Spontaneous lysosomal enzyme secretion by a murine macrophage-like cell line.
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ABSTRACT:
SUBMITTER: Jessup W
PROVIDER: S-EPMC1162168 | biostudies-other | 1980 Sep
REPOSITORIES: biostudies-other
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Project description:Rapid and parallel secretion of lysosomal beta-N-acetylglucosaminidase and preloaded fluorescein-labelled dextran was initiated in macrophages by agents affecting intracellular pH (methylamine, chlorpromazine, and the ionophores monensin and nigericin). In order to evaluate the relative role of changes in lysosomal and cytosolic pH, these parameters were monitored by using pH-sensitive fluorescent probes [fluorescein-labelled dextran or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein]. All agents except chlorpromazine caused large increases in lysosomal pH under conditions where they induced secretion. By varying extracellular pH and ion composition, the changes in lysosomal and cytosolic pH could be dissociated. Secretion was then found to be significantly modulated by changes in cytosolic pH, being enhanced by alkalinization and severely inhibited by cytosolic acidification. However, changes in cytosolic pH in the absence of stimulus were unable to initiate secretion. Dissociation of the effects on lysosomal and cytosolic pH was also achieved by combining stimuli with either nigericin or acetate. Further support for a role of intracellular pH in the control of lysosomal enzyme secretion was provided by experiments where bicarbonate was included in the medium. The present study demonstrates that an increase in lysosomal pH is sufficient to initiate lysosomal enzyme secretion in macrophages and provides evidence for a significant regulatory role of cytosolic pH.
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Project description:Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). CRISPR-Cas9 targeting to coding sequences of β-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.
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Project description:Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.
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Project description:PurposeTo assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383.MethodsFor all drugs, uptake at 5 μM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1-100 μM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated.ResultsBoth cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.ConclusionsThis study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.
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Project description:A receptor for beta-glucan was in the present study shown to mediate binding of zymosan particles to resident mouse peritoneal macrophages. Lysosomal enzyme secretion in response to zymosan was maximal at a low particle/cell ratio, continuous for at least 3 h after particle/cell contact and inhibitable by soluble glucan. Latex particles of various size caused no selective secretory response, but at high particle/cell ratios were toxic. By use of a fluorescent ligand, the macrophage beta-glucan receptor was shown to be trypsin-sensitive, Ca2+/Mg(2+)-independent, recirculating and also present in an intracellular mobilizable pool. Binding of ligand to the beta-glucan receptor and inhibition of the lysosomal secretory response to zymosan were both more efficient with glucans of larger size, indicating that clustering of glucan receptors at the cell surface occurs. Such clustering could stabilize ligand binding by multiple interactions and possibly trigger intracellular signaling events on binding of zymosan particles.
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