scholarly journals Down-regulation of mannosyl receptor-mediated endocytosis and antigen F4/80 in bacillus calmette-guerin-activated mouse macrophages. Role of T lymphocytes and lymphokines

1982 ◽  
Vol 155 (6) ◽  
pp. 1623-1637 ◽  
Author(s):  
AB Ezekowitz ◽  
S Gordon
Keyword(s):  
Plasma Membrane ◽  
Single Cell Analysis ◽  
Target Population ◽  
Peritoneal Macrophages ◽  
Membrane Receptors ◽  
Lymphocyte Function ◽  
Bacillus Calmette Guerin ◽  
Down Regulation ◽  
Receptor Mediated Endocytosis

Bacillus Calmette-Guerin (BCG) infection alters the surface and endocytic properties of mouse peritoneal macrophages (PM) compared with thioglycollate- elicited (TPM) or resident PM (RPM). Expression of Ia antigen (Ag) is enhanced up to fourfold, but plasma membrane receptors that mediate binding and uptake of mannosyl/fucosyl-terminated glycoconjugates (MFR), Fc receptors, and the macrophage (mφ)-specific Ag F4/80 are reduced by 50-80 percent. Levels of Mac-1 remain relatively stable. These changes are accompanied by enhanced secretion of O(2)(-), after further stimulation with phorbyl myristate acetate, and of plasminogen activator. Both these products are released by TPM, but not RPM. The characteristic surface phenotype of BCG-PM can also be induced by injection of C. parvum, another mφ- activating agent, but not by thioglycollate broth, lipopolysaccharide, or proteose peptone. Purified protein derivative (PPD) and N-acetylmuramyl-L- alanyl-D-isoglutamine. 2H(2)0 are soluble agents with partial activity. Alteration of mφ markers by BCG infection depends on T lymphocyte function, although studies with nude mice indicate that other pathways may also serve to modify the surface of the mφ. Mφ from uninfected animals displayed all markers of activation after adoptive transfer of specifically-sensitised lymphocytes with PPD, intraperitoneally, or after co- cultivation. Treatment of primed lymphocytes with anti-Thy-1 antibody and complement ablated this effect. Lymphokines obtaned by Ag or mitogen stimulation induced similar changes in TPM and RPM. Mannose-specific endocytosis decayed rapidly, time 1/2 approximately equal to 16 h and stabilized at approximately 25 percent of control values. Single-cell analysis showed that residual MFR activity was uniform in the target population. Loss of Ag F4/80 after activation by lymphocyte and PPD was less marked than after infection (35 percent vs 80 percent), unlike MFR activity, which declined to a similar extent. Induction of mφ Ia by lymphokine reached a peak after 2-3 d and was lost within 2 d of its removal. Recovery of MFR and F4/80 was incomplete under these conditions. These studies establish that activated mφ known to display enhanced antimicrobial/anticellular activity express markedly different surface properties distinct from elicited or resident cells. The role of antigen- stimulated T cell products in regulating mφ function is confirmed, and down-regulation of mannosyl-receptor-mediated endocytosis provides a sensitive, quantitative, and cell-specific new marker to study their properties and mechanism of action. Extensive, but selective remodeling of mφ plasma membrane structure could play an important role in controlling recognition and effector mechanisms of the activated mφ.

scholarly journals Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane.

1975 ◽  
Vol 142 (5) ◽  
pp. 1263-1282 ◽  
Author(s):  
F M Griffin ◽  
J A Griffin ◽  
J E Leider ◽  
S C Silverstein
Keyword(s):  
Plasma Membrane ◽  
Sheep Erythrocyte ◽  
Peritoneal Macrophages ◽  
Membrane Receptors ◽  
Macrophage Receptors ◽  
Specific Receptors ◽  
Particle Ingestion ◽  
Plasma Membrane Receptors ◽  
Mouse Peritoneal Macrophages

These experiments were designed to evaluate the role of macrophage plasma membrane receptors for the third component of complement (C) and for the Fc portion of IgG in the ingestion phase of phagocytosis. Sheep erythrocyte (E) were coated with anti-E IgG [E(IgG)]; these E(IgG) were then attached to cultivated monolayers of mouse peritoneal macrophages under conditions which reversibly inhibit ingestion of E(IgG). The E(IgG)-macrophage complexes were further incubated under similar conditions with an antimacrophage IgG fraction which blocks Fc receptor-mediated ingestion but has no effect upon ingestion mediated by other phagocytic receptors. When these cultures were subsequently incubated under conditions optimal for particle ingestion, phagocytosis of the IgG-coated erythrocytes did not occur; the erythrocytes remained bound to the Fc receptors of the macrophage plasma membrane. To determine whether ligands must cover the entire surface of an attached particle to permit ingestion of that particle, C-coated E [E(IgM)C] were bound to the C receptors of thioglycollate-induced (activated) macrophages at 4 degrees C. E(IgM)C-macrophage complexes were then trypsinized at 4 degrees C, a procedure which resulted in cleavage of erythrocyte-bound C3b molecules to a form of C3 not recognized by the macrophage receptors for C3b. Under the conditions used, trypsin did not affect the attachment of E(IgM)C to the macrophage surface or the macrophage receptors for C3b. When these trypsin treated E(IgM)C-macrophage complexes were incubated at 37 degrees C, the bound E(IgM)C were not ingested; the erythrocytes remained attached to the macrophage plasma membrane via the macrophage's C receptors. These results indicate that attachment of a particle to specific receptors on the macrophage plasma membrane is not sufficient to trigger ingestion of that particle. Rather, ingestion requires the sequential, circumferential interaction of particle-bound ligands with specific plasma membrane receptors not involved in the initial attachment process.

Effects of bafilomycin A1 on cytosolic pH of sheep alveolar and peritoneal macrophages: evaluation of the pH-regulatory role of plasma membrane V-ATPases.

1995 ◽  
Vol 198 (8) ◽  
pp. 1711-1715 ◽  
Author(s):  
T A Heming ◽  
D L Traber ◽  
F Hinder ◽  
A Bidani
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Initial Rate ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Cytosolic Ph ◽  
Phi Regulation

The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (m theta) from sheep. Cytosolic pH was measured using 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir < 6.8) by approximately 35%. Recovery of pHi was abolished by co-treatment of m theta with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal m theta from sheep. Sheep m theta also appear to possess a Na+/H+ exchanger. However, Na+/H+ exchange either is inactive or can be effectively masked by V-ATPase-mediated H+ extrusion at physiological pHi values.

scholarly journals Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase IIIα

2008 ◽  
Vol 19 (2) ◽  
pp. 711-721 ◽  
Author(s):  
Andras Balla ◽  
Yeun Ju Kim ◽  
Peter Varnai ◽  
Zsofia Szentpetery ◽  
Zachary Knight ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Small Interfering Rna ◽  
Type Ii ◽  
Measurable Effect ◽  
Down Regulation ◽  
Type Iii ◽  
Hormone Sensitive ◽  
Phosphatidylinositol 4 ◽  
Interfering Rna

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca2+ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2 levels in 32P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2 resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.

Spatiotemporal Regulation of C-Kit Signaling through Lipid Rafts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 819-819 ◽  
Author(s):  
Thomas Jahn ◽  
Stacie Gooch ◽  
Jaqueline Rogerio ◽  
Kenneth Weinberg
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Membrane Fraction ◽  
Time Course ◽  
Src Family Kinases ◽  
Membrane Receptors ◽  
Negative Regulator ◽  
Spatiotemporal Regulation

Abstract Recently, the understanding of membrane receptors has been transformed by studies characterizing the topology of the plasma membrane. The T-cell receptor (TCR) has been most studied for interactions of receptors with the lipid bilayer. TCR signaling has been shown to be dependent on the localization of the TCR complex to specific cholesterol- and sphingolipid-rich membrane subdomains, also called microdomains or lipid rafts. The definition of lipid rafts as assembly platforms to initiate membrane receptor signaling has induced a novel view of the plasma membrane as a compartmentalized structure. To investigate the role of lipid rafts in the signal transduction by the prototype receptor tyrosine kinase (RTK) c-kit, lipid raft, plasma membrane and cytosol fractions were obtained by subcellular fractionation of Mo7e cells widely used to study c-kit signaling. The purity of fractions was verified by the exclusive presence of marker proteins in their respective fraction. Time course experiments using non-stimulated and kit ligand (KL)-stimulated cells harvested after 5 and 20 minutes (′) revealed that non-activated c-kit was mainly localized within the membrane and that KL-induced activation of c-kit resulted in the redistribution of c-kit protein from the membrane fraction into lipid rafts. Activated c-kit was seen exclusively in lipid rafts at 5′ of KL-stimulation and was redistributed to the membrane after 20′. Analysis of downstream targets of c-kit revealed that various src-family kinases previously shown to be crucially involved in c-kit activation were predominantly present within the lipid raft fraction independently of c-kit activation. Investigating the main survival/proliferation pathway activated by c-kit we found that the p85 subunit of PI3-K was recruited to lipid rafts at 5′ of c-kit stimulation and was redistributed to the membrane fraction after 20′. Accordingly, PTEN, the central negative regulator of PI3-K, was present in lipid rafts in non-activated cells and was withdrawn from lipid rafts upon c-kit stimulation. PKB/Akt was not detected within lipid rafts but accumulated within the membrane fraction after 20′ of c-kit activation. Like PKB/Akt, PKC, Plcγ as well as PDK and adaptor molecules like Grb2, Grb4/Nckβ and Grb10 were predominantly localized in the cytosol and accumulated in the membrane fraction at 20′ of c-kit activation. To determine the biological role of lipid rafts in c-kit signaling we analyzed the effect of non-toxic concentrations of methyl-beta-cyclodextrin (MBCD) on c-kit dependent proliferation. MBCD has been shown to disrupt lipid rafts by removal of cholesterol from the plasma membrane. MBCD treatment of Mo7e cells resulted in complete inhibition of KL-mediated growth of Mo7e cells without inhibiting tyrosine phosphorylation of c-kit. We conclude that c-kit signaling is initiated in lipid rafts and that c-kit mediated proliferation is dependent on the integrity of lipid rafts. The predominant presence of src-family kinases in lipid rafts prior to activation of c-kit supports a crucial role for these signaling molecules in the initiation and amplification of c-kit signaling. The recruitment of p85 to lipid rafts and the synchronous withdrawal of PTEN from lipid rafts suggests that lipid rafts are the location of c-kit mediated activation of PI3-K. We propose a significant role for lipid rafts in the spatiotemporal regulation of c-kit signaling and hypothesize, that cell type- and cell state-specific compositional and topological variations of lipid rafts significantly influence the signaling outcome of c-kit and other RTKs.

Down-regulation in vivo of PGE receptors and adenylate cyclase stimulation

1980 ◽  
Vol 239 (1) ◽  
pp. E75-E80
Author(s):  
R. P. Robertson ◽  
K. R. Westcott ◽  
D. R. Storm ◽  
M. G. Rice
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Binding Affinity ◽  
Membrane Receptors ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Down Regulation ◽  
Liver Plasma Membrane ◽  
Plasma Membrane Receptors

Down-regulation in vivo of liver plasma membrane receptors for prostaglandin E (PGE) was investigated in Sprague-Dawley rats using the 16,16-dimethyl analogue of PGE2, This analogue was used for subcutaneous injections because it escapes the rapid pulmonic degradation characteristic of PGE and was recognized well by liver plasma membrane receptors. Following treatment with the analogue, the concentration of PGE receptors was significantly decreased (-37%, P less than 0.001), but the binding affinity was not altered. There was no evidence for carry-through of the analogue into the isolated plasma membrane preparation. It was also demonstrated that GTP decreased the binding affinity between PGE and its receptor. Down-regulation of receptor concentration was associated with a significant decrease (P less than 0.001) in PGE1-stimulated plasma membrane adenylate cyclase activity. These data provide the novel demonstration that rat liver plasma membrane receptor for PGE can be down-regulated in vivo and that this causes a corresponding decrease in PGE-induced plasma membrane adenylate cyclase activity.

scholarly journals Augmentation of macrophage complement receptor function in vitro. IV. The lymphokine that activates macrophage C3 receptors for phagocytosis binds to a fucose-bearing glycoprotein on the macrophage plasma membrane.

1984 ◽  
Vol 160 (4) ◽  
pp. 1206-1218 ◽  
Author(s):  
F M Griffin ◽  
P J Mullinax
Keyword(s):  
Plasma Membrane ◽  
Peritoneal Macrophages ◽  
Surface Glycoprotein ◽  
Complement C3 ◽  
Macrophage Receptors ◽  
C3 Receptors ◽  
Binding Lectin ◽  
Third Component Of Complement

Macrophage receptors for the third component of complement (C3) are normally immobilized and unable to diffuse within the cell's plasma membrane and, even though they promote avid particle binding, are unable to promote phagocytosis of C3-coated particles. We have previously identified a lymphokine that activates macrophage C3 receptors for phagocytosis and have found that it acts by freeing the receptors so that they can diffuse within the macrophage plasma membrane. It seemed likely to us that the initial lymphokine-macrophage interaction would occur at the macrophage surface, perhaps via a specific lymphokine receptor. Since the binding of many ligands to cells is mediated by cell surface glycoproteins, we examined the protein and sugar requirements for murine peritoneal macrophages to respond to the lymphokine. Macrophages treated with the neutral protease Dispase lost the ability to respond to the lymphokine, and inclusion of L-fucose in the incubation medium containing lymphokine and macrophages inhibited markedly the macrophages' response to the lymphokine, suggesting that the lymphokine exerts its effects by first binding to fucose residues on a glycoprotein receptor on the macrophage surface. Further evidence for the essential role of macrophage surface fucose was obtained by demonstrating that pretreatment of macrophages with either fucosidase or gorse lectin, a fucose-binding lectin, strikingly disabled the cells from responding to the lymphokine. All treatments that prevented lymphokine activation of macrophage C3 receptors for phagocytosis also prevented lymphokine-induced C3 receptor mobility. These results strongly suggest that the lymphokine binds to a fucose-bearing macrophage surface glycoprotein, perhaps a specific lymphokine receptor. They also strengthen our hypothesis that, for a receptor to be able to promote phagocytosis, it must be able to diffuse within the macrophage plasma membrane.

scholarly journals Single-cell analysis reveals new subset markers of murine peritoneal macrophages and highlights macrophage dynamics upon Staphylococcus aureus peritonitis

2016 ◽  
Vol 22 (5) ◽  
pp. 382-392 ◽  
Author(s):  
Solène Accarias ◽  
Clémence Genthon ◽  
David Rengel ◽  
Séverine Boullier ◽  
Gilles Foucras ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Immune Surveillance ◽  
Peritoneal Macrophages ◽  
Aureus Infection ◽  
Inflammatory Monocytes ◽  
Peritonitis Model

Resident macrophages play a central role in maintaining tissue homeostasis and immune surveillance. Here, we used single cell-based qPCR coupled with flow cytometry analysis to further define the phenotypes of large and small resident peritoneal macrophages (LPMs and SPMs, respectively) in mice. We demonstrated that the expression of Cxcl13, IfngR1, Fizz-1 and Mrc-1 clearly distinguished between LPMs and SPMs subsets. Using these markers, the dynamics of peritoneal macrophages in a Staphylococcus aureus-induced peritonitis model were analyzed. We found that S. aureus infection triggers a massive macrophage disappearance reaction in both subsets. Thereafter, inflammatory monocytes rapidly infiltrated the cavity and differentiated to replenish the SPMs. Although phenotypically indistinguishable from resident SPMs by flow cytometry, newly recruited SPMs had a different pattern of gene expression dominated by M2 markers combined with M1 associated features (inos expression). Interestingly, S. aureus elicited SPMs showed a robust expression of Cxcl13, suggesting that these cells may endorse the role of depleted LPMs and contribute to restoring peritoneal homeostasis. These data provide information on both resident and recruited macrophages dynamics upon S. aureus infection and demonstrate that single-cell phenotyping is a promising and highly valuable approach to unraveling macrophage diversity and plasticity.

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