Depletion of secondary lysosomes in mouse macrophages infected with Leishmania mexicana amazonensis: a cytochemical study

Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes of L. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reappeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5 degrees C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.     

Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana-infected macrophages.

Secondary lysosomes of cultured mouse peritoneal macrophages were labeled with the electron-dense colloid saccharated iron oxide; the identity of the labeled structures was checked by the Gomori reaction for acid phosphatase. Amastigotes of Leishmania mexicana mexicana derived from mouse lesions were used to infect these macrophages in vitro. In electron micrographs of thin sections of infected macrophages the labeled secondary lysosomes were seen fused with the parasitophorous vacuoles without preventing subsequent multiplication of the parasites. A similar fusion probably occurs in vivo, and may provide a pathway through which not only nutrients but also drugs and host antibodies could reach the intracellular parasite.     

Fusion of Host Cell Secondary Lysosomes with the Parasitophorous Vacuoles of Leishmania mexicana-inlected Macrophages

SYNOPSIS. Secondary lysosomes of cultured mouse peritoneal macrophages were labeled with the electron-dense colloid saccharated iron oxide; the identity of the labeled structures was checked by the Gomori reaction for acid phosphatase. Amastigotes of Leishmania mexicana mexicana derived from mouse lesions were used to infect these macrophages in vitro. In electron micrographs of thin sections of infected macrophages the labeled secondary lysosomes were seen fused with the parasitophorous vacuoles without preventing subsequent multiplication of the parasites. A similar fusion probably occurs in vivo , and may provide a pathway through which not only nutrients but also drugs and host antibodies could reach the intracellular parasite.     

Cytochemical study of macrophage lysosomal inorganic trimetaphosphatase and acid phosphatase

Cytochemical investigations have associated acid inorganic trimetaphosphatase (TMPase) activity with the lysosomes of certain cell types. We have used the modified staining technique of Berg to show that this enzyme activity is present in normal mononuclear phagocytes and macrophage cell lines. We have found this enzyme activity to be present in murine RAW264 macrophages, in human U937 macrophages, in normal human blood monocytes, and in guinea pig peritoneal macrophages. All of the RAW264 and U937 macrophages showed intense TMPase activity. Many of the human monocytes and most of the guinea pig macrophages were labeled by this method. The reaction product was associated with the lysosomes of these cell types. The lysosomal staining-pattern was similar to that of acid phosphatase. Differences with regard to Golgi staining were noted. This indicates that TMPase is a lysosomal enzyme of mammalian macrophages. The distinction between TMPase and acid phosphatase activity has been demonstrated by measuring the pH optimum of each enzyme. Using substrates identical to those of the ultrastructural cytochemistry, we show that the pH optimum of TMPase is 4.0 and that of acid phosphatase is 5.0. The enzymatic activities are therefore ultrastructurally and biochemically distinct. Following phagocytosis of latex, yeast (Saccharomyces cerevisiae), or Corynebacterium parvum, TMPase has been found to be associated with phagosomes. This enzyme may take part in the degradation of phagocytosed materials, particularly microorganisms which contain inorganic polyphosphates and metaphosphates.     

Differentiation of a cell line of mouse myeloid leukemia : I. Simultaneous induction of lysosomal enzyme activities and phagocytosis

Induction of phagocytic activity in the Ml cell line of mouse myeloid leukemia, on being exposed to a conditioned medium from cultured embryo cells, was accompanied by an increment in the activities of both lysosomal acid phosphatase and acid protease. The activity of these lysosomal enzymes, as well as that of phagocytosis, was not induced when Ml cells were incubated either with the conditioned medium subjected to heat treatment or in the presence of 5-bromodeoxyuridine (BUdR). The levels of these induced enzyme activities in Ml cells were comparable to those in normal mouse peritoneal macrophages. The lysosomal enzyme activity in Mm-1 cells, which were spontaneously differentiated from Ml cells and exhibiting a higher phagocytic activity, were reminiscent of those in peritoneal macrophages. Based on these observations, it was concluded that both phagocytosis and lysosomal enzyme activity occur simultaneously during the course of differentiation. This differentiation, morphological or functional, in Ml cells in the presence of the conditioned medium was further supported by biochemical evidence.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

Trypanosoma cruzi: phagolysosomal fusion after invasion into non professional phagocytic cells

Parasite-containing endocytic vacuoles are formed during the process of in vitro interiorization of the trypomastigote forms of Trypanosoma cruzi by primary culture of mouse fibroblasts, heart and skeletal muscle cells. Fusion of these vacuoles with host cell lysosomes takes place. The process of T. cruzi-muscle cell interaction was analysed by ultrastructural cytochemistry. Two lysosomal enzymes, acid phosphatase and aryl sulphatase and the fusion of peroxidase-labeled secondary lysosomes with the parasitophorus vacuoles were studied. These finding indicate that the basic mechanism of interaction of T. cruzi with the so called non phagocytic cells is similar to that which occurs with phagocytic cells.     

Leishmania mexicana: acid phosphatase ultrastructural cytochemistry of infected mouse macrophage cultures treated with phenazine methosulfate

Bone marrow-derived mouse macrophage cultures infected with Leishmania mexicana amazonensis amastigotes were given a 2-hr pulse with 10 microM phenazine methosulfate (PMS), a cationic electron carrier which destroys the intracellular parasites. Cultures were fixed at different times after the PMS pulse and processed for the detection of acid phosphatase (AcP) activity at the electron microscopic level. Only a small proportion of nontreated, infected macrophages stained for AcP. In contrast, 2 to 6 hr after exposure to PMS, many infected cells displayed AcP-positive lysosomes and parasitophorous vacuoles. This increased AcP reactivity paralleled the reduction in the percentage of morphologically intact parasites. In addition, qualitative observations indicated that while nontreated infected cells contained only few recognizable lysosomes, the lysosomal complement noticeably increased a few hours after exposure to PMS. Most intact intracellular amastigotes were not stained, but damaged parasites were often positive for AcP. Twenty hours after the PMS pulse, the percentage of AcP-positive macrophages dropped to the levels initially present in noninfected cultures and all of the parasites were destroyed. Exposure of noninfected macrophages to PMS did not affect their AcP reactivity.     

Isolation and properties of lysosomes from dark-grown potato shoots

Summary A method is described for the isolation of lysosomal fractions from dark-grown potato shoots using a single stage separation on a Ficoll gradient. Peaks of acid hydrolase activity consisting of acid phosphatase, phosphodiesterase, ribonuclease, carboxylic esterase and -glycerophosphatase were well separated from peaks of mitochondrial and glyoxysomal enzymes. A heavy lysosomal fraction with particle diameters from 0.1 to 1.6 and density of 1.10 g cm^-3 containing relatively low hydrolase activity was distinguishable from a light fraction with diameters 0.025 to 0.6 and density of 1.07 g cm^-3 with a higher level of hydrolase activity. Both fractions appeared heterogeneous by electron microscopy, but the fine structure of the membranes of both heavy and light lysosomes was similar. The heavy lysosomal fraction was rich in autophagic vacuoles (secondary lysosomes) containing organelles and amorphous cytoplasmic material. Both fractions were rich in ribonucleic acid.Freezing and thawing, high speed blending and ultrasonication either singly or in combination solubilised a maximum of ca. 30% of the acid phosphatase from crude lysosomal fractions derived from dark-grown potato shoots. Treatment with Triton X-100 and deoxycholate released appreciably more enzyme activity but acetone and carbon tetrachloride failed to solubilise any acid phosphatase. Only detergent treatments gave marked overrecovery of enzyme and indicated structure-linked latency. Liberation of enzyme from lysosomes varied with pH and was almost complete at both extremes of pH. Crude snake venom was rapid and effective in solubilising acid phosphatase from lysosomal preparations, purified phospholipase A was less effective and phospholipases C and D had negligible effects. Phospholipase and venom mediated release of acid phosphatase was accompanied by the coincident release of an acid end-product. Gel filtration of acid phosphatase liberated from heavy and light lysosomal fractions by snake venom digestion revealed that each of these fractions was characterised by the presence of distinct molecular forms of the enzyme. The nature of the association of acid phosphatase with potato shoot lysosomes is discussed.     

The Correlation of Digestive Vacuole pH and Size with the Digestive Cycle in Paramecium caudatum1

ABSTRACT. The temporal changes in the size and pH of digestive vacuoles (DV) in Paramecium caudatum were reevaluated. Cells were pulsed briefly with polystyrene latex spheres or heat-killed yeast stained with three sulfonphthalein indicator dyes. Within 5 min of formation the intravacuolar pH declined from ～7 to 3. With the exception of a transient and early increase in vacuolar size, vacuole condensation occurred rapidly and paralleled the acidification so that vacuoles reached their lowest pH and minimal size simultaneously. Neutralization and expansion of vacuole size began when vacuoles were GT8 min old. No labeled vacuoles were defecated prior to 21 min after formation but almost all DV were defecated within 1 h so that the digestive cycle of individual vacuoles ranged from 21 to 60 min. Based on these size and pH changes, the presence of acid phosphatase activity, and membrane morphology, digestive vacuoles can be grouped into four stages of digestion. The DV-I are GT6 min old and undergo rapid condensation and acidification. The DV-II are between 4 to 10 min old and are the most condensed and acidic vacuoles. The DV-III range in age from 8 to ～20 min and include the expanding or expanded vacuoles that result from lysosomes fusing with DV-II. The DV-IV are GD21 min old, and since digestion is presumably completed, they can be defecated. The rise in intravacuolar pH that accompanies vacuole expansion suggests that lysosomes play a role in vacuole neutralization in addition to their degradative functions. The acidification and condensation processes in DV-I appear to be unrelated to lysosomal function, as no acid phosphaiase activity has been detected at this stage, but may be related to phagosomal functions important in killing food organisms, denaturing proteins prior to digestion, and preparing vacuole membrane for fusion with lysosomes.     

Symbiotic Chlorella vulgaris of the ciliate Paramecium bursaria plays an important role in maintaining perialgal vacuole membrane functions

Treatment of symbiotic alga-bearing Paramecium bursaria cells with a protein synthesis inhibitor, cycloheximide, induces synchronous swelling of all perialgal vacuoles at about 24h after treatment under a constant light condition. Subsequently, the vacuoles detach from the host cell cortex. The algae in the vacuoles are digested by the host's lysosomal fusion to the vacuoles. To elucidate the timing of algal degeneration, P. bursaria cells were treated with cycloheximide under a constant light condition. Then the cells were observed using transmission electron microscopy. Results show that algal chloroplasts and nuclei degenerated within 9h after treatment, but before the synchronous swelling of the perialgal vacuole and appearance of acid phosphatase activity in the perialgal vacuole by lysosomal fusion. Treatment with cycloheximide under a constant dark condition and treatment with chloramphenicol under a constant light condition induced neither synchronous swelling of the vacuoles nor digestion of the algae inside the vacuoles. These results demonstrate that algal proteins synthesized during photosynthesis are necessary to maintain chloroplastic and nuclear structures, and that inhibition of protein synthesis induces rapid lysis of these organelles, after which synchronous swelling of the perialgal vacuole and fusion occur with the host lysosomes.     

Cytochemical localization of acid phosphatase in light- and dark-adapted eyes of a polychaete worm,Nereis limnicola

Summary The amount and distribution of the lysosomal enzyme acid phosphatase in light- and dark-adapted eyes of the brackish-water annelid Nereis limnicola were studied by standard cytochemical techniques. Precipitate from the acid phosphatase reaction was observed in Golgi-endoplasmic reticulum-lysosomal complexes, primary lysosomes, and secondary lysosomes, formed by fusion of primary lysosomes with phagocytic and pinocytic vesicles containing products of presumed rhabdomeric degradation. The acid phosphatase reaction occurred in these organelles in both sensory and supportive cells of both light- and darkadapted ocelli. Secondary lysosomes were more abundant in sensory cells of illuminated ocelli than in those maintained in the dark. Sparse reaction product was found in Golgi cisternae, none in rough endoplasmic reticulum. We suggest that the increase of lysosomal activity in light-adapted eyes is correlated with the breakdown of photosensory microvilli upon exposure to light. A diagram of our interpretation of recycling of photoreceptoral membrane in N. limnicola is presented.     

Effects of some biologically active compounds on phagosome-lysosome fusion in peritoneal macrophages of mice.

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P-L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P-L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes. All three compounds tested increase F-actin content in peritoneal macrophages. Thus, the effect of BR on P-L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P-L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.     

In vitro effects of hormones and autacoids on the activity of acid phosphatase in the lysates of endotoxin-activated rat peritoneal and bronchoalveolar macrophages

Peritoneal and bronchoalveolar macrophages activated in vitro by endotoxin, exhibit alterations in the acid phosphatase activity of cell lysates when certain hormones or autacoids are present in the culture medium. They also show morphological changes concerning general appearance and acid phosphatase cytochemistry. Certain agents known to increase the intracellular levels of cyclic AMP, such as dopamine and prostaglandin E2, decreased this enzyme activity in the lysates of peritoneal macrophages. Adrenalin had no effect on this activity at 14 hours, but was found to increase the activity in the culture medium at the initial hours of incubation. Glucagon decreased whereas insulin increased acid phosphatase activity in bronchoalveolar macrophages. Serotonin or histamine, known to activate phospholipase C, increased this activity in peritoneal or bronchoalveolar macrophages. The results of this study, taken together with previously published data (Kondomerkos et al., 2003), suggest that hormones and autacoids may control certain parameters of macrophage activation including acid phosphatase activity.     

Leishmania mexicana: a cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow-derived macrophages

The cellular localization and activity of the lysosomal enzymes acid phosphatase, trimetaphosphatase, and arylsulfatase were studied in rat bone marrow-derived macrophages infected with Leishmania mexicana amazonensis amastigotes. The specific activity of acid phosphatase normalized for protein content was similar in normal macrophages and in isolated amastigotes, whereas the latter were markedly deficient in trimetaphosphatase and arylsulfatase activities. It is thus likely that trimetaphosphatase and arylsulfatase activities detected in infected macrophages were of host cell origin. The activities of the three enzymes, assayed biochemically, varied independently in the infected macrophages. While arylsulfatase activity was unchanged after infection, the activity of acid phosphatase increased by 19, 40, and 94% at 6, 24, and 48 hr, respectively. Trimetaphosphatase activity rose only slightly during the first 24 hr after infection but increased by 74% at 48 hr. The rise in acid phosphatase activity could be accounted for only partially by multiplication of the amastigotes. Thus, as for trimetaphosphatase, these results suggest enhanced macrophage synthesis of acid phosphatase and/or reduced enzyme degradation by the infected macrophages. The reduction in host cell lysosomes previously described (Ryter et al. 1983; Barbieri et al. 1985) was confirmed but appearance of lysosomal enzyme activity in the parasitophorous vacuole is documented in the present report. Thus, Leishmania do not seem to reduce the amount and the activity of host lysosomal enzymes.     

Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages

, , and 1986. Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages. International Journal for Parasitology 16: 629–632. Prelabelling of lysosomes with acridine orange has been performed in order to verify whether metacyclic forms of Trypanosoma cruzi are capable of inhibiting lysosomal fusion during the first moments of interiorization in non-sensitized mouse peritoneal macrophages. Thus, the degree of degranulation (lysosomal fusion) in metacyclic forms is low while epimastigote forms present higher levels. When epimastigote forms are made to interact with the macrophages in the presence of various concentrations of the medium used for transformations of epimastigotes to metacyclic forms or when interaction was performed in the presence of NH₄Cl, the degree of degranulation was similar to that obtained when interaction was carried out with metacyclic forms.

The present results suggest that during the first moments of the interaction of T. cruzi, only the infective forms may increase the cytoplasmic pH value of the host phagocytic cell, avoiding lysosomal fusion and the subsequent destruction of the parasite.     

Electron microscopic study of intracellular digestion in intestinal cells of the ixodid tick Hyalomma asiaticum. I. Formation of primary and secondary lysosomes]

The formation of primary and secondary lysosomes in digestive cells of midgut of the tick H. asiaticum was investigated using ultracytochemical methods for acid phosphatase. This enzyme is synthesized in the rough endoplasmic reticulum cisternae to be concentrated in the Golgi complex. Vesicles 0.1-0.15 mum in diameter filled with the enzyme are propagated from the distal Golgi cisternae which are primary lysosomes. Secondary lysosomes are produced in result of fusion of primary lysosomes with heterophagosomes that appear during endocytosis. Another type of structures responsible for transport of lysosomal enzymes into heterophagosomes is represented by dense bodies 0.3-0.5 mum in size. These are rich in acid phosphatase being different stages of heterophagolysosomes and telolysosomes.     

Ultrastructural investigations on the lysosomal system of lymph node lymphocytes in transplantation immunity.

Activation of the lysosomal system in T lymphocytes is demonstrated in connexion with immune stimulation, by electron microscopic study of the lymph nodes draining skin allografts. The development of primary lysosomes from enlarged Golgi apparatus, their fusion with autophagic and secretory vacuoles forming secondary lysosomes, of the cytolysome and crinophagosome type, were identified by the ultrahistochemical acid phosphatase reaction, suggesting the involvement of these organelles either in blastic transformation or in the effector function of lymphocytes, in connexion with the elaboration of active factors and cytotoxic events.     

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.     

Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 macrophages

J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH4Cl induced a 12-fold increase in TGase secretion. NH4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.