Peritoneal Macrophage Membranes Research Articles

The Calcineurin B Subunit (CnB) Is a New Ligand of Integrin αM That Mediates CnB-Induced Apo2L/TRAIL Expression in Macrophages

We showed previously that the calcineurin B subunit (CnB) plays an important role in activation of peritoneal macrophage, but the underlying mechanism remained unknown. To examine whether there is a CnB receptor on peritoneal macrophages, we performed the radioligand binding assay of receptors. The receptor saturation binding curve demonstrated high-affinity and specific binding; the maximum binding was 1090 fmol/10(5) cells, and the K(d) was 70.59 pM. Then, we used a CnB affinity resin to trap potential receptors from highly purified peritoneal macrophage membranes. Mass spectrometry analysis showed that the binding protein was mouse integrin αM. We next performed a competition binding experiment to confirm the binding of CnB to integrin αM. This showed that FITC-CnB bound specifically to peritoneal macrophages and that binding was blocked by the addition of integrin αM Ab. We observed that CnB could induce TRAIL gene expression in peritoneal macrophages in vitro and in vivo. Integrin αM Ab blocking, RNA interference, and ligand competition experiments demonstrated that CnB-induced TRAIL expression is dependent on integrin αM. Furthermore, the tumoricidal activity of CnB-activated peritoneal macrophages is partially dependent on TRAIL. In addition, CnB treatment significantly prolongs the survival of mice bearing H22 ascites tumors, which has a positive correlation with the induction level of TRAIL. These results reveal a novel function of the CnB in innate immunity and cancer surveillance. They also point to a new signaling pathway leading to induction of TRAIL and suggest a possible application of CnB in cancer therapy.

Visualization of the Molecular Dynamics of Lipopolysaccharide on the Plasma Membrane of Murine Macrophages by Total Internal Reflection Fluorescence Microscopy

The molecular action of Alexa 594-labeled lipopolysaccharide (LPS) from Escherichia coli was examined on living peritoneal macrophages of C57BL/6 mice by total internal reflection fluorescence microscope (TIRFM), and the molecular kinetics of LPS was analyzed. TIRFM visualization of the action of fluorescence-labeled LPS revealed an increase in the mean fluorescence intensity of LPS on the plasma membrane of wild type macrophages at 60 min after administration, indicating the oligomerization of LPS after binding to the macrophages. Additionally, a time-dependent sharp decrease in the mean diffusion coefficient of LPS was observed. On the other hand, both mean fluorescence intensity and diffusion coefficient of LPS in cases of TLR4(-/-), MD2(-/-), MyD88(-/-), and TRIF(-/-) macrophages were significantly different from the corresponding values of wild type macrophage, whereas differences were also noticed among these molecule-deficient macrophages. Furthermore, statistical analysis indicated the major role of receptors (TLR4 and MD2) and intracellular signaling molecules (MyD88 and TRIF) in oligomerization and lowering of the diffusion rate of LPS on the plasma membrane of murine macrophages, respectively.

Functional characterization and mRNA expression of pituitary adenylate cyclase activating polypeptide (PACAP) type I receptors in rat peritoneal macrophages

The present work characterizes the mRNA expression of PACAP type I receptors in rat peritoneal macrophages but not in peritoneal lymphocytes by both retrotranscriptase and polymerase chain reaction (RT-PCR) and homologous Southern hybridization and the stimulation by PACAP27, PACAP38 and vasoactive intestinal peptide (VIP) of sn-1,2-diacylglycerol production in rat peritoneal macrophage membranes. The binding of [ 125 I ]PACAP27 was time and cell concentration dependent. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicates the existence of two classes of binding sites. The dissociation constant ( K d) was 0.64±0.08 nM and the maximal binding capacity ( B max) was 8.85±1.45 fmol/10 6 cells for the high affinity binding site. The low affinity binding site had a K d of 0.10±0.06 μM with a B max of 300±21.9 fmol/10 6 cells. Scatchard analysis of VIP displacement data indicated the presence of two classes of binding sites with a K d and B max different to those of PACAP27. These results suggest that PACAP binds to two binding sites, PACAP type I receptors and PACAP type II receptors. The PACAP27-stimulated diacylglycerol production was not affected by treatment with pertussis toxin. However, the presence of GTP partially inhibited this PACAP27 stimulation of 1,2-diacylglycerol in a dose dependent manner, although GTP alone stimulates diacylglycerol accumulation. In conclusion, for the first time we demonstrate by biochemical and molecular biology criteria the existence of PACAP type I receptors on rat peritoneal macrophages and the evidence for coupling with a pertussis toxin-insensitive G regulatory protein.

Characterization of VIP receptor-effector system antagonists in rat and mouse peritoneal macrophages

In the present study we show that the synthetic peptides [4-Cl- d-Phe 6,Leu 17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2 inhibit in a competitive manner the specific [ 125I]VIP binding to both rat and mouse peritoneal macrophages. In rat peritoneal macrophages, the order of potency of the different peptides, as expressed by the IC 50 values was: VIP (IC 50=1.90±0.16 nM)>[4-Cl- d-Phe 6,Leu 17]VIP (IC 50=125.8±13.2 nM)>[Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2 (IC 50=354.8±21.2 nM). In mouse peritoneal macrophages a similar pattern of potency was observed: VIP (IC 50=1.58±0.12 nM)>[4-Cl- d-Phe 6,Leu 17]VIP (IC 50=110.8±10.7 nM)>[Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2 (IC 50=251±19.2 nM). The behavior as VIP receptor antagonists of both [4-Cl- d-Phe 6,Leu 17]VIP and [Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2 in rat and mouse peritoneal macrophages was confirmed by: (a) the shift to the right of VIP dose-stimulated cyclic AMP production curves in the presence of the two antagonists; (b) the agreement between the order of efficacy of the two peptides in competition experiments with the corresponding inhibition of cyclic AMP production; (c) the inefficiency of the two antagonists on the stimulation of cyclic AMP production by the β-adrenoceptor agonist isoproterenol, which indicates the specificity of the interaction; (d) the synergic effect of VIP on isoproterenol-stimulated cyclic AMP production was completely abolished by [4-Cl- d-Phe 6,Leu 17]VIP or [Ac-Tyr 1, d-Phe 2]GRF-(1–29) -NH 2, suggesting that both antagonists acted via specific VIP receptors. Moreover, propranolol, a β-adrenoceptor antagonist, did not affect the VIP-stimulated cyclic AMP production and the antagonist role of [4-Cl- d-Phe 6,Leu 17]VIP or [Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2; (e) in cross-linking experiments, the intensity of the labeling of the [ 125I]VIP/receptor complexes was significantly lower with the antagonists than in the control experimental situation in both mouse and rat peritoneal macrophage membranes.

Composition of peritoneal macrophage membranes in autoimmune MRL lpr/lpr mice

Adult MRL lpr / lpr mice display phenotypic features that are consistent with both rheumatoid arthritis and systematic lupus erythematosus. Previous studies have reported that peritoneal macrophages harvested from this model have an increased propensity for both spontaneous and elicited release of prostaglandins and leukotrienes relative to immunologically normal control mice. To investigate whether one aspect of the differences in secretory potential between autoimmune and normal mice was at the level of increased substrate availability, gas chromatographic analysis of peritoneal macrophage membranes from autoimmune MRL lpr / lpr , young lpr, wild type ±/±, and immunologically normal mice was done. The results demonstrate enrichment of arachidonate in adult lpr macrophage membranes in all major phospholipid classes relative to young lpr, ±/± and immunologically normal C3H/HeN mice. Similarly, there was an increased mole % of arachidonic acid in lpr mice relative to controls. Elevated membrane arachidonate may contribute to the increased propensity of autoimmune strains to participate in the inflammatory process.

Characteristics of receptors for VIP in rat peritoneal macrophage membranes

Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [ 125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with K d values of 0.60 ± 0.08 and 275 ± 39 n M and binding capacities of 580 ± 71 and 72,500 ± 810 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. These pharmacological studies showed the following order of potency: VIP (IC 50 = 1 n M) > rGRF (IC 50 = 13 rmn M) > PHI (IC 50 = 421 n M) ⪢ secretin. Glucagon, somatostatin, insulin, octapeptide of cholecystokinin [CCK(26–33)], and pancreastatin were ineffective at concentrations up to 1 μ M. Binding of [ 125I]VIP to membranes is markedly reduced by increasing the ionic strength of incubation medium. Treatment of membranes with dithiothreitol, trypsin, and phospholipases A 2 and C resulted in a loss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The molecular characterization of VIP receptors in RPMM was performed after [ 125I]VIP cross-linking to membranes using the cross-linker dithiobis (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [ 125I]VIP-protein complexes of M r 55,000 ± 1700, 35,000 ± 900, and 22,000 ± 500.

Age-dependent modification of lipid composition and lipid structural order parameter of rat peritoneal macrophage membranes

The effect of aging on the lipid composition and fluidity of rat peritoneal macrophage membranes has been determined using young (3 months), mature (12 months) and aged (24 months) Wistar rats. In the aged animals, total phospholipid decreased significantly ( P < 0.05), whereas cholesterol increased ( P < 0.01), with an age-dependent increase in the molar ratio of cholesterol/phospholipid. The most marked change in phospholipid content was the significant ( P < 0.001) age-dependent increase of phosphatidylserine and cardiolipin and the significant decrease of phosphatidylcholine and phosphatidylinositol. During aging there was a considerable decrease in arachidonic acid and docosapentanoic acid (about 50% in both cases). In contrast, an increase in the levels of oleic, linolenic and docosahexanoic acid was observed. Steady-state fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as the probe was used to estimate the lipid structural order parameter of macrophage membranes. There was a highly significant ( P < 0.001) age-dependent increase in the lipid structural order parameter, which correlated well with the increased molar ratio of cholesterol/phospholipid in the membranes isolated from aged animals. The data suggests alteration in membrane lipid-protein interactions in aging, and are consistent with the hypothesis of the aging process.

Guanine nucleotide regulation of VIP binding to rat peritoneal macrophage membranes

In the present study, we have examined the effect of guanine nucleotides on VIP binding to rat peritoneal macrophage membranes. Both guanosine 5′-triphosphate (GTP) and its nonhydrolizable analog guanosine 5′-β, Y-imidotriphosphate [Gpp(NH)p] inhibited, in a dose-dependent manner, the VIP binding to its specific binding sites. Half-maximal inhibition (IC 50) was observed at 5.4 ± 0.5 μM GTP. The inhibitory effect of GTP was due to an increase of the dissociation rate of peptide bound to membranes. The specificity of the binding inhibition was assessed from the lack of action of the other nucleotides tested. These results directly suggest the coupling of VIP binding sites with guanine nucleotide binding proteins in rat peritoneal macrophage membranes.

Characterization of the beta-adrenergic receptor of the rat peritoneal macrophage.

The beta-adrenergic receptor was characterized on BCG-activated rat peritoneal macrophage membranes by radio-ligand binding studies. Saturable binding with [125I]iodocyanopindolol (125I-ICYP) was demonstrated. With Scatchard analysis, rat macrophages demonstrate approximately 1000 receptors per cell with a Kd of 5 X 10(-11) M for 125I-ICYP. Competition curves with (-) and (+) propranolol at concentrations below 10(-6) M confirmed stereospecificity. The potency of various ligands to compete for 125I-ICYP binding sites followed the order: propranolol greater than isoproterenol greater than epinephrine greater than norepinephrine with apparent Kd of 2.0 X 10(-9), 3.9 X 10(-7), 1.0 X 10(-5), and 2.5 X 10(-5) M, respectively. Isoproterenol-stimulated adenylate cyclase activity was two-fold above basal activity. The potential physiologic significance of a beta-adrenergic receptor on rat peritoneal macrophages was suggested by a dose-dependent decrease in phagocytosis of soluble, model immune complexes (aggregated gamma-globulin) by macrophages incubated with metaproterenol. We conclude that the rat macrophage has a beta-adrenergic receptor and that catecholamines may thereby modulate macrophage function.

The role of endogenous C1q in the membrane of peritoneal macrophages as trigger in polyanion-mediated macrophage stimulation

The role of endogenous C1q in the membrane of peritoneal macrophages as trigger in polyanion-mediated macrophage stimulation

Cytoskeletal control of concanavalin A receptor mobility in peritoneal macrophages

The role of microtubules and microfilaments in controlling the mobility of concanavalin A (ConA) receptors in the membrane of peritoneal macrophages was examined. Receptor mobility was assessed by counting the number of cells exhibiting cap formation following incubation with fluorescein-labelled ConA for 5 min at 37 °C. It was found that 1. 1. ConA binds to peritoneal macrophages in the form of multiple aggregates (patches), this being followed by rapid endocytosis. No spontaneous receptor capping is seen. 2. 2. Pretreatment of macrophages for 1 h at 37 °C with the microtubule disrupting drugs colchicine, vinblastine and podophyllotoxin induces ConA cap formation in 25–50% of cells. Cap formation is not induced by the glutathione-oxidizing agent diamide. 3. 3. Pretreatment of macrophages with cytochalasin B (CB), a drug interfering with microfilament function, does not facilitate capping. However, CB totally prevents the induction of cap formation by podophyllotoxin. 4. 4. Exposure of cells to the macrophage-specific lymphokine, migration inhibitory factor (MIF), partially prevents cap formation induced by microtubule disrupting drugs. It is concluded that in peritoneal macrophages, the mobility of membrane receptors for ConA is normally restricted by cytoplasmic microtubules and cap formation is only possible in the absence of microtubules. Cap formation also requires the presence of intact microfilaments. Drug-induced cap formation is antagonized by MIF, a mediator promoting microtubule assembly.