High Lysosomal Activity Research Articles

Macrophages allocate before apoptosis initiation and produce reactive oxygen species during interdigital phagocytosis.

During programmed cell death (PCD), it is commonly accepted that macrophages are recruited by apoptotic cells to complete cell degradation. Interdigital cell death, a classical model of PCD, contributes to digit individualization in limbs of mammals and other vertebrates. Here, we show that macrophages are present in interdigits before significant cell death occurs and remain after apoptosis inhibition. The typical interdigital phagocytic activity was not observed after a partial depletion of macrophages and was markedly reduced by engulfment/phagosome maturation inhibition, as detected by its association with high lysosomal activity. β-galactosidase activity in this region was also coupled with phagocytosis, against its relationship with cellular senescence. Interdigital phagocytosis correlated with high levels of reactive oxygen species (ROS), common in embryo regions carrying abundant cell death, suggesting that macrophages are the major source of ROS. ROS generation was dependent on NADPH oxidases and blood vessel integrity, but not directly associated with lysosomal activity. Therefore, macrophages prepattern regions where abundant cell death is going to occur, and high lysosomal activity and the generation of ROS by an oxidative burst-like phenomenon are activities of phagocytosis.

A new insight of cadmium-induced cellular evidence of autophagic-associated spermiophagy during spermatogenesis.

Autophagy plays a dynamic role in spermatozoa development during spermatogenesis. However, the disruption of autophagic flux induces cell death under metal toxicity and severe oxidative stress. Therefore, we hypothesized that cadmium-induced autophagy might be involved in this mechanism. To verify this hypothesis, we studied cadmium-induced cellular evidence of autophagic-associated spermiophagy within the testis. In the present study, treatment with cadmium caused nuclear depressive disorders and vacuolated mitochondrial damage of Sertoli cells. In addition, spermiophagy through the cellular evidence of spermatozoa phagocytosis, the high lysosomal activity (lysosome engulfment and phagolysosome), and autophagy activity (autolysosome and autophagosome) were observed in the Sertoli cells. The immunohistochemistry of lysosomal membrane protein (LAMP2) to target the phagocytosis of spermatozoa revealed that the immunoreactivity of LAMP2 was overstimulated in the luminal compartment of testis's seminiferous tubules. In addition, the immunohistochemistry and immunofluorescence of autophagy-related protein and microtubule-associated light chain (LC3) results showed the strong immunoreactivity and immunosignaling of LC3 in the Sertoli cells of the testis. Moreover, cadmium caused the overactivation of the expression level of autophagy-related proteins, autophagy-related gene (ATG7), (ATG5), beclin1, LC3, sequestosome 1 (P62), and LAMP2 which were confirmed by western blotting. In summary, this study demonstrated that hazards related to cadmium-induced autophagic-associated spermiophagy with the disruption of autophagic flux, providing new insights into the toxicity of cadmium in mammals and representing a high risk to male fertility.

Silicea terra and Zincum metallicum Modulate the Activity of Macrophages Challenged with BCG In Vitro.

The homeopathic medicines Silicea terra (Sil) and Zincum metallicum (Zinc) modulate macrophage activity and were assessed in an experimental study in-vitro for their effects on macrophage-BCG (Bacillus Calmette-Guérin) interaction. RAW 264.7 macrophages were infected with BCG, treated with different potencies of Sil and Zinc (6cH, 30cH and 200cH) or vehicle, and assessed 24 and 48 h later for bacilli internalization, hydrogen peroxide (H2O2) and cytokine production, and lysosomal activity. Treatment with vehicle was associated with non-specific inhibition of H2O2 production to the levels exhibited by uninfected macrophages. Sil 200cH induced significant reduction of H2O2 production (p < 0.001) compared with the vehicle and all other treatments, as well as higher lysosomal activity (p ≤ 0.001) and increased IL-10 production (p ≤ 0.05). Such effects were considered specific for this remedy and potency. The number of internalized bacilli was inversely proportional to Zinc potencies, with statistically significant interaction between dilution and treatment (p = 0.003). Such linear-like behavior was not observed for Sil dilutions: peak internalization occurred with the 30cH dilution, accompanied by cellular degeneration, and IL-6 and IL-10 increased (p ≤ 0.05) only in the cells treated with Sil 6cH. Sil and Zinc presented different patterns of potency-dependent effect on macrophage activity. Bacterial digestion and a balanced IL-6/IL-10 production were related to Sil 6cH, though reduced oxidative stress with increased lysosomal activity was related to Sil 200cH. Degenerative effects were exclusively related to Sil 30cH, and potency-dependent phagocytosis was related only to Zinc.

Confocal Laser Scanning Microscopy of Morphology and Apoptosis in Organogenesis-Stage Mouse Embryos.

After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde-fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, both of which correlate to apoptotic activity. Thus, LT is a good indicator of apoptosis visualized by confocal microscopy. Results of LT staining of apoptotic cell death correlate well with other whole mount apoptosis vital dyes such as Nile blue sulfate and neutral red, with the added benefit of being fixable in situ. Nile blue sulfate can also be used as a non-vital, nonspecific dye to visualize general morphology. Stains such as acridine orange can be used for surface staining of fixed embryos to yield confocal images that are similar to scanning electron micrographs. Mouse embryos were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Embryos are mounted on depression slides, and serial sections are imaged by confocal microscopy, followed by 3-D reconstruction. Embryos or tissues as thick as 500microns (μm) can be visualized after clearing with BABB. LysoTracker staining reveals apoptotic regions in organogenesis-stage mouse embryos. Morphological observation of tissue was facilitated by combining autofluorescence with Nile blue sulfate staining of fixed embryos or opaque surface staining with acridine orangestaining. The use of BABB for clearing LT vital-stained and fixed embryos matches the refractive index of the tissue to the suspending medium, allowing increased penetration of laser light in a confocal microscope. Nile blue sulfate used as a non-vital dye provides a nonspecific staining of fixed embryos that can then be cleared with methyl salicylate for confocal observation. Sample preparation and staining procedures described here, with optimization of confocal laser scanning microscopy, allow for the detection and visualization of morphological structure and apoptosis in embryos up to 500μm thick, and stained specimens can be fixed and mounted on depression slides.

Understanding macrophage diversity at the ontogenic and transcriptomic levels.

Macrophages are phagocytes characterized by high lysosomal activity and are involved in a wide range of biological processes. Consequently, macrophages have long been recognized for their critical roles in development as well as in healthy and pathological states. Our knowledge about macrophage biology has evolved greatly over the past several years. Significantly, it has now been demonstrated that monocytes are not direct precursors for most tissue-resident macrophages at the steady state. Only few tissue macrophage populations derive from monocytes during homeostasis; rather, monocytes give rise to inflammatory macrophages that infiltrate tissues during inflammation. Tissue-resident macrophages have recently been characterized at the transcriptional level, which provided the basis to uncover the molecular pathways controlling their functional diversity as well as to identify a core signature. Transcription factors controlling specific tissue-resident macrophage populations have been described, suggesting that diversity is under the control of specific regulatory programs. In this review, we discuss and summarize several of the new paradigms emerging in the field of macrophage biology. In particular, we emphasize new findings relevant to macrophage ontogeny, similarities and differences observed across macrophage populations, and gene regulatory programs controlling specialized aspects of tissue macrophage functions.

Pro-oxidant Treatment of Human Prostate Carcinoma (DU145) Induces Autoschizis Cell Death: Autophagosomes Build up out of Injured Endomembranes and Mitochondria

One hour after pro-oxidative treatment by either ascorbate (VC), menadione (VK3), or VC: VK3 combination followed by 24-h incubation in culture medium, DU145 human prostate carcinoma cells developed ultrastructural-dependent organelle damage with the sequence Sham > VC > VK3 > VC: VK3. Along the nuclear alterations and the cytoplasm self-excisions reducing cell size, other induced injuries concerned mitochondria and endomembranes that associated with lysosomes. Damaged organelles surrounded by specialized endoplasmic membranes formed autophagosomes out of phagophores that also captured pieces of glycogen-rich cytoplasm. Most autophagosomes amassed in the diminished-size perikarya and corroborated the enhanced cytotoxicity of the VC: VK3 treatment. These accumulations did not initiate cell death, instead were merely signs of excessive “recycling” of damaged organelles. These features may reflect that high lysosomal activities provided foodstuffs in an ultimate strategy of survival of the tumor cells already devastated by reactive oxidative species (ROS) energetic sites. As such they became transient markers preceding cell death induced to occur by autoschizis and not by apoptosis or other cell deaths. This report could provide more support for the usage of this vitamin combination named APATONE as inexpensive potent adjuvant or treatment in prostate cancers.

T1265 A Novel Near-Infrared (NIFR) Probe Reveals Lysosomal Protease Activity in Intestinal Dendritic Cells (DCS) By Intravital Microscopy

Background: Distinct DCs subsets populate the lamina propria (LP) of the small and large intestine and constantly sample and process food antigens, commensal microbiota and potentially intestinal pathogens. Antigen-processing capacity is known to depend on the lysosomal function of DCs. However, it remains to be determined which specific DCs subsets and immune compartments are responsible for antigen processing in the intestine. Aim: To develop a method for the In Vivo determination of lysosomal function in intestinal DCs by intravital microscopy in order to characterize antigen processing capacity for peptide antigens in different immune compartments of the intestine.Methods: Protease activity in the endosomal system was assessed using a protease-activated NIFR fluorescent probe, which can generate strong NIFR signals after enzyme activation by cathepsin-B, -L, -S and plasmin. In Vitro experiments were performed in RAW264.7 cells incubated with both NIFR probe and DQ-OVA. In Vivo experiments were conducted in CD11c/EYFP mice in which the CD11c promoter drives the expression of EYFP. NIFR probe was intravenously injected 24 hours before experiments or administered into intestinal loops. The uptake of food derived compounds was followed by their autofluorescence. Intravital confocal microscopy was carried out and antigen uptake and protease activity quantitated after 3D tissue reconstructions. Results: The NIFR probe was readily taken up by macrophage-like RAW cells within 1 hour and enriched in endosomal compartments which also contained DQ-OVA at different levels of enrichment. When injected into mice, the NIFR probe labelled the endosomal system of lamina propria DCs in the small intestine in conjunction with uptake of food derived compounds with strong enrichment in villus tips. In contrast, DCs present in PPs and ILFs and displayed low levels of endosomal activity although luminal antigen was collected by DCs in these structures. DCs in the cecal and colonic LP did not demonstrate significant protease activity or antigen uptake. When introduced into the intestinal lumen, the NIFR probe was detected in the endosomal compartment of LP DCs of the small intestine. Conclusion: Myeloid derived DCs throughout the lamina propria of the small intestine sample food antigen and are characterized by high lysosomal activity. In contrast, DCs in PP's and ILFs collect food compounds, but their lysosomal systems are distinctly regulated resulting slower antigens processing. It needs to be determined how the distinct antigen processing of LP DCs and PPs DCs translates into specific antigen presentation and T cell differentiation in these compartments.

Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis

After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays. The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB. Tissues as thick as 500 microm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide). The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red-emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500-microm thick.

Mammalian Apoptosis in Whole Neonatal Ovaries, Embryos and Fetal Limbs Using Confocal Microscopy

The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using a confocal microscope to image whole organs and embryos is the depth of penetration of the laser light into the tissue. We have optimized the confocal microscope performance and developed a sample technique that increases the optical resolution of the system.CLSM has been used to study cellular death (apoptosis) during GD 8-12 normal rat/mouse embryonic development, neonatal ovarian development (PD10-45) and fetal limb development (GD 11-15). LysoTracker Red (LT) was incorporated into the tissue prior to laser excitation. It is aldehyde fixable stain that concentrates into acidic structures or into cells that have high lysosomal activity.

High lysosomal activities in cystic fibrosis tracheal gland cells corrected by adenovirus-mediated CFTR gene transfer

Human tracheal gland serous (HTGS) cells are now believed to be a major target of cystic fibrosis (CF) gene therapy. To evaluate the efficiency of adenovirus-mediated gene transfer in these cells we tested the adenovirus construction containing β-galactosidase cDNA. We observed that the endogenous β-galactosidase activity in cultured CF-HTGS cells was too strong to allow us to detect any exogenous β-galactosidase activity. Immunohistological study on sections of human tracheal tissue confirmed the presence of β-galactosidase in the serous component of the submucosal glands. We then looked for other lysosomal activities in normal and CF-HTGS cells. We showed that normal cells already have elevated enzyme values and that CF-HTGS cells contained 2–4-fold more β-galactosidase, α-fucosidase, α-mannosidase and β-glucuronidase activities than normal cells. An analysis of their kinetic constants has shown that this difference could be attributed to a lower K m of CF lysosomal enzymes. More importantly, these differences are eliminated after adenovirus-mediated CFTR gene transfer and not after β-galactosidase gene transfer.

Involvement of the lysosomal system in yolk protein deposit and degradation during vitellogenesis and embryonic development in trout

AbstractIn adult female rainbow trout (Oncorhynchus mykiss), an immunocytochemical study of the oocyte has shown that a proteolytic enzyme, cathepsin D, is localized in multivesicular bodies (MVB) which begin to differentiate before the phase of vitellogenesis. Estrogens cause the liver to synthesize the protein vitellogenin (VTG), which then enters the systemic circulation. During vitellogenesis, endocytosed VTG is co‐localized with cathepsin D in the MVB. Assays of oocyte cathepsin activities have shown that the only proteolytic activity of note is that of cathepsin D. Along with the yolk proteins derived from VTG, this enzyme will be included in the central coalescent yolk mass.With the installation of the yolk syncytial layer and the surrounding yolk vascular system, embryonic development is characterized by high lysosomal activity, especially in the syncytial layer. At this level, proteolytic activity concerns a cathepsin L, secreted as a proenzyme. We propose the hypothesis that cathepsin D, along with the yolk proteins in the yolk globules that break away from the yolk mass in the vitellolysis zone, activates the proenzyme and leads to protein degradation, which then becomes very rapid. © Wiley‐Liss, Inc.

Mechanisms of Immunomodulation by Drugs

Immunomodulators are those extrinsic or intrinsic substances which regulate or alter the scope, type, duration or competency of the immune response. This paper presents an overview of the mechanisms of immunomodulation, and discusses selected chemical and biologic substances which are capable of modifying the immune or biologic response of the organism. The immunopharmacology, including in vivo and in vitro assays, of a novel acridine immunomodulator is discussed. This low molecular weight compound is an immunomodulator and anti-cancer adjuvant, which has been shown to induce high levels of circulating interferon in mice, protect mice against lethal viral infection, stimulate macrophage and NK cell cytotoxicity for tumor cells, partially restore humoral and cellular immune responses in tumor bearing immunosuppressed mice, and augment the cytotoxic T-lymphocyte response to syngeneic tumor cells. Tissue changes, consisting of presence of drug bound to lysosomal membranes, perivascular infiltrates in mouse liver, glomerular hyalinization in mouse kidney, and focal myocardial changes in mice are described. The compound persists intracellularly for extended periods of time in cells with high lysosomal activity. The tissue changes are interpreted to be a result of overloading of cellular mechanisms for elimination from the cells involved.

Enzyme activities in the rabbit cornea during immunogenic keratitis.

In rabbits inflammation of the cornea was induced by intrastromal injection of horse serum. Between 2 and 4 weeks after injection, infiltration of the cornea with leukocytes and neovascularization could be observed. During this period, the rabbits were killed and their corneas analyzed for protein, alkaline phosphatase, acid phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase. The enzyme activities in the inflamed corneal stroma reflect the high lysosomal activity, which probably originates from the leukocytes. The enzyme activities in the epithelium indicate that the tissue is abnormal and undergoing repair processes.

The effect of litoralon (gamma- l-glutamyl-taurine) on the lysosomal activity of mesenchymal cells and macrophages

1. 1. The metamorphosis influencing effect of litoralon becomes manifest partly in the enhancement of the lysosomal activity of mesenchymal cells, partly in the early appearance and partly in the increase in the number of macrophages. 2. 2. From the three developmental stages tested, stage 26 is characterized by the increase of lysosomal activity of macrophages. No effect was observed in stage 27. In stage 28 a cell type with high lysosomal activity appears at the margin and in the median area of the tail fin. 3. 3. Morphogenic processes are enhanced in stages 27 and 28.

The fine structure of lipofuscin in the human inner ear

Lipofuscin inclusions in the human membranous labyrinth were studied by electron microscopy. Lipofuscin is morphologically an irregularly shaped, membrane-bound inclusion consisting of an electron-dense structure. The most common component was a fine, granular, osmiophilic substance which was always associated with a homogenous, spherical structure resembling a lipid droplet. The combination of these two components was frequently observed in the human inner ear. Distended inclusions containing lipofuscin components were also observed within the supporting cells, saccular, utricular and ampullar wall, the epithelial cells of the transitional zone and in the dark cells. Lipofuscin is closely associated with lysosome and is known to accumulate in the tissue as a result of aging. The high lysosomal activity possibly may result in lipofuscin formation in the human inner ears. Also some other unknown metabolic conditions may provide the deposits of lipofuscin.