Trafficking and release of mycobacterial lipids from infected macrophages

Analysis of infected macrophages revealed that lipid-containing moieties of the mycobacterial cell wall are actively trafficked out of the mycobacterial vacuole. To facilitate the analysis of vesicular trafficking from mycobacteria-containing phagosomes, surface-exposed carbohydrates were labeled with hydrazide-tagged markers. The distribution of labeled carbohydrate/lipid moieties and subsequent interaction with cellular compartments were analyzed by immunoelectron microscopy and by fluorescence microscopy of live cells. The released mycobacterial constituents were associated with several intracellular organelles and were enriched strikingly in tubular endocytic compartments. Subcellular fractionation of infected macrophages by density gradient electrophoresis showed temporal movement of labeled bacterial constituents through early and late endosomes. Thin layer chromatography analysis of these subcellular fractions confirmed their lipid nature and revealed five dominant bacteria-derived species. These mycobacterial lipids were also found in extracellular vesicles isolated from the medium and could be observed in un-infected 'bystander' cells. Their transfer to bystander cells could expand the bacteria's sphere of influence beyond the immediate confines of the host cell.     

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.     

Mycobacterial lysocardiolipin is exported from phagosomes upon cleavage of cardiolipin by a macrophage-derived lysosomal phospholipase A2

Pathogenic mycobacteria are able to survive and proliferate in phagosomes within host macrophages (Mphi). This capability has been attributed in part to their cell wall, which consists of various unique lipids. Some of these are important in the host-pathogen interaction, such as resistance against microbicidal effector mechanisms and modulation of host cell functions, and/or are presented as Ags to T cells. Here we show that two lipids are released from the mycobacterial cell wall within the phagosome of infected Mphi and transported out of this compartment into intracellular vesicles. One of these lipids was identified as lysocardiolipin. Lysocardiolipin was generated through cleavage of mycobacterial cardiolipin by a Ca2+-independent phospholipase A2 present in Mphi lysosomes. This result indicates that lysosomal host cell enzymes can interact with released mycobacterial lipids to generate new products with a different intracellular distribution. This represents a novel pathway for the modification of bacterial lipid Ags.     

Intracellular or extracellular calcium can be used to trigger C5a-stimulated enzyme secretion from rabbit neutrophils

The ability of C5a to stimulate lysosomal enzyme release and 45Ca2+ efflux from rabbit neutrophils was studied. C5a stimulated beta-glucuronidase release from cytochalasin B-treated neutrophils either in the presence or absence of extracellular calcium. Depletion of cell calcium by pretreatment with the calcium ionophore A23187 blocked both the ability of C5a to elicit enzyme release in the absence of extracellular calcium and its ability to stimulate 45Ca2+ efflux. Both actions were dose-dependent over the same concentration range (10(-8)-10(-6) M ionophore A23187). In contrast, ionophore pretreatment had no effect on C5a-stimulated enzyme release in the presence of extracellular calcium. These results suggest that (a) release of cell calcium is required for enzyme secretion in the absence of extracellular calcium, and (b) C5a can trigger near-maximal enzyme release by using calcium from either of two sources: the extracellular space or an intracellular site.     

MHC class I endosomal and lysosomal trafficking coincides with exogenous antigen loading in dendritic cells

Background

Cross-presentation by dendritic cells (DCs) is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined.

Methodology/Principal Findings

In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I) from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing.

Conclusions/Significance

We conclude that DCs have ‘hijacked’ and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place.     

Why intracellular parasitism need not be a degrading experience for Mycobacterium.

The success of mycobacteria as pathogens hinges on their ability to infect and persist within the macrophages of their host. However, activation of host macrophages by cytokines from a productive cellular immune response can stimulate the cells to kill their resident pathogens. This suggests that the interaction between host cell and microbe is in delicate balance, which can be tipped in favour of either organism. Biochemical analysis of mycobacterial vacuoles has shown them to be integral to the host cell''s recycling endosomal system. As such they show limited acidification and hydrolytic activity despite possession of known lysosomal constituents such as cathepsins D, B and L, and LAMP 1. Even in established infections, they remain dynamic compartments accessible to several plasmalemma-derived constituents. Once the macrophage has been activated by IFN-gamma and TNF-alpha the vacuoles coalesce and acidify. This marks a distinct alteration in vacuole physiology and leads to stasis and death of the mycobacteria. Mycobacteria have developed several strategies to avoid this outcome. Most notably, live bacilli-induce sustained release of IL-6 from infected macrophages. IL-6 blocks the ability of both polyclonal primary T cells and T-cell hybridomas to respond to appropriate stimuli. Such an activity could render the centres of infection foci, such as granulomas, anergic and thus avoid release of macrophage-activating cytokines. This paper discusses both the mechanisms by which mycobacteria try to ensure their success as intracellular pathogens and the relevance of these strategies to the overall understanding of mycobacterial diseases.     

Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.     

Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance

By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages.     

Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exocytic system of Toxoplasma gondii

Regulated exocytosis allows the timely delivery of proteins and other macromolecules precisely when they are needed to fulfil their functions. The intracellular parasite Toxoplasma gondii has one of the most extensive regulated exocytic systems among all unicellular organisms, yet the basis of protein trafficking and proteolytic modification in this system is poorly understood. We demonstrate that a parasite cathepsin protease, TgCPL, occupies a newly recognized va cuolar c ompartment (VAC) that undergoes dynamic fragmentation during T. gondii replication. We also provide evidence that within the VAC or late endosome this protease mediates the proteolytic maturation of proproteins targeted to micronemes, regulated secretory organelles that deliver adhesive proteins to the parasite surface during cell invasion. Our findings suggest that processing of microneme precursors occurs within intermediate endocytic compartments within the exocytic system, indicating an extensive convergence of the endocytic and exocytic pathways in this human parasite.     

IL-1 beta maturation: evidence that mature cytokine formation can be induced specifically by nigericin.

Mouse peritoneal macrophages stimulated with LPS produce large amounts of pro-IL-1 beta. When these cells were pulse-labeled with [35S]methionine, however, little labeled cytokine appeared in the medium after a chase, and that which was externalized was not processed to its mature biologically active form. In an effort to promote proteolytic maturation of IL-1 beta, macrophages were treated with agents that were expected to compromise their viability. The calcium ionophore A23187 and the detergent saponin caused complete release of nonprocessed 35-kDa pro-IL-1 beta and liberation into the extracellular medium of the cytoplasmic marker enzyme LDH and the lysosomal enzyme beta-N-acetylglucosaminidase. Hypotonic lysis resulted in the release of a 20-kDa IL-1 beta species that was distinct from the 17-kDa mature species. Importantly, incubation of the murine macrophages with the potassium/proton ionophore nigericin led to a quantitative conversion of pro-IL-1 beta to a 17-kDa species. The N-terminus of this nigericin-derived product possessed the amino acid sequence expected for mature biologically active IL-1 beta. Monensin, an ionophore similar to nigericin, did not induce release or proteolysis of IL-1 beta. Complete release of mature IL-1 beta required concentrations of nigericin in excess of 2 microM and a minimum of 10 min of treatment. Mature 17-kDa IL-1 beta was observed within the nigericin-treated cells before their lysis. Nigericin's effect was not limited to mouse peritoneal macrophages, inasmuch as the ionophore also induced release and proteolytic maturation of IL-1 beta produced by LPS-stimulated human peripheral blood monocytes. Treatment of macrophages with LPS and nigericin, therefore, results in a unique series of intracellular events that promote formation of mature 17-kDa IL-1 beta.     

TI-VAMP/VAMP7 is required for optimal phagocytosis of opsonised particles in macrophages

Phagocytosis relies on extension of plasmalemmal pseudopods generated by focal actin polymerisation and delivery of membranes from intracellular pools. Here we show that compartments of the late endocytic pathway, bearing the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7), are recruited upon particle binding and undergo exocytosis before phagosome sealing in macrophages during Fc receptor (FcR)-mediated phagocytosis. Expression of the dominant-negative amino-terminal domain of TI-VAMP or depletion of TI-VAMP with small interfering RNAs inhibited phagocytosis mediated by Fc or complement receptors. In addition, inhibition of TI-VAMP activity led to a reduced exocytosis of late endocytic vesicles and this resulted in an early blockade of pseudopod extension, as observed by scanning electron microscopy. Therefore, TI-VAMP defines a new pathway of membrane delivery required for optimal FcR-mediated phagocytosis.     

Effects of hyposmotic stress on exocytosis in isolated turbot, Scophthalmus maximus, hepatocytes

The effect of hyposmotic shock on exocytosis was examined in isolated hepatocytes of turbot, a marine flatfish, using the molecular probe FM1-43. Sudden exposure to a reduced osmolality caused an increase in cell exocytic activity related to the osmotic gradient between intra- and extracellular fluids. Cytoskeletal microtubules could contribute to this hyposmotic-induced exocytosis since colchicine inhibited the process. Protein kinase C, phosphatidylinositol-3 kinase, phospholipases A2, C and D could constitute key enzymes in the mechanism since their inhibition by specific agents altered the hyposmotic-induced exocytic activity. Moreover, arachidonic acid and derivates from the 5-lipoxygenase pathway as well as calcium could participate in the process. As regulatory volume decrease (RVD) exhibited by turbot hepatocytes following hyposmotic stimulation involves similar features, a potential role of exocytosis in volume regulation is suggested. In particular, exocytosis could serve RVD by contributing to ATP release since this latter process similarly appeared to be phospholipase D-dependent and related to the osmotic gradient. This study provides the first evidence of a volume-sensitive exocytosis that could aim at volume constancy in a marine teleost fish cell type.     

Endocytosis, recycling, and regulated exocytosis of glucose transporter 4

Glucose transporter 4 (GLUT4) is responsible for the uptake of glucose into muscle and adipose tissues. Under resting conditions, GLUT4 is dynamically retained through idle cycling among selective intracellular compartments, from whence it undergoes slow recycling to the plasma membrane (PM). This dynamic retention can be released by command from intracellular signals elicited by insulin and other stimuli, which result in 2-10-fold increases in the surface level of GLUT4. Insulin-derived signals promote translocation of GLUT4 to the PM from a specialized compartment termed GLUT4 storage vesicles (GSV). Much effort has been devoted to the characterization of the intracellular compartments and dynamics of GLUT4 cycling and to the signals by which GLUT4 is sorted into, and recruited from, GSV. This review summarizes our understanding of intracellular GLUT4 traffic during its internalization from the membrane, its slow, constitutive recycling, and its regulated exocytosis in response to insulin. In spite of specific differences in GLUT4 dynamic behavior in adipose and muscle cells, the generalities of its endocytic and exocytic itineraries are consistent and an array of regulatory proteins that regulate each vesicular traffic event emerges from these cell systems.     

Plasma membrane phospholipid translocation in the mouse peritoneal macrophage: differential response to stimulation of eicosanoid production.

The metabolism and translocation of exogenously introduced plasma membrane phosphatidylcholine (PC) having the fluorescent fatty acid analog aminocaproyl NBD (N-nitrobenzo-2-oxa-1,3 diazole) (NBD-PC), in the sn2 position was studied in cultured murine peritoneal macrophages using biochemical and morphological techniques. Following labeling of the cell plasma membrane at 2 degrees C by vesicle lipid exchange, macrophages were warmed in the presence or absence of pharmacological stimuli of eicosanoid production and release. Fluorescence microscopy indicated that the phospholipid was translocated to an internal cellular pool upon stimulation with zymosan. In contrast, the membrane PC analog was primarily metabolized and released after being found diffusely associated with the cytoplasm in macrophages stimulated with the calcium ionophore A23187. Evidence obtained by double labeling zymosan-treated macrophages with NBD-PC and a monoclonal antibody directed against a lysosomal membrane protein demonstrated that the fluorescent lipid is internalized in association with the zymosan particles and both are found in lysosomes. The results suggest that multiple pathways exist in peritoneal macrophages which target plasma membrane PC into different cellular compartments for hydrolysis and conversion to eicosanoid products and release from cells.     

Rab7 controls lipid droplet-phagosome association during mycobacterial infection

Lipid droplets (LDs) are organelles that have multiple roles in inflammatory and infectious diseases. LD act as essential platforms for immunometabolic regulation, including as sites for lipid storage and metabolism, inflammatory lipid mediator production, and signaling pathway compartmentalization. Accumulating evidence indicates that intracellular pathogens may exploit host LDs as source of nutrients and as part of their strategy to promote immune evasion. Notably, numerous studies have demonstrated the interaction between LDs and pathogen-containing phagosomes. However, the mechanism involved in this phenomenon remains elusive. Here, we observed LDs and PLIN2 surrounding M. bovis BCG-containing phagosomes, which included observations of a bacillus cell surrounded by lipid content inside a phagosome and LAM from mycobacteria co-localizing with LDs; these results were suggestive of exchange of contents between these compartments. By using beads coated with M.tb lipids, we demonstrated that LD-phagosome associations are regulated through the mycobacterial cell wall components LAM and PIM. In addition, we demonstrated that Rab7 and RILP, but not Rab5, localizes to LDs of infected macrophages and observed the presence of Rab7 at the site of interaction with an infected phagosome. Moreover, treatment of macrophages with the Rab7 inhibitor CID1067700 significantly inhibited the association between LDs and LAM-coated beads. Altogether, our data demonstrate that LD-phagosome interactions are controlled by mycobacterial cell wall components and Rab7, which enables the exchange of contents between LDs and phagosomes and may represent a fundamental aspect of bacterial pathogenesis and immune evasion.     

Modified lysosomal compartment as carrier of slowly and non-degradable tracers in macrophages

A previous immunocytochemical study of macrophages infected with Bacillus subtilis showed that a cell wall antigen could be detected for several days in a population of small vesicles randomly distributed within the cells and apparently distinct from perinuclear lysosomes. These observations suggested the possibility that these vesicles might constitute a "storage" compartment for non-degradable compounds. In the present report we compared in pulse-chase experiments the location and fate of a series of degradable and non-degradable pinocytic tracers within the macrophages. The tracers, detected by fluorescent microscopy, were bovine serum albumin (BSA), hen egg ovalbumin (OVA), horseradish peroxidase (HRP). Lucifer Yellow, fluorescent dextran, and levan. BSA and OVA remained located in perinuclear lysosomes during the chase period until their disappearance occurring within 3 h. In contrast, the other tracers, although initially located in perinuclear lysosomes, were found after a 3 to 5-h chase in small vesicles homogeneously distributed in the macrophage cytoplasm where they remained visible for 2 to 3 days. The use of markers for different cell organelles indicated that these dispersed vesicles exhibited several of the lysosomal features. They were acidic, they contained the 100 kDa and the 120 kDa lysosomal proteins as well as some acid proteases albeit these markers were in lesser concentrations than in the perinuclear lysosomal compartment. The addition of bacteria to the macrophages previously loaded with fluorescent dextran showed that all dispersed vesicles have the same fusion property as lysosomes and that slowly degraded or non-degradable tracers turn over through the perinuclear lysosomal compartment by using the endocytic pathway. Measurement of the release of some of the tracers into the culture medium suggested that the "dispersed vesicles" were probably not implicated in exocytosis of the tracers.     

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.     

MHC class II compartments in human dendritic cells undergo profound structural changes upon activation

Immature dendritic cells efficiently capture exogenous antigens in peripheral tissues. In an inflammatory environment, dendritic cells are activated and become highly competent antigen-presenting cells. Upon activation, they lose their ability for efficient endocytosis and gain capability to migrate to secondary lymphoid organs. In addition, peptide loading of MHC class II molecules is enhanced and MHC class II/peptide complexes are redistributed from an intracellular location to the plasma membrane. Using immuno-electron microscopy, we show that activation of human monocyte-derived dendritic cells induced striking modifications of the lysosomal multilaminar MHC class II compartments (MIICs), whereby electron-dense tubules and vesicles emerged from these compartments. Importantly, we observed that MHC class II expression in these tubules/vesicles transiently increased, while multilaminar MIICs showed a strongly reduced labeling of MHC class II molecules. This suggests that formation of the tubules/vesicles from multilaminar MIICs could be linked to transport of MHC class II from these compartments to the cell surface. Further characterization of endocytic organelles with lysosomal marker proteins, such as the novel dendritic cell-specific lysosomal protein DC-LAMP, HLA-DM and CD68, revealed differential sorting of these markers to the tubules and vesicles .     

Intracellular calcium does not appear to be essential for arachidonic acid release from stimulated macrophages as shown by studies with Quin-2

The present investigation was undertaken to study the potential role of intracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages activated by inflammatory stimuli. The intracellular calcium concentration, as measured using fluorescent probe Quin-2, was 112 +/- 8.4 nM. The chelation of intracellular calcium with Quin-2 did not affect the release of arachidonic acid from macrophages upon stimulation with phorbol myristate acetate, opsonized zymosan or calcium ionophore A23187. However, the removal of calcium from the extracellular medium resulted in a 30-50% decrease in arachidonic acid release from phorbol myristate acetate- and zymosan-stimulated macrophages and also the stimulation of arachidonic acid release from calcium ionophore-stimulated cells were nullified. These studies indicated the existence of calcium-dependent and independent mechanisms modulating the release of arachidonic acid from macrophages subjected to inflammatory stimuli.