyl phenylalanine (FMLP) induces within 30 s large enhancements in the influxes of both 22Na+ and 45Ca+2 and an increase in the cellular pool of exchangeable calcium. The magnitude of the changes induced by cytochalasin B and FMLP exceeds that induced by FMLP or cytochalasin B alone, and cannot be explained on the basis of an additive effect of the two agents. However, these compounds either separately or together produce much smaller enhancements in 45Ca efflux. The divalent cation ionophore A23187 also produces a rapid and large increase in the influxes of both 22Na and 45Ca+2 in the presence and absence of cytochalasin B. We have also found an excellent correlation between calcium influx and lysosomal enzyme release. 42K influx is not significantly affected by any of these compounds. On the other hand, a large and rapid increase of 42K efflux is observed under conditions which give rise to lysosomal enzyme release. A flow diagram of the events that are thought to accompany the stimulation of polymorphonuclear leukocytes (PMNs) by chemotactic or degranulating stimuli is presented.     

Inhibition by phorbol esters of antiimmunoglobulin-induced calcium signalling and B-cell activation

The inhibitory effect of phorbol dibutyrate (PDB) on B-cell stimulation was evaluated using a model in which activation is induced by modest doses of antiimmunoglobulin antibody (anti-Ig) and progression to DNA synthesis is induced by cytochalasin. PDB preferentially inhibited anti-Ig-induced activation and did so during brief (2 hr) preincubation with anti-Ig. Activation was inhibited whether PDB was added before or shortly after anti-Ig. Since activation for cytochalasin responsiveness appears to be mediated by Ca2+, the effect of PDB on the anti-Ig-induced rise in intracellular Ca2+ was evaluated. PDB (and other phorbol esters that activate protein kinase C) inhibited the rise in Ca2+ normally associated with anti-Ig treatment; moreover, PDB reversed an established anti-Ig-induced Ca2+ response. These data suggest that phorbol esters inhibit B-cell activation by interfering with the elevated levels of intracellular Ca2+ produced by cross-linking of surface immunoglobulin by anti-Ig. This could represent a "feedback inhibition" type of response, but it remains to be seen if this occurs under physiological conditions of protein kinase C activation.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

PGBX, a prostagandin derivative, mimics the action of the calcium ionophore A23187 on human neutrophils

The interactions have been studied of a water-soluble, polymeric derivative of prostaglandin B1, PGBX, with human polymorphonuclear leukocytes (PMN). PGBX, which is a potent ionophore of divalent cations, provoked superoxide anion (O2.-) generation and lysosomal enzyme release in cytochalasin B-treated PMN in the presence of extracellular divalent cations (Ca2+, Sr2+, Mg2+, Mn2+, Ba2+). Kinetic and dose-response studies showed that PGBX mimicked te action of ionophore A23187 in PMN. Both ionophores induced superoxide generation and release of enzymes from specific and azurophil granules (lysozyme > beta-glucuronidase) without provoking release of the cytoplasmic marker enzyme lactic dehydrogenase. In contrast, the precursor of PGBX, prostaglandin B1 (PGB1), and arachidonate did not mimic ionophore-induced stimulation of PMN. PGBX induced enzyme release both in the presence of extracellular Ca2+ and Ba2+ (both of which it translocates in model liposomes), whereas A23187 showed specificity for Ca2+ (which it translocates preferentially over Ba2+). These studies indicate that the actions of a water-soluble polymer (PGBX) derived from a naturally occurring prostaglandin (PGB1) on human neutrophils resemble those of a classical ionophore (A23187). Moreover, they provide additional evidence that increments in the intracellular levels of divalent cations may signal stimulus-secretion coupling in human neutrophils.     

Immunoregulatory factors from a human macrophage-like cell line. II. A human T-cell lymphokine-induced suppressor factor for lymphocyte proliferation

It has been demonstrated that the human histiocytic lymphoma-derived cell line U937, which has monocytoid characteristics, responds to a concanavalin A-induced T-cell-derived suppressor supernatant (T-SFS) with the release of a factor markedly suppressing mitogen-stimulated proliferation of normal peripheral blood lymphocytes. The suppressor material is not dialyzable, appears within 2 hr of exposure of U937 cells to the T-SFS, persists for at least 24 hr, and has a Mr of approximately 40,000 by gel chromatography. The suppressor factor does not affect the proliferation of continuous T- and B-lymphoid cell lines, distinguishing it from the inhibitor of DNA synthesis also released by U937, but appears to be specific for a stage of activation of normal lymphocytes that is independent of (a) utilization of interleukin-2 and (b) inhibition of production of interleukin-2.     

Induction of suppression by a murine nonspecific suppressor-inducer cell line (M1-A5)

A murine nonspecific suppressor-inducer cell line (M1-A5) was established by the limiting dilution method from the spleen cells of a mouse bearing an advanced methylcholanthrene-induced fibrosarcoma. Indirect immunofluorescence studies demonstrated that the M1-A5 cells were Thy-1-, sIg-, Ly-5+, MAC-, and 45% asialo GM1+. The M1-A5 cells were able to activate suppressor cells from unprimed, syngeneic normal spleen cells. These activated cells inhibited antibody production by cocultured syngeneic lymphoid cells. Induction of suppression by the M1-A5 cells was via the release of a suppressor-inducing factor, which was found to be protein in nature. Kinetic studies showed that when M1-A5 cells were separated from NSC by a dialysis tubing in Marbrook vessels, the M1-A5 cells required a minimum of 8 hr incubation period before suppressor cell activity could be demonstrated in precursor cells. On the other hand, induction of suppression by the suppressor-inducing factor required a minimum of 3 hr exposure of the precursor cells to the factor.     

Regulation of magnesium-inhibited cation current by actin cytoskeleton rearrangement

Magnesium-inhibited, non-selective cation current (I(MIC)) is activated by depletion of intracellular Mg(2+) and ATP. I(MIC) transports various divalent cations including Mg(2+) and Ca(2+), and is involved in cell viability. We investigated the effect of actin dynamics on I(MIC). Formation of a stable cortical actin network by calyculin A inhibited the activation of I(MIC), while the actin depolymerizing reagent, cytochalasin D, reversed the inhibition. Induction of a dense cortical actin layer by transfecting the constitutively active form of RhoA also inhibited the activation of I(MIC). These results suggest that the activation of I(MIC) may be dynamically regulated by actin cytoskeleton rearrangement.     

Inhibitory activity of interleukin B on the suppressor T cell hybrid T2D4

Interleukin B (IL-B), a product of unstimulated B cells, is defined by its ability to selectively prevent the differentiation of suppressor T lymphocytes from precursors into effectors. The present study was undertaken to determine whether IL-B could also be active in modulating the activity of the T cell hybrid T2D4, which produces immunoglobulin-binding suppressor factors. T2D4 cells can be selectively induced by incubation with various isotypes of antibody to express isotype-specific Fc receptors and to release soluble factors that suppress production of the corresponding isotype. The data presented here demonstrate that IL-B is greatly effective in inhibiting T2D4 activities. Either pretreatment with IL-B or continuous exposure to IL-B prevents isotype activation of T2D4. As a result, T2D4 cells do not express isotype receptors and do not produce detectable amounts of isotype-specific suppressor factors. This IL-B regulatory activity on T2D4 is temperature dependent and is inhibited by cytochalasin B. These findings provide new insights on the mechanism by which IL-B enhances antibody responses, and they offer a conceptual framework for analyzing IL-B activity on suppressor T cells.     

Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration

T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca(2+) ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca(2+)-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.     

Production of platelet-activating factor by stimulated human polymorphonuclear leukocytes. Correlation of synthesis with release, functional events, and leukotriene B4 metabolism

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation that is synthesized by several human cell types including polymorphonuclear leukocytes (PMN). We examined the synthesis and release of PAF by stimulated human PMN under several conditions, assayed by the incorporation of [3H]acetate into PAF and by bioassay. PAF synthesis was induced by calcium ionophore A23187 (IoA), opsonized zymosan (OpsZ), and N-formyl-methionyl-leucyl-phenylalanine (FMLP) with the relative order of potency IoA much greater than OpsZ greater than FMLP. A variety of other agonists, including phorbol myristate acetate, an activator of protein kinase C and of PMN functional responses, did not stimulate PAF synthesis. PAF synthesis by PMN in response to IoA, OpsZ, and FMLP was concentration- and time-dependent but release of the phospholipid was not: little PAF (1 to 10%) was released from PMN in suspension regardless of the total amount produced, the agonist, its concentration, the time of incubation, or the concentration of extracellular albumin. This was also the case with functionally altered neutrophils that had been "primed" with cytochalasin B or lipopolysaccharide or that had adhered to surfaces. PAF synthesis was tightly coupled with leukotriene B4 production by adherent PMN as well as by neutrophils in suspension, supporting the hypothesis that the two lipid autacoids may be derived from a common precursor. However, PAF synthesis could be dissociated from aggregation and surface adhesion, indicating that it is not absolutely required for these responses of activated PMN. The total amount of PAF that accumulated, but not the percentage that was released, was altered in adherent PMN compared to cells in suspension. These experiments demonstrate that PAF production and its subsequent processing by human neutrophils are highly regulated events. PAF synthesis is associated with PMN activation, but it is not a requisite for early adhesive responses of neutrophils. Because little of the PAF produced by stimulated PMN is released from the cells, it appears that PAF has an intracellular role in PMN function and/or that it may have novel intercellular effects that do not require release into the fluid phase.     

Platelet-derived growth factor stimulates phospholipase C-gamma 1, extracellular signal-regulated kinase, and arachidonic acid release in rat myometrial cells: contribution to cyclic 3',5'-adenosine monophosphate production and effect on cell proliferation

In the present study, we examined downstream signaling events that followed exposure of cultured rat myometrial cells to platelet-derived growth factor (PDGF) and their effect on cell proliferation. PDGF-BB induced tyrosine phosphorylation of PDGF-beta receptors and increased inositol trisphosphate production via the tyrosine phosphorylation of phospholipase (PL)C-gamma 1. PDGF-BB also increased cAMP synthesis. This increase was potentiated by forskolin and reduced by indomethacin, a cyclooxygenase inhibitor, reflecting a Gs protein-mediated process via prostaglandin biosynthesis. The prostaglandin produced by PDGF was characterized as prostacyclin (PGI(2)). PDGF-BB increased arachidonic acid (AA) release, which, similarly to cAMP accumulation, was abolished in the presence of AACOCF3, a cytosolic PLA(2) inhibitor, and in the absence of Ca(2+). U-73122, a potent inhibitor of PLC activity, blocked both the production of inositol phosphates and the AA release triggered by PDGF-BB. Extracellular signal-regulated kinases (ERKs) 1 and 2 are expressed in myometrial cells, and PDGF-BB selectively activated ERK2. PD98059, an inhibitor of the ERK-activating kinase, blocked PDGF-BB-mediated ERK2 activation, AA release, and cAMP production. The results demonstrate that PDGF-BB stimulated cAMP formation through both PLC activation and ERK-dependent AA release and PGI(2) biosynthesis. PDGF-BB also increased cell proliferation and [(3)H]thymidine incorporation. This was abolished by PD98059, demonstrating that the ERK cascade is required for the mitogenic effect of PDGF-BB. Forskolin, which potentiated the cAMP response to PDGF-BB, attenuated both DNA synthesis and ERK activation triggered by PDGF-BB, suggesting the presence of a negative feedback regulation.     

Cytochalasin B-sensitive, sodium ion-dependent glucose transport in intestinal microvillous membrane

It was found that sodium ion-dependent glucose uptake by microvillous membrane (MVM) vesicles was partially inhibited by cytochalasin B with a half-maximum inhibition at ca. 10 microM. The MVM was photolabeled with [3]cytochalasin B. The Kd value and the maximum number of binding sites for cytochalasin B were ca. 8 microM and 70 pmol/mg protein, respectively. SDS-PAGE of the photolabeled MVM revealed 2 binding components. One was 86 K in Mr and the other 42 K. The binding of cytochalasin B to the 86 K component was affected neither by cytochalasin E nor by the presence of 0.5 M NaCl, but was depressed in the presence of 2-deoxy-D-glucose or phlorizin, which had no effect on the labeling of the 42 K component. These and other data suggested that the 86 K component might be responsible for a cytochalasin B-sensitive glucose transport in intestinal epithelial MVM.     

Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.     

Dissociation between aggregation and superoxide production in human granulocytes

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.