Molecular Vision 2012; 18:103-113 <http://www.molvis.org/molvis/v18/a13>
`Received 5 August 2011 | Accepted 12 January 2011 | Published 17 January 2012
`
`© 2012 Molecular Vision
`
`Accumulation and autofluorescence of phagocytized rod outer
`segment material in macrophages and microglial cells
`
`Lei Lei,1,2 Radouil Tzekov,1 Shibo Tang,2 Shalesh Kaushal1
`
`1The Department of Ophthalmology, University of Massachusetts Medical School, Worcester, MA; 2State Key Laboratory of
`Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
`
`Purpose: To explore the ability of macrophages and microglial cells to phagocytize rod outer segments (ROSs) in a cell
`culture and characterize the resulting lipofuscin-like autofluorescence (LLAF).
`Methods: Either regular or modified ROSs or ROS components (11-cis-retinal, all-trans-retinal, lipids) were fed to
`macrophages and microglial cells for 4 days. Afterwards, autofluorescence was detected by fluorescence-activated cell
`sorting (FACS) at two different wavelengths (533 nm and 585 nm), and the cells were imaged by confocal and electron
`microscopy. Fluorescein isothiocyanate (FITC)-labeled ROSs were added to macrophage and microglial cell cultures for
`1–24 h to determine the kinetics of phagocytosis in these cell lines.
`Results: Feeding with different ROSs or ROS components led to a significant increase in LLAF in both microglia and
`macrophages. The 4-hydroxynonenal (HNE)-modified ROSs gave rise to the highest increase in LLAF at both 533 nm
`and 585 nm. Application of 11-cis-retinal or all-trans-retinal resulted in higher LLAF at 585 nm, compared to application
`of 9-cis-retinal or liposomes. Fluorescein isothiocyanate-labeled ROSs co-localized well with lysosomes in both types of
`cells. HNE-modified ROSs were phagocytized more rapidly by both types of cells, compared to unmodified ROSs.
`Electron microscopy demonstrated inclusion bodies containing whorls of membranes in all types of cells fed with ROSs.
`Conclusions: Both macrophages and microglia have the ability to phagocytize ROSs, and this results in increased
`autofluorescence. Oxidation of ROSs results in faster phagocytosis, higher levels of LLAF, and the appearance of more
`inclusion bodies inside the cells. Results from the present study suggest that both types of cells accumulate lipofuscin-
`like material under physiologically relevant conditions. Such accumulation could interfere with their ability to clear cellular
`debris and could be part of the pathogenetic mechanism for age-related macular degeneration and other lipofuscinopathies.
`
`Lipofuscin is a polymorphous substance consisting of
`granular yellow-brown pigment granules composed of lipid-
`containing residues of lysosomal digestion, which are
`autofluorescent and accumulate in many tissues during
`senescence [1]. Excessive accumulation of lipofuscin could
`compromise essential cell function and, therefore, contribute
`to many age-related diseases, including age-related macular
`degeneration (AMD) [2]. There is clear clinical and
`pathological evidence for the age-dependent accumulation of
`lipofuscin in the retinal pigment epithelium (RPE). Recently,
`some studies reported that microglial cells and macrophages
`are also involved in the process of lipofuscin accumulation
`[3]. Specifically, the authors reported that subretinal microglia
`containing autofluorescent granules accumulated in an age-
`dependent manner in the subretinal space of adult normal mice
`and the number of autofluorescent microglial cells was higher
`compared to the number of autofluorescent cells at the same
`age. The autofluorescence emission fingerprints were similar
`between these cells and RPE cells that accumulate lipofuscin.
`Similarly, another recent report found accumulation of
`
`to: Shalesh Kaushal, The Department of
`Correspondence
`Ophthalmology, University of Massachusetts Medical School, 381
`Plantation Street, Worcester, MA, 01605; Phone: (352) 362-6058;
`FAX: (508) 856-1552; email: shalesh.kaushal@umassmemorial.org
`
`autofluorescent subretinal macrophages in aging ccl-2
`knockout mice that have some phenotypic features resembling
`human AMD [4]
`The ability of RPE cells to phagocytize the tips of rod
`outer segments (ROSs) as part of the outer segment daily
`renewal process is very important for the normal functioning
`of the retina and has been reported and discussed widely [5].
`However, very little is known about the ability of either
`macrophages or microglia to phagocytize and degrade ROS
`material. Nor is there much known about the contributions of
`different natural components of ROSs to lipofuscin-like
`autofluorescence (LLAF) from these cells. Therefore, we
`examined the phagocytic ability of macrophages and
`microglia for unmodified and modified ROSs and the
`resulting change in LLAF in a cell culture model. An
`accumulation of undegraded ROS material and a proportional
`increase in LLAF was observed in both types of cells.
`
`METHODS
`Murine macrophage and MyD/Trif DKO microglial cell
`cultures and treatment: The macrophage cell line was the
`immortalized mouse macrophage cell line A3.1A [6]. The
`microglial cell line was derived from immortalized microglial
`cells from MyD88/TRIF double KO, which consists of
`microglial cells that retain their morphological and functional
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`Molecular Vision 2012; 18:103-113 <http://www.molvis.org/molvis/v18/a13>
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`© 2012 Molecular Vision
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`Figure 1. Spectrum of rhodopsin in rod
`outer segments. The characteristic
`rhodopsin absorbance at 498 nm was
`used to quantitate pigment yields. Note
`the absorbance difference at
`that
`wavelength between bleached rod outer
`segments (ROSs; green trace) and
`unbleached ROSs (orange trace). The
`modified rod outer segments (HNE-
`ROSs) at 5 mM (red trace) showed
`decrease in absorbance compared to
`unbleached ROSs.
`
`characteristics [7]. The cells were grown in high glucose
`Dulbecco s Modified Eagle Medium (DMEM, Cellgro/
`Mediatech, Manassas, VA) supplemented with 10% heat-
`inactivated fetal calf serum (FCS, Sigma-Aldrich, St. Louis,
`MO), 1% penicillin/streptomycin (Gibco, Grand Island, NY),
`1% HEPES, 1% non-essential amino acid solution (NEAA),
`and 1:1,000 ciprofloxacin at 37 °C in the presence of 5%
`CO2. Cells cultured in 10 cm plate were trypsinized and plated
`in 24 well plates or an 8-well chamber slide at a confluent
`density of 1.66×105/cm2. ROSs were obtained from Invision
`BioResources (Seattle, WA) and isolated by a method similar
`to the one described by Papermaster [8]. All ROS preparations
`were incubated on a shaker at room temperature, overnight.
`After an additional culturing for 3 days, different types of ROS
`at 2 μg/ml were added every day for 4 days. The types of ROS
`were unbleached ROSs (prepared in the dark), bleached ROSs
`(exposed to 700 lux white light for 1 h), and 4-hydroxynonenal
`(HNE)-modified ROSs (see below). Additionally, several
`ROS components were added separately: 11-cis-retinal
`(10 μM, National Eye Institute, Bethesda, MD), all-trans-
`retinal (10 μM, Sigma-Aldrich, St. Louis, MO), 9-cis-retinal
`(10 μM, Sigma-Aldrich). Liposomes were prepared as
`described below and fed at concentration of 10 μM.
`Modification of rod outer segments: The 4-hydroxynonenal
`(Cayman Chemical, Ann Arbor, MI) was prepared as
`previously described [9]. ROSs were incubated with 5 mM
`HNE at room temperature overnight on a shaker. Unbound
`HNE was removed by repeated washes in PBS 1× strength
`solution (derived from 10× solution by dilution with distilled
`deionized water, PBS 10×, Fisher BioReagents, BP3994;
`Fisher Scientific, Pittsburg, PA). The protein content of ROS
`preparations was measured using a BioRad BC kit (Bio-Rad,
`
`Hercules, CA). Modified ROSs were stored at −80 °C until
`use.
`Extrusion of liposomes: Phosphatidylethanolamine (PE,
`Avanti Polar Lipids, Alabaster, AL) and phosphatidylcholine
`(PC, Avanti Polar Lipids) were mixed at a ratio of 60%:40%
`to
`a
`final
`concentration
`of
`10 mM.
`The
`phosphatidylethanolamine used was 1,2-didocosahexaenoyl-
`sn-glycero-3-phosphoethanolamine (22:6 PE), and
`the
`phosphatidylcholine used was 1,2-dioleoyl-sn-glycero-3-
`phosphocholine (18:1 [Δ9-Cis] PC). The solvent was dried out
`by argon at room temperature and 1x PBS was added.
`Liposomes were extruded with Avanti Mini-Extruder (Avanti
`Polar Lipids) to extrude liposomes to an average diameter of
`100 nm. Extruded vesicles were stored at 4 °C for 3–4 days.
`Flow cytometry: Cells were cultured in 24-well plates and
`incubated with different components as described above (see
`Murine macrophage and MyD/Trif DKO microglia cell
`cultures and treatment). Cells were repeatedly washed,
`detached with trypsin, and analyzed on a C6 flow cytometer
`(Accuri Cytometers, Ann Arbor, MI). A gate was set to
`exclude cell debris and cell clusters, and 10,000 gated events
`were recorded. Experiments were performed in triplicates.
`Two channels were used: the FITC/GFP channel (excitation
`laser wavelength, 488 nm; detection filter wavelength, 533/30
`nm) and the PE/PI channel (excitation laser wavelength, 488
`nm; detection filter wavelength, 585/40 nm). For each
`condition and cell type, the fluorescence detected from the
`control samples was averaged, and the fluorescence detected
`from the test samples was expressed as a fraction of the
`averaged control value. Thus, the values are presented as a
`ratio (autofluorescence ratio [AF ratio]) compared to the
`fluorescence recorded from the control.
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`of
`2. Autofluorescence
`Figure
`macrophages and microglial cells after
`4 day feeding with different rod outer
`(ROSs). A: This
`segments
`is a
`fluorescence-activated
`cell
`sorting
`(FACS) analysis of the fluorescein
`isothiocyanate
`(FITC)
`channel
`(detection filter, 533/30 nm) of the
`microglial cells
`(left panel) and
`macrophage cells (right panel) fed with
`different ROSs. Note the large increase
`of AF in cells fed with modified rod
`outer segments (HNE-ROSs) compared
`to the other two groups. B: FACS
`analysis in the PE channel (detection
`filter, 585/40 nm) of the microglial cells
`(left panel) and macrophage cells (right
`panel) fed with different ROSs. Note the
`slight increase in the bleached ROS
`group and the decrease in HNE-ROSs
`compared to the corresponding AF
`registering at 533 nm. Abbreviation key:
`BL-ROSs=bleached
`rod
`outer
`segments; UB-ROSs=unbleached rod
`outer segments. Each bar reflects the
`average value obtained from nine
`samples. Asterisks indicate statistical
`t-test;
`significance
`(one
`sample
`***=p<0.001).
`
`Fluorescence labeling of lysosomes in cultured microglial
`cells and macrophages: One mM LysoTracker Red DND-99
`(Molecular Probes, Junction City, OR) stock solution was
`diluted to 75 nM in the growth medium. Macrophage and
`microglial cell cultures were maintained in the LysoTracker
`Red DND-99-containing medium for 2 h and then replenished
`with fresh medium. The 561 nm wavelength was used to
`identify and observe the lysosomes by confocal microscopy.
`Fluorescent labeling of rod outer segments: Two mg/ml stock
`solution of FITC (Molecular Probes) in 0.1 mol/1 sodium
`bicarbonate at pH 9.0–9.5, was prepared under dim red light,
`filter-sterilized, and stored in aliquots at −20 °C. The FITC
`stock was added to the ROS solution (final concentration,
`10 μg/ml), and incubation was continued for 1 h at room
`temperature in the dark. The FITC-stained ROSs (FITC-
`ROSs) were pelleted in a microcentrifuge (4 min at 5,000× g)
`and resuspended in growth medium.
`Incubation of cultured macrophages and microglial cells with
`fluorescein
`isothiocyanate rod outer segments: After
`culturing for 3 days, macrophage and microglial cells were
`fed with FITC-ROSs at 4 μg/cm2 and incubated at 37 °C from
`1 h to 24 h. At the end of the incubation time, unattached FITC-
`ROSs were removed and the cells were washed. To quench
`external bound ROSs, samples were incubated with 0.4%
`trypan blue for 10 min.
`
`Confocal microscopy: Cells were cultured in eight-well
`microscopy glass slides (Lab-Tek Chamber Slide, Nunc,
`Langenselbold, Germany) and treated with different ROSs as
`described. After 4 day feeding or 1 day feeding of FITC-
`ROSs,
`cells were
`repeatedly washed
`to
`remove
`noninternalized ROSs, fixed with 4% paraformaldehyde
`(PFA), stained with 1 μg/ml Hoechst Stain solution H6024
`(Sigma-Aldrich) for 5–7 min, and mounted in Vectashield
`mounting medium (Vector Laboratories, Burlingame, CA).
`Confocal microscopy was performed with a Solamere
`Technology Group CSU10B Spinning Disk Confocal System
`that consisted of a Yokogawa CSU10 spinning disk confocal
`scan head attached to a Nikon TE2000-E2 inverted
`microscope (Nikon Instruments, Melville, NY) with a custom
`acousto-optic tunable filter (AOTF)-controlled laser launch
`with 405 nm, 488 nm, 561 nm, and 636 nm lasers.
`Electron microscopy: Cells were cultured in six-well plates
`and incubated with different ROSs as described previously.
`The cells were then fixed by adding 1 ml of 2.5%
`glutaraldehyde (v/v) in 0.75 M Na phosphate buffer (pH 7.2)
`to each of the wells in the culture plate and allowed to fix
`overnight at 4 °C. The next day, the fixed samples were
`washed three times in 0.75 M Na phosphate buffer (pH 7.2).
`The cells were then scraped off the bottom of the wells with
`a soft plastic spatula, collected in a microfuge tube, pelletted,
`briefly rinsed in DH2O, and postfixed for 1 h in 1% osmium
`
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`© 2012 Molecular Vision
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`Figure 3. Confocal microphotographs of macrophages and microglial cells after 4 day feeding with different rod outer segments (ROS). Shown
`are laser-scanning confocal micrographs of macrophages and microglial cells fed with different ROSs. Microglial cells fed with no ROSs
`(control), bleached ROSs, unbleached ROSs, and HNE-modified ROSs are presented on the top row. Macrophages tested under the same
`conditions are presented on the bottom row. Each group demonstrates different extents of green-yellow autofluorescence at the FITC channel
`(excitation, 488 nm; detection, 530 nm). Panels marked as “HNE-ROS (A)” (middle panels) indicate microphotographs of cells fed with HNE-
`modified ROSs taken at an original magnification of 400×. All other photographs were taken with original magnification 200×. Scale bar=10
`μm. Abbreviations are the same as in Figure 2.
`
`tetroxide (w/v) in DH2O. The fixed cells were then washed
`again in the DH2O and dehydrated through a graded ethanol
`series of 20% increments, before two changes in 100%
`ethanol. Samples were then infiltrated first with two changes
`of propylene oxide and then in a mixture of 50% propylene
`oxide/50% SPIpon 812-Araldite epoxy resin and left
`overnight to infiltrate. The following morning, the cell pellets
`were transferred through three changes of fresh SPIpon 812-
`Araldite epoxy resin and finally embedded in molds filled with
`the same resin and polymerized for 48 h at 70 °C. The epoxy
`blocks were then trimmed, and ultrathin sections were cut on
`a Reichart-Jung ultramicrotome
`(Leica Microsystems,
`Buffalo Grove, IL) using a diamond knife. The sections were
`collected and mounted on copper support grids and contrasted
`with lead citrate and uranyl acetate. The samples were
`examined on Philips CM 10 and FEI Tecani 12 BT (FEI,
`Hillsboro, OR) transmission electron microscopes using a 80
`kV accelerating voltage. Images were captured using a Gatan
`TEM CCD camera (Gatan, Pleasanton, CA).
`
`RESULTS
`Autofluorescence changes from feeding with modified and
`unmodified rod outer segments: Bleaching the ROSs for 1 h
`resulted in a rhodopsin content decrease of approximately
`80%, compared to unbleached ROSs. In contrast, modifying
`the ROSs with HNE resulted in only an approximately 25%
`decrease in rhodopsin content (Figure 1). When macrophage
`or microglial cells were incubated daily with either bleached,
`
`unbleached, or HNE-modified ROSs at doses of 2 μg/cm2 for
`4 days, increased LLAF was observed by FACS at both
`wavelengths (Figure 2). The LLAF in cells fed with HNE-
`modified ROSs increased around 6–7 fold, compared to a
`twofold increase in bleached or unbleached ROSs at 533 nm
`(Figure 2A). Interestingly, LLAF in cells fed with bleached
`ROSs increased slightly at 585 nm, while the LLAF of cells
`fed with either unbleached or HNE-modified ROSs decreased
`slightly at the same wavelength, compared to the change at
`533 nm (Figure 2A,B). In general, the LLAF generated by
`adding HNE-modified ROSs was approximately two to three
`times higher, compared to either adding bleached or
`unbleached ROSs, and the difference was highly statistically
`significant for every condition (p<0.0001; Mann–Whitney
`test).
`Meanwhile, confocal microscopy demonstrated the
`formation of yellow-green inclusions in cells when incubated
`daily with different ROSs for 4 days (Figure 3). The inclusions
`were most numerous and prominent in cells fed with HNE-
`modified ROSs (Figure 3). This was confirmed, through
`transmission electron microscopy, by the presence of
`numerous inclusion bodies containing membrane swirls in
`cells fed with bleached, unbleached, and HNE-modified
`ROSs (Figure 4, Figure 5, and Figure 6).
`The contribution of different components of rod outer
`segments
`to
`the
`formation of autofluorescence: The
`composition of ROSs is varied, including retinoids (11-cis-
`retinal, all-trans-retinal), proteins (e.g., opsin), phospholipids
`
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`© 2012 Molecular Vision
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`Figure 4. Transmission electron microscopy of microglial cells after 4 day feeding with ROSs. A: Electron micrographs of microglial cells
`(control). B: Electron micrograph of microglial cells after 4 day feeding with bleached ROSs. Magnification: 6,000×. A higher magnification
`of the intracellular inclusion body region is presented in the micrograph below (magnification 26,000×). C. Electron micrograph of microglial
`cells after 4 day feeding with unbleached ROSs. Magnification: 4,200×. A higher magnification of the intracellular inclusion body region is
`presented in the micrograph below (magnification 43,000×). White arrows indicate intracellular inclusion bodies in the higher magnification
`photographs in B and C.
`
`(phosphatidylethanolamine [PE], phosphatidylcholine [PC]),
`and so forth. Previous work has focused on in vitro formation
`of fluorophores from oxidized lipid-protein complexes or
`combinations of PE and all-trans-retinal in RPE cells [9-11].
`However, studies examining the parallel or competitive
`contribution of the different ROS elements in the formation
`of fluorophores from various components are lacking for RPE
`cells, as well as for macrophages or microglia. Therefore, we
`tested the effect of feeding bleached, unbleached, and HNE-
`modified ROSs and compared the contribution of different
`components of ROSs by FACS. Among retinoids, LLAF
`induced by applying 11-cis-retinal was slightly higher,
`compared to LLAF induced by feeding with all-trans-retinal
`and 9-cis-retinal, especially at 585 nm (Figure 7). Feeding
`with liposomes containing PE and PC (6:4 ratio) induced
`relatively low LLAF, compared to the retinoid.
`
`Phagocytosis of rod outer segments by macrophages and
`microglial cells: To further investigate the finding of
`increased LLAF in cells fed with HNE-modified, unbleached
`and bleached ROSs, we performed a phagocytosis assay in
`these three groups. FITC-stained ROSs appeared inside the
`lysosome and co-localized well with it after 24 h incubation
`in macrophage and microglial cells. The rate of phagocytosis
`was considerably higher in both macrophages and microglial
`cells fed with HNE-modified ROSs, compared to the levels of
`phagocytosis observed in the other two groups (Figure 8 and
`Figure 9).
`
`DISCUSSION
`The presence of both macrophages and microglial cells has
`been documented in human and other mammalian retinas and
`subretinal spaces under normal and diseased conditions. For
`example, microglial cells are ubiquitous in humans, being
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`© 2012 Molecular Vision
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`Figure 5. Transmission electron microscopy of macrophages after 4 day feeding with rod outer segments. A: Electron micrographs of
`macrophages (control). B: Electron micrograph of macrophages after 4 day feeding with bleached ROSs. Magnification: 6,000×. A higher
`magnification of the intracellular inclusion body region is presented in the micrograph below (magnification 26,000×). C: Electron micrograph
`of macrophages after 4 day feeding with unbleached ROSs. Magnification: 4,600×. A higher magnification of the intracellular inclusion body
`region is presented in the micrograph below (magnification 25,000×). White arrows indicate intracellular inclusion bodies in the higher
`magnification photographs in B and C.
`
`found in every layer of the retina and in several layers in the
`mouse retina [12,13]. In contrast, macrophages are only
`occasionally found in a normal healthy retina, mostly
`perivascularly distributed [14]. However, they can increase in
`numbers with normal aging [15]. Additionally, infiltration of
`macrophages
`from
`the blood circulation and para-
`inflammation, induced to repair and remodel the tissue
`through microglial, macrophage, and complementary
`activation, has been proposed as part of the pathogenic
`mechanism in some retinal diseases, including AMD [16].
`The ability of both macrophages and microglial cells to
`phagocytize dying cells was established long ago and has been
`studied quite extensively, but the potential for both types of
`cells to phagocytize photoreceptor outer segments, and thus
`to contribute to the LLAF associated with aging and some
`maculopathies, has received very little attention. Gery and
`O’Brien were the first to document ROS and dystrophic retinal
`debris uptake in macrophages from RCS and Sprague Dawley
`
`to
`limited
`their observation was
`rats [17], although
`establishing the fact and measuring early uptake (up to 3 h).
`Finnemann and Rodriguez-Boulan [18] explored the role of
`surface receptors (αvβ3 and αvβ5) and protein kinase C
`activation in recognizing and initiating the phagocytosis of
`ROSs and apoptotic cells in macrophages and RPE cells,
`focusing their work on this aspect of the process. More
`recently, it was shown that subretinal microglia contains
`phagocytozed rod outer segment discs after intense light
`exposure [19]. It was also demonstrated that, similar to
`microglia, macrophages could selectively eliminate apoptotic
`photoreceptors from their physiologic localization in the
`subretinal space and phagocytoze them [20]. Only recently,
`this capability was linked directly to AMD-like phenotypes in
`models of retinal diseases [21-23]. However, many aspects of
`this process remain uncharacterized. For example, what is the
`relative contribution of
`the different outer segment
`components (retinoids, proteins, phospholipids) to the LLAF
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`Figure 6. Transmission electron microscopy of macrophages and microglial cells after 4 day feeding with HNE-modified ROSs. A: Electron
`micrograph of microglial cells after 4 day feeding with HNE-modified ROSs. Magnification: 6,000×. A higher magnification of the intracellular
`inclusion body region is presented in the micrograph below (magnification 26,000×). B: Electron micrograph of macrophages after 4 day
`feeding with HNE-modified ROSs. Magnification: 8,000×. A higher magnification of the intracellular inclusion body region is presented in
`the micrograph below (magnification 43,000×). White arrows indicate intracellular inclusion bodies in the higher magnification photographs
`in A and B.
`
`emission from macrophages or microglia? What role could
`excessive oxidation of outer segments play in the capacity of
`microglial cells or macrophages to phagocytize outer
`
`segments and in the spectral signature of the resulting LLAF?
`Are there any differences between both types of cells in their
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`of
`7. Autofluorescence
`Figure
`macrophage and microglial cells after 4
`day feeding with different preparations
`A: FACS analysis in FITC channel
`(detection filter wavelength, 533/30 nm)
`of the microglial cells and macrophage
`cells fed with different preparations. B:
`FACS analysis in PE channel (detection
`filter wavelength, 585/40 nm) of the
`microglial cells and macrophage cells
`fed with different preparations. Note the
`LLAF
`increase
`in cells fed with
`retinoids,
`especially
`the
`increase
`observed after feeding with 11-cis-
`retinal at 585 nm compared to that at 533
`nm. Abbreviation key: ATRS=all-trans-
`retinal; 9-cis=9-cis retinal; 11-cis=11-
`cis-retinal; Lipo=liposome preparation.
`Each bar reflects the average value
`obtained from three samples. Asterisks
`indicate statistical significance (one
`sample t-test; * p<0.05, ** p<0.01).
`
`ability to phagocytize and their autofluorescence (AR)
`spectral signature?
`Both macrophages and microglial cell lines exhibited a
`very similar ability to phagocytize ROSs: the resulting
`autofluorescent properties measured at 533 nm and at 585 nm
`were almost identical for both types of cells. Several published
`works have provided evidence that microglial cells are the
`resident macrophage of the neural tissue [24]. The main
`differences between the two cell types is the that microglial
`cells lack MHC class I/MHC class II, as well as other proteins
`that are characteristic for macrophages [25]. Our observation
`strengthens the case for similarities between macorphages and
`microglial cells, but it is limited because of the use of cell lines.
`Although the effect of phospholipid peroxidation by
`oxidizing agents like HNE on the ability of ROSs to increase
`LLAF in RPE cells has been explored and reported recently
`[26,27], it is unclear whether the same effect is present in
`macrophages or microglial cells. It is also unclear if the HNE-
`modified ROSs preserve enough rhodopsin to remain a
`physiologically relevant model. The rhodopsin analysis in our
`study indicates that HNE-modified ROSs contain enough
`rhodopsin, with levels similar to that of unbleached ROSs, to
`retain physiologic relevance.
`Our results confirm that both macrophages and microglial
`cells exhibited substantial increases in LLAF after being fed
`with HNE-modified ROSs, compared to the LLAF registered
`from control cells or cells fed with either bleached or
`
`unbleached ROSs. Interestingly, the LLAF in cells fed with
`HNE-modified ROSs was slightly higher at 533 nm than at
`585 nm, which suggests an LLAF spectral fingerprint slightly
`different compared to what typical LLAF spectral patterns
`from human RPE lipofuscin granules or fundus LLAF, where
`the LLAF peaks are 580–620 nm [27,28]. The electron
`microscopy imaging (EM) of both cell types fed with HNE-
`modified ROSs showed numerous
`inclusion bodies
`containing membrane
`swirls. Additionally, confocal
`microscopy revealed
`the autoflourescent material was
`localized within lysosomes. Finally, the rate of phagocytosis
`was increased after feeding with HNE-modified ROSs,
`compared to feeding with nonmodified ROSs.
`Even though several hypotheses have been advanced over
`the years about the role of the main components of the ROSs
`in RPE LLAF generation, there has been little direct evidence
`to support those claims. Similarly, this problem remains open
`for other phagocytic cells, such as macrophage or microglial
`cells. We have approached this problem by supplying
`equimolar amounts of 11-cis-retinal, 9-cis-retinal, all-trans-
`retinal, and similar quantities of
`liposomes
`to both
`macrophages and microglial cells. Of note, the ROS-
`component concentrations used in this experiment were
`similar to the ROS composition in vivo, the latter having a
`lower concentration of retinoids and a slightly different ratio
`of the phospholipids [29,30]. Based on our results, LLAF was
`increased more after application of 11-cis-retinal and all-
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`Molecular Vision 2012; 18:103-113 <http://www.molvis.org/molvis/v18/a13>
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`© 2012 Molecular Vision
`
`Figure 8. Rod outer segment phagocytosis by macrophage cells. A: Confocal micrographs of macrophage cells incubated with FITC-modified
`HNE-ROSs at 1 h (top, second) and at 24 h (top, third), and bleached rod outer segments (ROSs) at 24 h (top, right). FITC-ROSs co-localized
`well with macrophage cells that stained with a fluorescent acidotropic probe of Lysotracker Red, which show as yellow spots in
`microphotographs. There was more co-localization in cells fed with HNE-ROSs than in cells fed with bleached-ROSs at 24 h. B: Color channel
`separation for the cells fed with HNE-modified ROSs at 24 h. Scale bar=10 μm.
`
`trans-retinal, compared to the other compounds. This increase
`was much more pronounced at 585 nm, compared to at 533
`nm, typical of the RPE lipofuscin spectral signature [27,28,
`31]. Thus, we were able to establish a major contribution to
`microglial or macrophage LLAF from the two main retinoids
`present in the outer segments, namely 11-cis-retinal and all-
`trans-retinal. It is very likely that the same process may take
`place in vivo and that extracellular retinoids could accumulate
`in macrophages or microglial cells and lead to an increased
`LLAF. Since these cells do not possess the enzymatic
`machinery necessary for retinoid recycling as the RPE cells
`do, they may be more susceptible to adverse effects from
`retinoids. This may explain why subretinal microglia
`containing autofluorescent granules accumulated in an age-
`dependent manner in the subretinal space of adult normal mice
`and why the number of autofluorescent microglial cells was
`higher compared to the number of RPE autofluorescent cells
`at the same age [19].
`The fate of the undigested ROS material is uncertain. In
`our EM images, we show internalized ROSs with intact
`membrane swirls. We wonder whether these ROSs (and other
`autofluorescent material) will ultimately be degraded. This is
`an interesting topic for future studies.
`One of the challenges in the interpretation of the current
`data are related to the uncertainty over the degree to which
`
`increased autofluorescence can be attributed to enhanced
`phagocytosis or to differential fluorescence between parent
`species. Further work is warranted for establishing a
`quantitative ratio from
`the
`two possible sources of
`autofluorescence.
`Our observation that the application of HNE-modified
`ROSs stimulated a higher degree of LLAF underscores the
`importance of oxidative stress as a factor in retinal/RPE
`lipofuscinogenesis; however, further work is warranted for
`quantifying
`in more detail
`the differences between
`contributions from oxygen and retinoids to lipofuscin buildup.
`The enhanced rate of phagocytosis observed in the
`current study, resulting from the application of HNE-modified
`(oxidized) ROSs, is similar to that observed in the
`phagocytosis stimulation process during the auto-oxidation
`and oligomerization of protein S on the apoptotic cell surface
`[32]. Similarly, it has been demonstrated that H2O2, which is
`a potent oxidizing agent, activates MerTK, a major regulator
`and initiator of phagocytosis [33]. Our observations are
`consistent with those findings and support the role of oxidative
`stress in stimulating phagocytosis.
`One of the limitations of the present study is the use of
`cell lines. Further research would benefit from focusing on
`local retinal microglia or macrophage populations in their
`natural environment.
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`Molecular Vision 2012; 18:103-113 <http://www.molvis.org/molvis/v18/a13>
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`© 2012 Molecular Vision
`
`Figure 9. Rod outer segment phagocytosis by microglial cells. A: Confocal micrographs of microglial cells incubated with FITC-modified
`HNE-ROSs at 1 h (top, second) and at 24 h (top, third), and bleached-ROSs at 24 h (top, right). FITC-ROSs co-localized well with microglial
`cells that stained with a fluorescent acidotropic probe of Lysotracker Red, which show as yellow spots in pictures. There was more co-
`localization in HNE-ROSs than bleached-ROSs at 24 h. B: Color channel separation for the cells fed with HNE-modified ROSs at 24 h. Scale
`bar=10 μm.
`
`In conclusion, the present study demonstrates that
`microglial cells and macrophages behave in a very similar way
`by phagocytizing ROSs, which leads to increased LLAF.
`They also appear to be sensitive to the addition of free
`retinoids such as 11-cis-retinal and all-trans-retinal that also
`increase LLAF. In addition, phospholipid peroxidation of
`ROS material can increase LLAF and lead to the accumulation
`of undigested material inside the cells. Collectively, these
`findings strengthen the proposed role of microglial cells and
`