Phorbol ester induces elevated oxidative activity and alkalization in a subset of lysosomes
© Chen; licensee BioMed Central Ltd. 2002
Received: 2 April 2002
Accepted: 6 August 2002
Published: 6 August 2002
Lysosomes are acidic organelles that play multiple roles in various cellular oxidative activities such as the oxidative burst during cytotoxic killing. It remains to be determined how lysosomal lumen oxidative activity and pH interact and are regulated. Here, I report the use of fluorescent probes to measure oxidative activity and pH of lysosomes in live macrophages upon treatment with the tumor promotor phorbol 12-myristate 13-acetate (PMA), and provide novel insight regarding the regulation of lysosomal oxidative activity and pH.
The substrate used to measure oxidative activity was bovine serum albumin covalently coupled to dihydro-2', 4,5,6,7,7'-hexafluorofluorescein (OxyBURST Green H2HFF BSA). During pulse-chase procedures with live macrophages, this reduced dye was internalized via an endocytic pathway and accumulated in the lysosomes. Oxidation of this compound resulted in a dramatic increase of fluorescence intensity. By using low-light level fluorescence microscopy, I determined that phorbol ester treatment results in increased oxidative activity and pH elevation in different subsets of lysosomes. Furthermore, lysosomes with stronger oxidative activity tended to exclude the acidotropic lysosomal indicator, and thus exhibit higher alkalinity.
Results indicate that there is a regulatory mechanism between lysosomal oxidative activity and pH. Activation of lysosomal Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase by phorbol ester may result in increase of intralysosomal O2•- and H2O2, concurrent with pH elevation due to consumption of H+ and generation of OH-. Furthermore, the effect of phorbol ester on elevated oxidative activity and pH is heterogeneous among total lysosomal population. Higher oxidative activity and/or pH are only observed in subsets of lysosomes.
The lysosome is a pivotal organelle responsible for a diverse spectrum of cellular functions including protein and lipid catabolism, vesicular transport and sorting . Abnormal lysosomal physiology may result in cellular dysfunction, and eventually lead to a variety of diseases. For example, deficiencies of various enzymes responsible for lipid metabolism result in lipid accumulation in lysosomal storage diseases such as Niemann-Pick and Gaucher diseases . Alternatively, modulation of lysosomal lumen pH may alter membrane trafficking and associated metabolic processes. It has been reported that elevated lysosomal pH is associated with the genetic lysosomal disorder, Mucolipidosis type IV disease where various sphingolipids and mucolipids accumulate in the lysosome . Finally, lysosomes may play multiple roles in cellular oxidative metabolism . The oxidative activity in lysosomes may regulate the modification of proteins and lipids, resulting in disruption of lysosomal membrane integrity .
The tumor promoter phorbol myristate acetate (PMA) has been shown to activate the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and initiate the oxidative burst response in various phagocytes, including neutrophils and the macrophage-like cell line J774A.1 [6, 7]. During the oxidative burst, generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and superoxide anion (O2•-), is involved in degranulation, secretion of lysosomal enzymes and subsequent cytotoxic killing of invading pathogens. Although activation of the NADPH oxidase complex in phagolysosomal or lysosomal membranes has been reported, in situ generation of either H2O2 or O2•- in these organelles in live cells has not been demonstrated . This is partly due to the lack of appropriate indicators that can monitor the formation of these ROS and the concurrent change of oxidative activity in these vacuoles. Furthermore, since the charged free radical O2•- should have difficulty leaking across hydrophobic membranes, formation of this anion radical inside the lysosome may have an immediate impact on pH in this organelle. The question addressed in the present study is: what is the distribution of oxidative activity and pH changes in the lysosome when cells are stimulated with PMA. A fluorogenic substrate, OxyBURST™ Green H2HFF Bovine Serum Albumin (OxyBURST Green H2HFF BSA), was used to detect lysosomal oxidative activity. OxyBURST Green H2HFF BSA consists of bovine serum albumin coupled to dihydro-2',4,5,6,7,7'-hexafluorofluorescein (H2HFF), a reduced dye with improved chemical stability. The non-fluorescent BSA conjugate is internalized into cells through the endocytic pathway, and accumulates in lysosomes where the extent of its oxidation depends on the oxidative activity in this organelle. The spatial distribution of lysosomal pH in cells after PMA treatment was investigated using both fluorescein-conjugated dextran and a selective lysosomal probe, LysoTracker™ Red. Finally, possible mechanisms of regulation between pH and oxidative activity in lysosomes are discussed.
Results and discussion
Effect of phorbol ester on the oxidative activities of lysosomes
Effect of PMA on the oxidative activity and pH of total lysosomes
Oxidative activity a (gray level/pixel area)
82.1 ± 14.4 (100%)
4.37 ± 0.05
123.9 ± 20.3 (150%)
4.70 ± 0.21
Effect of phorbol ester on lysosomal pH
Regulation of lysosomal pH and oxidative activities
The observation that there are elevations of both oxidative and pH activities in only a subset of lysosomes (Figures 2 and 4) suggests that pH and oxidative activity may be coupled in this organelle. In order to demonstrate this relationship in live cells, the oxidative activities and pH in lysosomes were simultaneously monitored by OxyBURST Green H2HFF BSA and LysoTracker Red DND-99 which is able to qualitatively indicate acidic lysosomes.
One concern regarding to the use of OxyBURST Green H2HFF BSA in the present study is that the accumulation of OxyBURST Green H2HFF BSA itself may drive the increase in pH. If this is the case, then lysosomes containing OxyBURST Green H2HFF BSA should have more alkaline pH. However, this possibility is not consistent with one observation that was presented in Figure 9 where I showed that distribution of OxyBURST Green H2HFF BSA was very homogenous among lysosomes, nevertheless not every lysosomes exhibited elevated lumen pH.
Another novel finding in the present study is the heterogeneous distribution of oxidative activity and pH in lysosomal population responding to PMA treatment. Heterogeneous distribution of low molecular weight redox-active iron has been reported in lysosomal population within cells, which may contribute to differing stability of lysosomal membranes to oxidative stress as shown by Brunk and colleagues . Although the regulation of this heterogeneity is too complicated and beyond the scope of the present study, it does open up another window for investigating lysosomal trafficking in various diseases. For example, the accumulation of lipids in lysosomal storage diseases appears to be heterogeneous [unpublished data]. Oxidative activity and pH distribution may be used as in situ indexes to improve our understanding of lipid accumulation in subsets of lysosomes. Understanding the regulation mechanism will be helpful in developing new treatment strategy for these diseases.
Using a novel indicator for oxidative activity, OxyBURST Green H2HFF BSA, I am able to study the distribution of lysosomal oxidative burst activity and its concurrent pH changes in live macrophage-like J744A.1 cell. The present result indicates that oxidative activity and pH in lysosomes are inter-regulated. Higher oxidative activity results in higher alkalinity while lower lysosomal pH is associated with lower oxidative activity. This approach provides a better opportunity to study the dynamic changes of oxidation and pH in phagolysosomes during phagocytosis, which can not be resolved by conventional biochemical analyses.
Reagents and cell cultures
OxyBURST Green H2HFF BSA, dextrans (10 K, lysine fixable) conjugated with either Cascade Blue or fluorescein fluorophores, LysoTracker Red DND-99 and 5-(and -6)-carboxyfluorescein were from Molecular Probes, Inc. (Eugene, OR). Nigericin, Monensin, Phorbol 12-myristate 13-acetate (PMA), horseradish peroxidase (HRP, type IV) and hydrogen peroxide (H2O2) were purchased from Sigma (St. Louis, MO). Potassium superoxide (KO2) was from Aldrich (Milwaukee, WI). All buffered solutions and other reagents for organic synthesis were analytical grade and freshly prepared.
Murine macrophage-like cell line (J774A.1) was from American Type Culture Collection (Manassas, VA). They were routinely grown in a monolayer (30%-50% confluence) on a glass cover slide at 37°C in DMEM (Dulbecco's Modified Eagle Medium), supplemented with 1% L-glutamine, 1% HEPES [4-(2-hydroxyethyl)piperazine-1-(2-ethanesulfonic acid)], 0.5% gentamicin, 1% penicillin/streptomycin and 10% Fetal Bovine Serum (Gibco, NY).
Measurement of oxidative activity in lysosomes using OxyBURST Green H2HFF BSA
The lysosomal population was first labeled with a Cascade Blue-conjugated dextran as previously described . The cell monolayer was then incubated with 200 μg/mL OxyBURST Green H2HFF BSA in complete culture medium at 37°C for 2 hours. Afterwards, excess dye was removed by washing with phosphate-buffered saline (PBS) and the cells were incubated in complete culture medium in the presence or absence of 200 nM PMA at 37°C for an additional 0.5 hour. The cell monolayer was then washed with PBS and the fluorescent staining of the oxidized OxyBURST Green H2HFF BSA and Cascade Blue-conjugated dextran were then recorded by low-light level fluorescence microscopy with appropriate filters. The fluorescent intensity of the oxidized OxyBURST in the lysosomes was then quantified using the staining of Cascade Blue-conjugated dextran as a mask, as described previously . The average fluorescence intensity (gray level/pixel area) in the mask region was then calculated and represents the relative oxidative activity in lysosomes. This procedure was repeated on images collected from seven or eight different cells in the same culture dish.
Measurement of Lysosomal pH
The pH of the lysosomal population in the J774A.1 cells was measured by a dual excitation ratiometric measurement according to established procedures using the fluorescein-conjugated dextran [3, 8]. Namely, cells were incubated with 2–5 mg/mL fluorescein-conjugated dextran for approximately 18 hours in culture medium. Cells were washed and then incubated for an additional 0.5 hour in fresh culture medium in the presence or absence of 200 nM PMA. Samples were then washed with cold (4°C) MES (4-morpholineethanesulfonic acid) buffer solutions containing 5 mM NaCl, 115 mM KCl, 1.2 mM MgSO4 and 25 mM MES twice, and then observed under fluorescence microscope. Two low light level images of each cell were obtained at green emission region (λem = 515–565 nm), under sequential excitation by first at λex 485 nm, and then at λex 440 nm (10-nm band width). Background fluorescence was corrected by images of blank region of the same culture dish at both excitation wavelengths.
Imaging analysis to calculate the ratio of green fluorescence in lysosomes under excitation by 485 and 440 nm (" [F535(λ485)/F535 (λ440)]") was carried out as previously described . The ratiometric data were converted to pH using a calibration curve from the lysosomes of ionophore-clamped cells. These cells were treated with 10 μM Monensin and 10 μM Nigericine and equilibrated for 2 min with MES (4-morpholineethanesulfonic acid) calibration buffer with pHs from 4.0 to 7.0 prior to image acquisition.
Selective staining of acidic lysosomes
In order to stain acidic lysosomes, LysoTracker Red DND-99 was used. The LysoTracker Red DND-99 is a fluorescent analog of chloroquine, which selectively accumulates in lysosomes . Cells were incubated with a 25 nM concentration of the dye at 37°C for 5 min. Afterwards, excess dye was removed by washing with PBS. The fluorescence distribution was then viewed using a rhodamine filter.
Fluorescence microscopy and image processing
Fluorescence microscopy was performed with an inverted microscope (DIAPHOT-TMD, Nikon) equipped with a Plan Apo 60X (1.4 N.A.) objective and epifluorescence optics for various fluorophores including fluorescein (also for OxyBURST), Cascade Blue and rhodamine. A CCD camera (Quantix, Photometrics Ltd., AR) was used to obtain the microscopic images. Thirty-two fluorescence video images of cells were digitized (16 bits) and averaged using the Metamorph Image Processing system (Universal Imaging Corp., Media, PA).
Stock solutions of OxyBURST Green H2HFF BSA were prepared in phosphate buffered saline (PBS) and stored under nitrogen at 4°C. The HRP-catalyzed oxidation of this compound was determined spectroscopically as follows. To a PBS solution containing 50 μg/mL of OxyBURST Green H2HFF BSA, HRP was added to a final concentration of 4 μg/mL. The resulting solution was incubated at room temperature for 5 minutes to consume any endogenous H2O2. Afterwards, H2O2 was added, with stirring, to a final concentration of 10 nM. The spectral change in emission resulting from the oxidation of this compound was then recorded on a spectrophotometer (F-4500, Hitachi Instruments, Inc., Tokyo, Japan).
To study the effect of pH on the fluorescence of oxidized OxyBURST Green H2HFF BSA and 5-(and -6)-carboxyfluorescein, 78 μg/mL of the oxidized dye or 4.2 μM 5-(and -6)-carboxyfluorescein were prepared in MES (4-morpholineethanesulfonic acid) buffer solutions containing 5 mM NaCl, 115 mM KCl, 1.2 mM MgSO4 and 25 mM MES (pHs from 4.5 to 7.5). The fluorescence emission ratio at λ535 by excitation with λ485 over that by excitation with λ440 was then measured (i.e. F535(λ485)/F535 (λ440)). The effect of pH on the fluorescence of LysoTracker Red DND-99 (final concentration is 2 μM) was similarly performed, except that the emission fluorescence at λ592 was recorded upon excitation at λ574.
The effects of H2O2 and O2•- on the fluorescence of LysoTracker Red DND-99 were studied by preparing 2 μM solutions of the dye in MES buffer solutions at pH 4.5 or 7.5, containing various concentrations of H2O2 and KO2. After incubation at room temperature for 10 min, the emission fluorescence at λ592 was recorded as previously described.
The effect of oxidation on pH-dependency of fluorescein-conjugated dextran was tested in vitro by the HRP/H2O2 oxidant-generating system. Briefly, fluorescein dextran (final concentration at 30 μg/ml) was prepared in MES calibration buffers with pHs ranging from 3.0 to 7.3. These solutions were then incubated at 37°C for 30 min, in the absence or presence of H2O2 and HRP (final concentrations are 1 μM and 4 μg/mL, respectively). The fluorescence emission ratio, F535(λ485)/F535 (λ440), was then measured using SPEX Fluorolog 3 (Jobin Yvon Inc., Edison, NJ).
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