Extracellular fusion of macrophage phagosomes with lysosomes

Following ingestion of micro-organisms by phagocytes, lysosomes fuse with the resulting phagocytic vacuoles. This process is presumed to assist in the destruction of the intracellular organisms. We have previously studied morphological and biochemical correlates of this subcellular fusion in macrophages and now report the observation of a similar fusion between cell-free organelles. Evidence so far obtained indicates that this fusion closely resembles its intracellular counterpart.     

BORC coordinates encounter and fusion of lysosomes with autophagosomes

Whereas the mechanisms involved in autophagosome formation have been extensively studied for the past 2 decades, those responsible for autophagosome-lysosome fusion have only recently begun to garner attention. In this study, we report that the multisubunit BORC complex, previously implicated in kinesin-dependent movement of lysosomes toward the cell periphery, is required for efficient autophagosome-lysosome fusion. Knockout (KO) of BORC subunits causes not only juxtanuclear clustering of lysosomes, but also increased levels of the autophagy protein LC3B-II and the receptor SQSTM1. Increases in LC3B-II occur without changes in basal MTORC1 activity and autophagy initiation. Instead, LC3B-II accumulation largely results from decreased lysosomal degradation. Further experiments show that BORC KO impairs both the encounter and fusion of autophagosomes with lysosomes. Reduced encounters result from an inability of lysosomes to move toward the peripheral cytoplasm, where many autophagosomes are formed. However, BORC KO also reduces the recruitment of the HOPS tethering complex to lysosomes and assembly of the STX17-VAMP8-SNAP29 trans-SNARE complex involved in autophagosome-lysosome fusion. Through these dual roles, BORC integrates the kinesin-dependent movement of lysosomes toward autophagosomes with HOPS-dependent autophagosome-lysosome fusion. These findings reveal a requirement for lysosome dispersal in autophagy that is independent of changes in MTORC1 signaling, and identify BORC as a novel regulator of autophagosome-lysosome fusion.     

Tubular lysosomes accompany stimulated pinocytosis in macrophages

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.     

Tubular lysosomes in ruffle-ended ameloblasts associated with enamel maturation in rat incisor

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.     

A role for the PhoP/Q regulon in inhibition of fusion between lysosomes and Salmonella-containing vacuoles in macrophages

After uptake by murine macrophages, Salmonella typhimurium is able to survive and replicate within specialized phagosomes called Salmonella -containing vacuoles (SCVs), which are segregated from the late endocytic pathway. The molecular basis of this process and the virulence factors required are not fully understood. In this study, we used confocal fluorescence microscopy to evaluate interactions between the endocytic pathway of the murine macrophage cell line RAW 264.7 and different S. typhimurium strains. The analysis was carried out using the fluid-phase marker Texas red–ovalbumin and antibodies against the lysosomal enzyme cathepsin D, the late endosomal lipid lysobisphosphatidic acid and the adaptor proteins AP-1 and AP-3. Less than 10% of wild-type SCVs were associated with these markers at 24 h after uptake by macrophages. A similar low level of association was observed for vacuoles containing mutant strains affected in the function of the Salmonella pathogenicity island (SPI)-2 type III secretion system or the virulence plasmid spv operon. However, at this time point, the proportion of vacuoles containing phoP ⁻ mutant bacteria that were associated with each of the markers ranged from 25% to 50%. These results show that the regulon controlled by the PhoP/Q two-component system makes a major contribution to trafficking of the SCV in macrophages. Segregation of SCVs from the endocytic pathway was also found to be dependent on bacterial proteins synthesized between 15 min and 4 h after uptake into macrophages. However, after this time, protein synthesis was not required to maintain the segregation of SCVs from late endosomes and lysosomes.     

Effect of polyamine synthesis inhibitors separately and in combination with epidermal growth factor on fusion of lysosomes with phagosomes and F-actin level in mouse peritoneal macrophages

Effects of polyamine (PA) synthesis inhibitors--alpha-difluoromethylornithinchloride (DFMO) and alpha-methylornithinchloride (MO)--separately or in combination with the epidermal growth factor (EGF)--on lysosome-phagosome fusion (P-LF) and F-actin content in murine peritoneal macrophages were studied using fluorescent dye Acridine orange for lysosome labelling, FITC-phalloidin for F-actin, and yeast cells as a target. DFMO and MO significantly inhibited P-LF and decreased F-actin content in murine peritoneal macrophages. A combination of DFMO and MO with EGF failed to inhibit P-LF or to decrease F-actin content in these cells. The results obtained with DFMO and MO suggested new cellular targets of their effects. These results may be extended to cancer research to provide a rationale for clinical trials using combinations of EGF with DFMO or MO.     

Acetylated microtubules are required for fusion of autophagosomes with lysosomes

Background  

Autophagy is a dynamic process during which isolation membranes package substrates to form autophagosomes that are fused with lysosomes to form autolysosomes for degradation. Although it is agreed that the LC3II-associated mature autophagosomes move along microtubular tracks, it is still in dispute if the conversion of LC3I to LC3II before autophagosomes are fully mature and subsequent fusion of mature autophagosomes with lysosomes require microtubules.     

LAMP proteins are required for fusion of lysosomes with phagosomes

Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other.     

Rapid-freezing cytochemistry: preservation of tubular lysosomes and enzyme activity

We show that tubular structures present in phorbol ester-stimulated macrophages are sensitive to commonly used chemical fixatives (i.e., they usually become fragmented during fixation). These structures are well preserved in macrophages that are physically fixed by rapid-freezing and subsequent freeze-substitution in osmium-acetone. We have developed methods that combine rapid-freezing, freeze-substitution, and enzyme cytochemistry for preservation of these tubular structures and for detection of endocytosed material (i.e., horseradish peroxidase). This method of rapid-freeze cytochemistry may be useful in other situations where chemical fixation does not adequately preserve cell structures, particularly of membrane compartments.     

Thymocyte maturation following interaction with thymus-derived macrophages.

Murine C57BL/6 thymocytes were cultivated together with syngeneic thymus-derived macrophages (TDM phi) for up to 96 hr to determine whether TDM phi participate in thymocyte maturation. The expression level of H-2b and Thy-1.2 antigens served as thymocyte differentiation surface markers as analyzed by flow cytometry. Indirect immunofluorescent staining profiles of the thymocytes demonstrate a dramatic increase in H-2b expression and a profound decrease in Thy-1.2 expression during cultivation with TDM phi. A similar phenomenon was observed when enriched populations of immature thymocytes were cocultivated with TDM phi. These changes were not observed when thymocytes were cultivated alone or with trypsin-treated TDM phi; neither were they observed when cortisone-resistant thymocytes manifesting mature characteristics were cultivated together with TDM phi. These findings suggest that interaction of thymocytes with TDM phi, involving binding and engulfment, results in the appearance of mature thymocyte subsets.     

The uniformity of phagosome maturation in macrophages

Many studies of endocytosis and phagocytosis presume that organelles containing a single kind of internalized particle exhibit invariant patterns of protein and phospholipid association as they mature inside cells. To test this presumption, fluorescent protein chimeras were expressed in RAW 264.7 macrophages, and time-lapse ratiometric fluorescence microscopy was used to measure the maturation dynamics of individual phagosomes containing IgG-opsonized erythrocytes. Quantitative analysis revealed consistent patterns of association for YFP chimeras of beta-actin, Rab5a, Rab7, and LAMP-1, and no association of YFP chimeras marking endoplasmic reticulum or Golgi. YFP-2xFYVE, recognizing phosphatidylinositol 3-phosphate (PI(3)P), showed two patterns of phagosome labeling. Some phagosomes increased labeling quickly after phagosome closure and then lost the label within 20 min, whereas others labeled more slowly and retained the label for several hours. The two patterns of PI(3)P on otherwise identical phagosomes indicated that organelle maturation does not necessarily follow a single path and that some features of phagosome maturation are integrated over the entire organelle.     

Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes

The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets.These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells.     

Multivesicular bodies isolated from rat hepatocytes. Cytochemical evidence for transformation into secondary lysosomes by fusion with primary lysosomes

Plasma lipoproteins (and other ligands) are endocytosed by hepatocytes and appear in multivesicular bodies (MVBs) in the Golgi-lysosome region of the cell prior to their degradation. We have isolated MVB fractions from livers of estradiol-treated rats, permitting studies of their properties (Hornick et al. 1985). Here we report our cytochemical studies of lysosomal enzyme activity in partially and highly purified MVB fractions and in MVBs in hepatocytes in situ. Only about 15% of partially or highly purified MVBs were positive for acid phosphatase and arylsulfatase, consistent with the prelysosomal nature of this compartment. Partially purified MVB fractions contained small round vesicles, 70-120 nm in diameter, which stained intensely for these enzymes; occasionally these vesicles appeared to fuse with MVBs, suggesting that these structures are primary lysosomes. Such stained vesicles were rarely seen in highly purified MVB preparations. Acid phosphatase reaction product with cerium as capture reagent appeared as uniform precipitates surrounding endocytosed plasma lipoproteins in positively stained MVBs. Arylsulfatase reaction product, however, appeared as distinctive arc or plaque-like deposits just inside the MVB-limiting membrane, often in continuity with intense reaction product contained in a fusing primary lysosome. Similar putative primary lysosomes were occasionally observed in isolated, "intact" Golgi fractions from the same livers. Similar histochemical reactivities of MVBs and putative primary lysosomes were observed in thin sections of hepatocytes in situ. These observations support the conclusion that, in hepatocytes, MVBs represent the immediate prelysosomal compartment in the endocytic pathway of macromolecular catabolism, and suggest that MVBs are converted to secondary lysosomes by direct fusion with primary lysosomes arising from closely adjacent Golgi compartments.     

Cytolysin-dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes (Lm) evades the antimicrobial mechanisms of macrophages by escaping from vacuoles to the cytosol, through the action of the cytolysin listeriolysin O (LLO). Because of heterogeneities in the timing and efficiency of escape, important questions about the contributions of LLO to Lm vacuole identity and trafficking have been inaccessible. Expression of cyan fluorescent protein (CFP)-labelled endocytic membrane markers in macrophages along with a yellow fluorescent protein (YFP)-labelled indicator of Lm entry to the cytosol identified compartments lysed by bacteria. Lm escaped from Rab5a-negative, lysosome-associated membrane protein-1 (LAMP1)-negative, Rab7-positive, phosphatidylinositol 3-phosphate [PI(3)P]-positive vacuoles. Lm vacuoles did not label with YFP-Rab5a unless the bacteria were first opsonized with IgG. Wild-type Lm delayed vacuole fusion with LAMP1-positive lysosomes, relative to LLO-deficient Lm. Bacteria prevented from expressing LLO until their arrival into LAMP1-positive lysosomes escaped inefficiently. Thus, the LLO-dependent delay of Lm vacuole fusion with lysosomes affords Lm a competitive edge against macrophage defences by providing bacteria more time in organelles they can penetrate.     

Tubular lysosomes and their drug reactivity in cultured resident macrophages and in cell-free medium

Lysosomes were assessed in normal living resident mouse peritoneal macrophages, using mainly phase-contrast microscopy (PCM), darkfield microscopy (DFM), and fluorescence microscopy (FM) after terminal acridine orange (AO) staining; these procedures avoided dyes during experimentation. After a few hours of culture a variable proportion of the normal spherical lysosomes began to assemble in a linear fashion. Fully formed tubular structures, with appearances generally recognized as characteristic of tubular lysosomes (TL), could be seen by PCM or, after labeling, by FM, at 2-5 days (best usually at 4-5 days). This peak was followed by a reduction, and at 8-10 days most of the TL had disappeared, leaving only spherical lysosomes. Renewal of the medium at this stage was followed by a temporary reappearance of TL, suggesting that the medium was a major factor in their initial development also. Formation of TL was enhanced by chloroquine (Cq), though to a lesser degree than by phorbol ester (PMA); in contrast NH4Cl (like Cq a weakly basic amine) caused their disassembly into spherical lysosomes. Manual disruption of the monolayer macrophages enabled TL to be transferred to a cell-free medium, in which they remained apparently stable for several hours. Two known microtubule depolymerizers caused disassembly of TL in the intact cells, reinforcing the idea that the TL are associated with the cytoplasmic microtubule (MT) system; but these agents were inactive in vitro, suggesting that disorganization of the system was responsible for this change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular transport of cholesteryl esters from lysosomes to cytoplasm in macrophages

The mechanism through which nonmembranous lipid inclusion bodies consisting of cholesteryl esters accumulate in the cytoplasm was studied. Most lipid inclusion bodies in macrophages after 24 h incubation with anisotropic cholesteryl oleate liquid crystals were surrounded by a limiting membrane. The limiting membrane, however, could not be observed after further incubation for 48 h in the presence of esterastin, which is known to be an inhibitor of lipase and esterase. Under these conditions, the levels of hydrolysis and re-esterification of cholesteryl esters were less than 15% and 5% of the control ones, respectively. These results suggest that the inclusion bodies were transferred from lysosomes to the cytoplasm, with partial hydrolysis of cholesteryl esters, in addition to through the pathway via microsomes.     

Leaky lysosomes in lung transplant macrophages: azithromycin prevents oxidative damage

Background

Lung allografts contain large amounts of iron (Fe), which inside lung macrophages may promote oxidative lysosomal membrane permeabilization (LMP), cell death and inflammation. The macrolide antibiotic azithromycin (AZM) accumulates 1000-fold inside the acidic lysosomes and may interfere with the lysosomal pool of Fe.

Objective

Oxidative lysosomal leakage was assessed in lung macrophages from lung transplant recipients without or with AZM treatment and from healthy subjects. The efficiency of AZM to protect lysosomes and cells against oxidants was further assessed employing murine J774 macrophages.

Methods

Macrophages harvested from 8 transplant recipients (5 without and 3 with ongoing AZM treatment) and 7 healthy subjects, and J774 cells pre-treated with AZM, a high-molecular-weight derivative of the Fe chelator desferrioxamine or ammonium chloride were oxidatively stressed. LMP, cell death, Fe, reduced glutathione (GSH) and H-ferritin were assessed.

Results

Oxidant challenged macrophages from transplants recipients without AZM exhibited significantly more LMP and cell death than macrophages from healthy subjects. Those macrophages contained significantly more Fe, while GSH and H-ferritin did not differ significantly. Although macrophages from transplant recipients treated with AZM contained both significantly more Fe and less GSH, which would sensitize cells to oxidants, these macrophages resisted oxidant challenge well. The preventive effect of AZM on oxidative LMP and J774 cell death was 60 to 300 times greater than the other drugs tested.

Conclusions

AZM makes lung transplant macrophages and their lysososomes more resistant to oxidant challenge. Possibly, prevention of obliterative bronchiolitis in lung transplants by AZM is partly due to this action.