Properties of prostaglandin-sensitive adenylate cyclase system of a murine macrophage-like cell line (P388D1)

The properties of basal and prostaglandin (PG)-stimulated adenylate cyclase of membrane preparations of P388D1 cells were investigated. Three partially purified membrane fractions were obtained by sucrose density gradient centrifugation at the final step of purification from crude homogenate. About 96% of the basal and 89% of PGE2-stimulated adenylate cyclase activity in the homogenate were recovered in three membrane fractions. Two lighter membrane fractions (I and II), which were enriched 11-fold and 8.4-fold in adenylate cyclase activity over crude homogenate, were pooled and subjected to various studies. Results suggested that the basal activity of the membrane preparations has, as in many other cell types, a relatively broad pH optimum (pH 7.5 to 8.5), requires Mg2+, which must be present in excess ATP, and is inhibited by Ca2+. Highly reactive sulfhydryl group(s), which may be present in the lipid bilayer, is required for the adenylate cyclase activity. Because both fluoride ions and GTP augment the enzymatic activity, P388D1 cell membrane adenylate cyclase must possess stimulatory guanine nucleotide-binding protein. The membrane preparations respond to exogeneously added PG by 1.5-fold to 3-fold increase in adenosine 3'-5' cyclic monophosphate (cAMP) production. The magnitude of PG-responsiveness was dependent on the types of PG and the order of potency in stimulation was PGE1 greater than PGE2 greater than PGI2. PGA1, B1, B2, F1 alpha, and F2 alpha stimulated adenylate cyclase only at the highest concentration tested.     

Interferon-γ down-regulates membrane adenylate cyclase activity of a murine macrophage-like cell line (P388D1)

Effects of recombinant murine interferon-γ (rIFN-γ) on the membrane adenylate cyclase of a murine macrophage cell line (P388D₁) were investigated in order to explore the nature of a signal transmitted by IFN-γ receptor. Following the incubation of P388D₁ cells with 40 U/ml of rIFN-γ, the intracellular level of cAMP gradually increased about twofold over the control level within 60 min, and then began to gradually decline to about half the control level by 24 h incubation. The initial rise in cAMP level appeared to be due to the modest activation of adenylate cyclase and not due to the inhibition of cAMP-phosphodiesterase. Later decrease of intracellular cAMP may be due to quantitative down-regulation of the adenylate cyclase system. The basal enzymatic activity of the membrane prepared from P388D₁ cells exposed to IFN-γ for 24 h was found to be reduced to about 20% of that of the control membrane. However, the quality of the adenylate cyclase system appeared unchanged, because the relative degree of the response of the down-regulated membrane adenylate cyclase to prostaglandin PGE₂, NaF, guanylimidodiphosphate (GppNHp), cholera toxin (CT), or forskolin was found to remain unchanged. This quantitative down-regulation of adenylate cyclase must be due to the action of rIFN-γ, since the prior treatment of rIFN-γ with either acid (pH 2) or monoclonal anti-IFN-γ antibody inhibited the ability of IFN-γ to induce the down-regulation. The rIFN-γ-induced down-regulation is a reversible process, since the adenylate cyclase activity of the membrane was found to be restored when the rIFN-γ-exposed cells were cultured for 72 h in the absence of rIFN-γ. In addition, the 48 h-incubation of P388D₁ cells with rIFN-β or IFN-α was found not to significantly affect the membrane adenylate cyclase system.     

PGE2-induced desensitization of adenylate cyclase of a murine macrophage-like cell line (P388D1)

The mode of PGE2-induced desensitization of the adenylate cyclase of a murine macrophage-like cell line, P388D1 was investigated. The exposure of cells to PGE2 for 60 min induced PGE2-specific desensitization of the adenylate cyclase system which still responded normally to other specific ligand such as isoproterenol, 5'-guanylimidodiphosphate (Gpp(NH)p), or forskolin. The exposure of the cells to PGE2 for 6 hr induced heterologous desensitization, as the responses of adenylate cyclase to PGE2 as well as to isoproterenol or Gpp(NH)p were significantly reduced. The lowest concentration of PGE2 to induce both early homologous and late heterologous desensitization was found to be about two-fold over the KD of the low affinity PGE2-binding sites of P388D1 cells. The early homologous desensitization appeared to be due in part to the reduction in number of PGE2 receptors from the cell surface. The late heterologous desensitization may involve functional and/or structural alteration of Gs proteins, in addition to the reduction of PGE2 receptors from the cell surface.     

Prostaglandin-sensitive adenylate cyclase of a murine macrophage-like cell line (P388D1): II. Isolation and partial characterization of PGE2-binding proteins

Properties of prostaglandin (PG) E2 binding sites of a murine macrophage cell line (P388D1) were investigated. The specific binding of [3H]-PGE2 to intact P388D1 cells at 4 degrees C in the presence of cytochalasin D (10 micrograms/ml) approached saturation at concentration greater than 7.5 X 10(-9) M, and could be displaced most effectively by unlabeled PGE2 and less effectively by unlabeled PGI2. The Scatchard analysis of the binding data clearly indicated the heterogeneity with respect to the PGE2 binding affinity and showed the presence of about 3.9 fmol/10(8) cells of the high affinity sites (KD = 1.1 X 10(-9) M) and about 24 fmol/10(8) cells of the low affinity sites (KD = 2 X 10(-8) M). PGE2-binding proteins were isolated from the detergent lysate of the radiolabeled P388D1 cells by affinity chromatography on Sepharose 4B coupled to PGE2. The affinity-isolated materials were further purified by successive use of Sephadex G-100 gel filtration and isoelectric focusing in the presence of dithiothreitol (1 mM) and Triton X-100 (0.5%). The final step yielded about 0.25% of the original radioactivity, which sharply focused as a single peak at pH near 6.5. The electrofocused PGE2-binding proteins migrated as a single band with a m.w. of 95,000 during SDS-PAGE. The electrofocused PGE2-binding proteins bound specifically to [3H]-PGE2 but showed again the heterogeneity with respect to their affinity.     

Phospholipase activities of the P388D1 macrophage-like cell line

The murine macrophage (M phi) cell line, P388D1, was employed as a source of M phi phospholipases in order to characterize the enzymatic properties and subcellular localization of these enzymes because of their importance for prostaglandin biosynthesis. Phospholipase activity was assessed with dipalmitoylphosphatidylcholine (DPPC) as substrate. Phospholipases were characterized with respect to divalent cation dependence, pH optima, and localization in subcellular compartments using linear sucrose gradients. By these criteria a number of different phospholipases were identified. Most importantly, a single Ca2+-dependent activity with a pH optimum of 8.8 was identified in membrane-rich fractions (plasma membrane, mitochondria, and endoplasmic reticulum) and could be clearly separated from the remaining activities, which are Ca2+ independent and exhibit pH optima of 7.5, 5.1, and 4.2. The phospholipases with acidic pH optima may be associated with subcellular components containing lysosomal enzymes and both phospholipase A1 and phospholipase A2 activities are observed. In contrast, the phospholipase activity with a pH optimum of 7.5 sediments with the cytosolic proteins and is inhibited by 5 mM Ca2+. No significant phospholipase C activity was detected in assays performed with or without added Ca2+ at pH's 4.2, 5.1, 7.5, or 8.8 using DPPC as substrate. However, the P388D1 cells do contain a lysophospholipase that is at least 20 times more active than the phospholipase A activities identified. Its presence must be taken into account in evaluating the positional specificities and properties of the macrophage phospholipases.     

Relationship between Fc gamma 2b receptor and adenylate cyclase of a murine macrophagelike cell line, P388D1

The relationship between Fc receptor specific for IgG2b (Fc gamma 2bR) and membrane adenylate cyclase was investigated. The specific binding of IgG2b immune complexes to P388D1 cell surface Fc gamma 2bR was found to inhibit the basal, forskolin-stimulated, and NaF-stimulated activities of membrane adenylate cyclase by 53%, 57%, and 31%, respectively. On the other hand, the binding of IgG2a immune complexes to cell surface Fc gamma 2aR increased the basal activity about 2.5-fold and the forskolin- and NaF-stimulated activities slightly. The fusion of liposomes containing Fc gamma 2bR, which was obtained as phosphatidylcholine (PC) binding protein as previously described, with the cyc- membrane preparations resulted in the marked suppression of membrane adenylate cyclase, whereas the fusion of liposomes containing Fc gamma 2a, which was obtained as IgG-binding protein, led to about a 2.7-fold increase. The Fc gamma 2bR-mediated inhibition of adenylate cyclase may be due to the temporary change of the lipid environment caused by the action of phospholipase A2, which was previously shown to be associated with Fc gamma 2bR, since (1) addition of snake venom phospholipase A2 or cholate-solubilized PC-binding protein to P388D1 membrane was found to inhibit adenylate cyclase in a dose-dependent manner, (2) prior treatment of snake venom phospholipase A2 or PC-binding protein with a specific inhibitor, p-bromophenacyl bromide, significantly reduced their inhibitory action, and (3) a product of phospholipase A2 action, arachidonic acid, was found to be an effective inhibitor of membrane adenylate cyclase, whereas the other product, lysophosphatidylcholine, was much less inhibitory than arachidonic acid. Arachidonic acid appeared to interfere with the functions of both guanine nucleotide-binding stimulatory (Gs) protein and the catalytic subunit of adenylate cyclase, since exogenously added arachidonic acid significantly suppressed the GTPase activity of P388D1 membrane and the forskolin response of the adenylate cyclase activity of Gs protein deficient cyc- membrane. The primary site of action of lysophosphatidylcholine is not clear but may be other than Gs protein and/or the catalytic subunit, since it did not change either GTPase activity of P388D1 membrane or the response to forskolin of adenylate cyclase of cyc- membrane. The Fc gamma 2bR/phospholipase A2 mediated inhibition of adenylate cyclase would be a transient event in viable cells, since phospholipase A2 did not inhibit adenylate cyclase in the presence of microsomal fraction, mitochondria, and coenzyme A, suggesting the occurrence of rapid acylation of CoA and reacylation of lysolecithin.     

Isolation of an Fcgamma-binding protein from the cell membrane of a macrophage-like cell line (P388D1) after detergent solubilization

Lactoperoxidase-catalyzed iodination, NP-40 lysis, and subsequent affinity chromatography on IgG-Sepharose were used in an attempt to define some of the molecular properties of the Fc receptor of P388D1, a macrophage-like mouse tumor line. Radioiodinated material retained on columns of Sepharose coupled either to monomeric mouse IgG2a or monomeric human IgG1 appeared on SDS polyacrylamide gel electrophoresis to contain principally three labeled components, a major band of about 57,000 m.w. and two minor bands of 28,000 and 24,000 m.w. The mobilities of these components changed little on reduction, which suggested that they represented single polypeptide chains, An identical pattern was obtained with Sepharose-linked Fc fragments of human IgG1, but neither Fab fragments of IgG1 nor IgM appeared to bind these components. Since the specificity of binding to the immobilized proteins is the same as that observed in vivo, it is postulated that these proteins represent either all or some portion of the P388D1 Fc receptor.     

Purification and characterization of a lysophospholipase from a macrophage-like cell line P388D1

Two lysophospholipase activities (designated I and II) were identified in the macrophage-like cell line P388D1. Lysophospholipase I was purified (8,500-fold) to homogeneity by DEAE-Sephacel, Sephadex G-75, Blue-Sepharose, and chromatofocusing chromatography. Lysophospholipase II was separated from the lysophospholipase I in the Blue-Sepharose step. The apparent molecular mass of lysophospholipase I and II are 27,000 and 28,000 daltons, respectively, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their pI values were 4.4 and 6.1 respectively, as determined by isoelectric focusing. Lysophospholipase I exhibited a broad pH optimum between 7.5-9.0. The double-reciprocal plot of the substrate dependence curve of the purified lysophospholipase I showed a break around the critical micelle concentration of the substrate (1-palmitoyl-sn-glycerol-3-phosphorylcholine). The apparent Km, determined from substrate concentrations above 10 microM was 22 microM, and the apparent Vmax was 1.3 mumol min-1mg-1. The purified enzyme did not have phospholipase A1, phospholipase A2, acyltransferase, or lysophospholipase-transacylase activity. No activity was detected toward triacylglycerol, diacylglycerol, p-nitrophenol acetate, p-nitrophenol palmitate, or cholesterol ester. The enzyme did, however, hydrolyze monoacylglycerol although at a rate 20-fold less than lysophospholipid, 0.06 mumol min-1mg-1. The lysophospholipase I was inhibited by fatty acids but not by glycerol-3-phosphorylcholine, glycerol-3-phosphorylethanolamine, or glyc-fjerol-3-phosphorylserine. A synthetic manoalide analogue 3(cis,cis,-7,10)hexadecadienyl-4-hydroxy-2-butenolide inhibited the enzyme with half-inhibition (IC50) at about 160 microM. Triton X-100 decreased the enzymatic activity, although this apparent inhibition can be explained by a "surface dilution" effect. The pure lysophospholipase I was stable for at least 5 months at -20 degrees C in the presence of glycerol and beta-mercaptoethanol. Lysophospholipid also demonstrated a protective effect during the later stage of purification.     

Prostaglandin-E2-induced activation of adenosine 3'-5' cyclic monophosphate-dependent protein kinases of a murine macrophage-like cell line (P388D1)

Changes in the activities of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinases in response to prostaglandin (PG)E2-induced elevation of intracellular cAMP level were investigated with a murine macrophage-like cell line, P388D1. Photoaffinity labeling with 8-azido-[32P]cAMP showed that untreated P388D1 cells possess two types of cAMP-binding proteins of m.w. 49,000 and 52,000, respectively, in the cytosol fraction in a ration of 1:8. They must represent regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinases, because affinity chromatography on a column of omega-aminohexyl-agarose of the cytosol fraction clearly separated two fractions that exhibited the enzymatic activities and cAMP-binding activities. Photoaffinity labeling of these fractions with 8-azido-[32P]cAMP confirmed the separation of two types of isoenzymes, because each cAMP-dependent protein kinase active fraction was associated with only one type of regulatory subunit. The exposure of P388D1 cells to exogenously added PGE2 (1 microM) caused about 7.5-fold increase in the intracellular cAMP level within 30 sec. The cAMP level then sharply dropped to about 100 pmol/10(7) cells, remained at this level for about 20 min, and then gradually increased to 200 pmol/10(7) (about fivefold over the control). The enzyme assay of the cytosol demonstrated that the activation of cAMP-dependent protein kinases closely follows the kinetics of the intracellular cAMP level. The activation of the enzyme was specific for PGE2 and was not triggered by 1 microM PGF2 alpha or PGD2 which have been shown to be unable to activate adenylate cyclase of P388D1 cells. The PGE2-induced increase in the intracellular cAMP level appeared to activate preferentially the type I isoenzyme, inasmuch as the enzymatic activity of this type separated by the affinity chromatography of the cytosol of PGE2-exposed cells was lower in the presence than in the absence of cAMP, whereas the type II enzyme activity remained responsive to exogenously added cAMP.     

Biochemical signal transmitted by Fc receptor for immunoglobulin G2a of a murine macrophage-like cell line, P388D1: mode of activation of adenylate cyclase mediated by immunoglobulin G2a binding proteins

The effects of immunoglobulin G2a binding proteins isolated from P388D1 cells on adenylate cyclase of cyc- cells were investigated to explore a potential role of Fc gamma 2a receptor in the activation of the adenylate cyclase system. Immunoglobulin G (IgG) binding proteins obtained from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose were separated into two fractions (denoted as IgG-B1 and IgG-B2) by Sephadex G-100 gel filtration in the presence of 6 M urea. Polyacrylamide gel electrophoretic analysis in the presence of sodium dodecyl sulfate revealed that the major component in the IgG-B1 fraction was a protein of molecular weight near 50 000, whereas the IgG-B2 fraction contained two major components of molecular weight near 25 000 and 17 000. Both IgG-B1 and -B2 proteins can be inserted into liposome consisting of phosphatidylcholine and phosphatidylethanolamine. Liposomes containing IgG-B1 proteins effectively inhibited EA2a, but not EA2b, rosetting by either S49 or P388D1 cells, suggesting their proper orientation within liposome, whereas IgG-B2-containing liposome failed to do so. Simultaneous fusion of the liposomes containing IgG-B1 and -B2 proteins with guanine nucleotide binding stimulatory (G/F) protein/Fc gamma 2aR-deficient cyc- cells resulted in the formation of the hybrid membrane whose adenylate cyclase responds to immune complex formed with IgG2a-subclass antibody (IC2a) by about a 2.7-fold increase in the activity over the control (hybrid membrane between cyc- cells and liposome containing no protein). The response appeared to be specific, since IC2b failed to stimulate the enzymatic activity of this hybrid membrane. Furthermore, IgG-B1 and -B2 proteins were able to confer their activating effects on the enzyme only in concert, since the fusion of liposomes containing either type of protein alone with cyc- cells did not result in the activation of adenylate cyclase of cyc- membrane. IgG-B1 and -B2 proteins could also confer their activating effects in concert to the enzyme in cholate-solubilized forms. Such activation was dependent on the concentration of IC2a, suppressed by the chelating agent ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and significantly inhibited by trifluoperazine, suggesting potential involvement of Ca2+ and calmodulin in the activating process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fc gamma 2b receptor-mediated prostaglandin synthesis by a murine macrophage cell line (P388D1)

The nature of signals transmitted by two types of Fc gamma receptors (one specific for IgG2b and the other for IgG2a) present on the surface of a murine macrophage cell line (P388D1) was investigated. Specific binding of IgG2b (presented as EA2b) to cell surface Fc gamma 2br triggered the release of 3H-arachidonic acid and 3H-prostaglandins (PG) from P388D1 cells that were prelabeled with 3H-arachidonate. The release of 3H-arachidonic acid, which increased in a dose-dependent manner, was enhanced by exogenous Ca++ (1.25 mM) and was completely blocked by ethylenediaminetetraacetate (EDTA) (4 mM) or a phospholipase A2 inhibitor, p-bromophenacylbromide (7 microgram/ml). A cyclooxygenase inhibitor, indomethacin (9 microgram/ml), reduced the 3H-arachidonic acid release and completely blocked the conversion of arachidonate into PG. Cytochalasin D (1 microgram/ml), which inhibited the phagocytosis of immune complexes by 90% of P388D1 cells, did not affect the Fc gamma 2bR-triggered release of arachidonic acid. Specific binding of IgG2a (presented as EA2a) to cell surface Fc gamma 2aR did not trigger the release of either 3H-arachidonic acid or 3H-PG from P388D1 cells. Our data demonstrate a signal for the activation of the arachidonic acid metabolic cascade is transmitted by Fc gamma 2bR, but not by Fc gamma 2aR, on the surface of P388D1 cells, probably through the initial activation of the phospholipase A2 activity associated with Fc gamma 2bR.     

Fc gamma receptor-mediated suppression of gamma-interferon-induced Ia antigen expression on a murine macrophage-like cell line (P338D1)

A variant (HS-1) of a murine macrophage cell line (P388D1) was obtained by cell cluster technique based on the Ia antigen expression induced by lymphokines. Receptors for both IgG2a and IgG2b but no detectable I-Ad are expressed on the surface of the majority of HS-1 cells. Exposure of HS-1 cell to concanavalin A supernatant or recombinant IFN-gamma resulted in the induction of I-Ad antigens on greater than 90% of the cells within 48 hr. The effects of lymphokines were transient and dependent on the synthesis of messenger RNA because the removal of lymphokines or the presence of actinomycin D both blocked Ia expression. The prior or simultaneous binding of monoclonal IgG2a or IgG2b antibodies complexed with sheep erythrocytes to respective cell surface Fc gamma R suppressed the Ia antigen inducing activity of lymphokines. Neither antibody nor antigen alone could suppress the effect of lymphokines. Inhibitors of phospholipase A2 or cyclooxygenase, which have been shown previously to suppress Fc gamma 2bR, but not Fc gamma 2aR, triggered activation of the adenylate cyclase system and reversed Fc gamma 2bR- but not Fc gamma 2aR-mediated suppression of IFN-gamma-induced Ia antigen expression.     

Protein kinase activity associated with Fc gamma 2a receptor of a murine macrophage like cell line, P388D1

The properties of protein kinase activity associated with Fc receptor specific for IgG2a (Fc gamma 2aR) of a murine macrophage like cell line, P388D1, were investigated. IgG2a-binding protein isolated from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose was found to contain four distinct proteins of Mr 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [gamma-32P]ATP. The autophosphorylation of Fc gamma 2a receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Casein was found to be a much better phosphate acceptor than histone in this system, as casein incorporated about 32-fold more 32P than histone did. Phosphorylation of casein catalyzed by Fc gamma 2a receptor complex was dependent on casein concentration (maximum phosphate incorporation being at 0.5 mg/mL), increased with time or temperature, was dependent on the concentration of ATP and Mg2+, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn2+ (greater than 25 mM) or KCl (greater than 100 mM) or by a small amount of heparin (greater than 10 units/mL) and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc gamma 2a receptor used ATP as substrate with an apparent Km of 2 microM as well as GTP with an apparent Km of 10 microM. Prior heating (60 degrees C for 15 min) or treatment with protease (trypsin or Pronase) of Fc gamma 2a receptor complex almost totally abolished casein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization, purification, and characterization of a membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line

The release of free arachidonic acid from membrane phospholipids is believed to be the rate-controlling step in the production of the prostaglandins, leukotrienes, and related metabolites in inflammatory cells such as the macrophage. We have previously identified several different phospholipases in the macrophage-like cell line P388D1 potentially capable of controlling arachidonic acid release. Among them, a membrane-bound, alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular interest because of the likelihood that the regulatory enzyme has these properties. This phospholipase A2 has now been solubilized from the membrane fraction with octyl glucoside and partially purified. The first two steps in this purification are butanol extractions that yield a lyophilized, stable preparation of phospholipase A2 lacking other phospholipase activities. This phospholipase A2 shows considerably more activity when assayed in the presence of glycerol, regardless of whether the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated vesicles or mixed micelles with the nonionic surfactant Triton X-100. Glycerol (70%) increases both the Vmax and the Km with both substrate forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater for vesicles than for mixed micelles. The lyophilized preparation of the enzyme is routinely purified about 60-fold and is suitable for evaluating phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps in the purification are acetonitrile extraction followed by high performance liquid chromatography on an Aquapore BU-300 column and a Superose 12 column. This yields a 2500-fold purification of the membrane-bound phospholipase A2 with a 25% recovery and a specific activity of about 800 nmol min-1 mg-1 toward 100 microM dipalmitoylphosphatidylcholine in mixed micelles. When this material was subjected to analysis on a Superose 12 sizing column, the molecular mass of the active fraction was approximately 18,000 daltons.     

Inductive effect of recombinant human interleukin-1 alpha and beta on differentiation of macrophage-like tumor cell line P388D1

We used the mouse monocyte/macrophage-like tumor cell line P388D1 to test whether or not interleukin-1 (IL-1) stimulates differentiation of monocyte/macrophage progenitors. Incubation of these cells with recombinant human interleukin-1 (rhIL-1) alpha and beta resulted in their increased adherence, stimulation of nonspecific esterase activity, and increased Fc rosette formation. rhIL-1s inhibited cell growth and stimulated Fc rosette formation in a dose-dependent fashion. The cell growth inhibition due to rhIL-1s depended on the concentration of serum in culture medium. Synergism between rhIL-1 and calcium ionophore A23187 was found for the cell growth inhibition and Fc rosette formation. The presence of ethylene glycol bis- (beta-aminoethyl ether) N,N,N,N,-tetraacetic acid(EGTA) in the medium abolished the stimulatory effect of rhIL-1 on Fc rosette formation of the cell line. These results demonstrate that rhIL-1s are a potent inducer of the differentiation of the macrophage-like tumor cell line P388D1.     

Fc gamma 2b receptor-mediated phagocytosis by a murine macrophage-like cell line (P388D1) and peritoneal resident macrophages. Up-regulation by the inhibitors of phospholipase A2 and cyclooxygenase

The mechanisms of Fc gamma R-mediated phagocytosis of immune complexes were investigated by the use of a murine macrophage-like cell line (P388D1) and murine peritoneal resident macrophages. About 40 to 80% of P388D1 cells phagocytosed SRBC coated with IgG2a subclass anti-SRBC mAb (EA2a) within 60 min, whereas only 10 to 20% of the cells phagocytosed EA2b during the same period. The treatment of P388D1 cells with inhibitors of phospholipase A2 (p-bromophenacylbromide, EGTA, or dexamethasone) or of cyclooxygenase (indomethacin or aspirin) significantly promoted the Fc gamma 2bR-mediated phagocytosis of EA2b, but did not affect the Fc gamma 2aR-mediated phagocytosis of EA2a. These results suggest that the activation of phospholipase A2 activity associated with Fc gamma 2bR may lead to the inhibition of phagocytosis of EA2b. This inhibition appeared to be due to the blockade of the interaction of Fc gamma 2bR with various cytoskeletal components, because the association of Fc gamma 2bR and these cytoskeletal components, which could be eliminated by cytochalasin D, was found to be increased by the inhibition of phospholipase A2 activity.     

Plasma membrane depolarization reduces nitric oxide (NO) production in P388D.1 macrophage-like cells during Leishmania major infection

In the present study, we compare changes in host cell plasma membrane potential (V(m)), K(+) fluxes, and NO production during K(+) channel blockade with those changes that occur during infection with Leishmania major. Infection of P388D.1 cells with L. major promastigotes or treatment with K(+) channel blockers (either 1mM 4-AP, 10mM TEA, or 200 microM quinine) suppressed NO production. Inhibition of NO production correlated with depolarization of the P388D.1 cell V(m). Infection of P388D.1 cells with L. major increased the unidirectional influx of rubidium (86Rb), a tracer for K(+) flux, that was comparable to that induced by K(+) channel blockade by 1mM 4-AP. The similar effects of K(+) channel blockers and L. major on NO production, K(+) influx, and V(m) suggest that K(+) channel activity and the maintenance of V(m) is important for NO production in these cells. We suggest that intracellular parasites employ a strategy to inhibit NO production by disrupting V(m) during the invasion/infection process by altering host cell K(+) channel activity.