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INTRODUCTION |
12/15-Lipoxygenase (LO)1
is a member of the LO family of enzymes that insert peroxyl groups into
double bonds of free and phospholipid-bound polyunsaturated fatty
acids. The exact role of these enzymes in biological processes has
remained elusive, but increasingly evidence has accumulated that they
play important roles in specific cellular functions. For example, 15-LO
activity in reticulocytes at the stage of organelle degradation may
contribute to membrane destabilization and contribute to pore formation
in intracellular membranes (1, 2). Fatty acid products of 12/15-LO are
powerful agonists for the nuclear receptor PPAR-
, which helps
regulate glucose metabolism and adipocyte and macrophage
differentiation and function (3). A remarkable feature of 12/15-LO is
that its expression is not constant during the cell life span but
rather turns on at certain points during cell development. While
circulating human monocytes do not express 15-LO, monocyte-derived
macrophages exposed to interleukin-4 or interleukin-13 express 15-LO
(4, 5). In addition, mouse macrophages residing for a long time in the
peritoneum (resident macrophages) also highly express the mouse
homologue, 12/15-LO, although the pathway leading to 12/15-LO
expression may differ somewhat from that which occurs with human
monocytes (6).
Macrophages of atherosclerotic lesions express high levels of 15-LO
(7), and recent evidence utilizing apoE
/
mice in which the 12/15-LO gene was disrupted demonstrated its importance in the pathogenesis of atherosclerosis (8). Another characteristic of atherosclerotic tissue but not of normal vascular wall is the high abundance of apoptotic cells (9). This fact might
reflect either an increased rate of formation of such cells, a
decreased rate of clearance, for example by arterial macrophages, or
both. In either case, phagocytosis and the degradation and metabolism
of the ingested contents of dying cells are crucial for preventing the
release of toxic cellular compounds and consequent inflammation.
Inhibition of efficient phagocytosis would presumably lead to the
accumulation of pro-inflammatory necrotic debris, plaque instability,
and thrombogenesis.
During the complex process of phagocytosis, major changes in the
cytoskeleton of the cell occur leading to the formation of filopodia
surrounding an apoptotic cell or a microorganism to be engulfed.
Changes in actin polymerization play a vital role in this process.
Remarkably, 12-LO products are found in many tumor cells and have been
suggested to have effects on actin polymerization (10) and cytoskeleton
reorganization during cell transformation (10, 11). Therefore, it was
tempting to propose that the activity of 12/15-LO in non-malignant
cells, such as macrophages, was also related to a cytoskeleton function
and to phagocytosis. Indeed, we now demonstrate that upon exposure to
apoptotic cells, 12/15-LO translocates from the cytosol to sites of
apoptotic cell binding and furthermore that actin polymerization itself
is dependent on activity of 12/15-LO.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Materials--
Peritoneal macrophages were
harvested from 8- to 10-week-old female mice, either Swiss Webster or
C57BL/6 strains. The latter were strain-, age-, and sex-matched to
12/15-LO knockout mice. Resident or thioglycollate-elicited macrophages
were plated in RPMI 1640 (BioWhittaker) supplemented with 10% fetal
bovine serum (FBS) (Omega Scientific). Murine fibroblast cell lines
overexpressing either human 15-LO (clone 12) or
-galactosidase
(LacZ) were cultured in Dulbecco's modified Eagle's
medium (BioWhittaker) with 10% FBS and 0.2 mg/ml G418 (Calbiochem) to
maintain selection (12). Thymocytes were harvested from the thymuses of
4-week-old mice of the same strain as used for macrophage isolation and
treated with 1 µM dexamethasone in 10% FBS/RPMI 1640 for
4 h to induce apoptosis (13). The appearance of condensed nuclei
was a marker for apoptosis. We previously demonstrated that
concentrations up to 20 µM of the specific lipoxygenase
inhibitor PD 146176 (a gift from J. Cornicelli of Parke-Davis) were
non-toxic for macrophages (14). Cytochalasin D was from Sigma, and
latrunculin A was from Molecular Probes.
13(S)-Hydroxyoctadecadienoic acid (13(S)-HODE), 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE),
12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE),
and linoleic acid were from Cayman Chemical.
Western Blot--
Cells were lysed on the plate with 5% SDS in
phosphate-buffered saline. Protein content was determined with a BCA
kit (Pierce), and 15-100 µg of the cell lysate was run on a pre-cast
4-12% gradient polyacrylamide gel (Novex) and then transferred to a
nitrocellulose membrane (Millipore). The membrane was blocked with 5%
non-fat milk and incubated with a protein A-purified polyclonal guinea pig anti-rabbit 15-LO antibody (7). This antibody cross-reacts not only
with human 15-LO (7) but also with mouse 12/15-LO; it stained the band
of 75 kDa, typical for 12/15-LO. It did not yield any such band when
lysates from 12/15-LO
/
mice were used.
Guinea pig preimmune IgG produced no specific staining.
Immunocytochemistry and Imaging--
Macrophages plated
overnight on coverslips were fixed with 3.7% paraformaldehyde for 10 min at 37 °C, permeabilized with 0.2% Triton X-100 for 5 min,
blocked with 0.8 µg/ml Fc block (PharMingen) in 5% non-fat milk,
0.2% Triton X-100, stained for 30 min with the guinea pig anti-rabbit
15-LO antibody and for another 30 min with a rhodamine red-X-conjugated
F(ab')2 fragment donkey anti-guinea pig Ig G (H+L) antibody
(Jackson ImmunoResearch). Alternatively, a guinea pig anti-rabbit ApoA1
antibody was used as a negative control. Filamentous actin (F-actin)
was stained by addition of 1.5 µM FITC-conjugated
phalloidin (Sigma) to the solution of the secondary antibody. Cytosol
was labeled by incubation of live cells with 0.5 µM,
5-chloromethylfluorescein diacetate (CellTracker green CMFDA
from Molecular Probes) in serum-free medium for 30 min followed by a
30-min incubation in the regular culture medium. This green staining
remained in fixed cells. Cell nuclei were stained blue with 1 µg/ml
Hoechst 33258 (Sigma) for 15 min. The coverslips were mounted on
microscopic glass slides with ProLong antifade medium (Molecular
Probes). Images were captured by deconvolution microscopy (15) using a
DeltaVision deconvolution microscopic system operated by SoftWorx
software (Applied Precision). Pixel intensities were kept in the linear
response range of the digital camera. Optical sections through the
samples were taken with increments of 0.2-0.5 µm depending on
magnification. The images were deconvolved and examined either section
by section or volume views were generated by combining areas of maximal
intensity of each optical section with SoftWorx programs. Data
Inspector application was used to quantitatively analyze the images.
Adobe Photoshop 6.0 software was used to design figures.
Flow Cytometry--
The relative content of F-actin in
macrophages activated by addition of apoptotic thymocytes was assessed
by flow cytometry as described in Ref. 16 with some modification. In
brief, at the end of incubation of the plated macrophages with
apoptotic thymocytes, 1 volume of the solution containing 1.6 µM FITC-phalloidin, 18% paraformaldehyde, and 0.8%
saponin (all from Sigma) was added to 3 volumes of the culture medium
and incubated for an additional 10 min. Cells were then washed, scraped
from the plate, filtered through a Nitex nylon mesh (Sefar America),
and analyzed on a FACScan (Becton Dickinson).
To examine the expression of cell-specific CD markers, the cells
attached to the plate were gently scraped, incubated in suspension for
30 min with either a FITC-conjugated anti-CD80 antibody, a FITC-conjugated anti-CD3 antibody, or a phycoerythrin-conjugated anti-CD19 antibody (all from PharMingen), washed, and analyzed on the FACScan.
Actin Polymerization--
Assays were performed as described
previously (17). This assay is based on the measurement of fluorescence
intensity of pyrene covalently linked to actin, which increases when
actin polymerizes. In brief, unlabeled and pyrene-labeled monomeric G-actin from rabbit muscle (kindly provided by K. Aman from the Salk
Institute) at the ratio of 95:5 were diluted in G-buffer (2 mM Tris, pH 8.0, 0.2 mM ATP, 0.1 mM
CaCl2, and 0.5 mM dithiothreitol) and then
converted to Mg-actin by adding 0.1 volume of 10 mM EGTA and 1 mM MgCl2. Polymerization was initiated by
addition of either macrophage lysates or 0.1 volume of 10× KMEI (500 mM KCl, 10 mM MgCl2, 10 mM EGTA, and 100 mM imidazole, pH 7.0). Lysates
were prepared from macrophages scraped from the plate in a lysis buffer (2 mM Tris, pH 8.0, 1 mM EGTA, 0.2 mM MgCl2, and protease inhibitors mixture
(Sigma)) by sonication and centrifugation at 10,000 × g for 30 min. Protein concentration was measured using a BCA
kit from Pierce. Spectra and time courses of pyrene fluorescence were measured on an LS50B luminescence spectrophotometer (PerkinElmer Life Sciences).
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RESULTS |
12/15-LO Expression and Phagocytic Activity of Elicited
Macrophages--
When non-septic inflammation in mice is induced by
intraperitoneal injection of thioglycollate, many monocytes are
recruited into the peritoneum, where they differentiate into
macrophages. Initially, these newly recruited, "elicited"
macrophages express very little 12/15-LO. This was evident from Western
blots of cell lysates made from elicited and resident macrophages.
Relative to total cell protein, there was 10-15-fold less 12/15-LO
expressed in the elicited macrophages as compared with the enzyme
content in resident macrophages (data not shown). Immunocytochemical
examination of the elicited macrophage population revealed two cell
populations, either positive or negative for 12/15-LO staining (Fig.
1a). The 12/15-LO-positive
cells (less than 10% of total) presumably originated from resident
macrophages. The 12/15-LO-negative cells were probably newly recruited
monocyte-macrophages that did not yet express the enzyme. This
observation is in agreement with an earlier report on heterogeneity of
elicited macrophages from immunodeficient mice (6).

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Fig. 1.
Phagocytosis of apoptotic thymocytes
by elicited macrophages. a, apoptotic thymocytes were added
to cultured thioglycollate-elicited macrophages for 15 min. Cells were
fixed and stained to visualize 12/15-LO (red), nuclei
(blue), and F-actin (green). Nuclei of apoptotic
thymocytes are the homogeneous round blue bodies of condensed
chromatin. Two microscopic fields were merged into the image shown.
Bar, 15 µm. b, 150-200 macrophages that were
12/15-LO-positive or -negative were counted, and the number of
apoptotic thymocytes bound (15 min of incubation, cross-hatched
columns) or engulfed (60 min of incubation, black
columns) for each macrophage was determined. Some macrophage
cultures were preincubated for 1 h with 0.5-20 µM
of the 12/15-LO inhibitor PD 146176 prior to addition of apoptotic
thymocytes. Data are mean ± S.D. *, p < 0.0001 compared with 12/15-LO( ); p numbers on the graph show
significance of the difference between apoptotic cells binding to
non-treated 12/15-LO(+) macrophages and those treated with specific
concentrations of PD 146176.
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To ensure that the thioglycollate-elicited cells that attached to the
plate overnight were indeed macrophages and not other cell types, these
cells were analyzed by flow cytometry for the presence of cell-specific
markers. Ninety eight percent of the attached cells were positive for
the macrophage marker CD80 and negative for the T-cell marker CD3 and
the B-cell marker CD19. (Splenocytes, a mixed population of all the
three cell types, were used as positive controls.) Thus, the majority
of the plate-attached cells harvested from peritoneum were macrophages,
and the difference in the 12/15-LO expression is probably a function of
the stage of macrophage differentiation.
When apoptotic thymocytes were incubated with the elicited macrophage
population, we noted a striking difference between 12/15-LO-positive and -negative cells in the ability to bind and engulf apoptotic thymocytes (Fig. 1a). Counting multiple microscopic fields
confirmed that following a 15-min incubation, the 12/15-LO-expressing
macrophages bound 20 times more apoptotic cells than the
12/15-LO-negative cells (cross-hatched columns in Fig.
1b). This ability of the 12/15-LO-positive cells to bind
apoptotic cells was significantly reduced when the elicited macrophages
were pretreated with the specific 12/15-LO inhibitor PD 146176 (18).
The effect of PD 146176 was dose-dependent. A statistically
significant decrease in apoptotic cells binding to 12/15-LO-positive
elicited macrophages was observed already at 0.5 µM PD
146176. This result corresponds well to the previously reported
IC50 values of 0.8 µM for the PD 146176 inhibition of 15-LO activity in cell culture (19). Following a 1-h
incubation, when most of the apoptotic thymocytes were already engulfed
by the macrophages, the same tendency was observed (black
columns in Fig. 1b). The correlation between the phagocytic function of elicited macrophages and the 12/15-LO activity suggests a role for 12/15-LO in phagocytosis.
Translocation of 12/15-LO in Phagocytosing Macrophages--
To
explore further a potential relationship of 12/15-LO to phagocytosis,
we examined the localization of 12/15-LO and F-actin in resident
macrophages, resting or phagocytosing apoptotic thymocytes. Nearly all
(more than 95%) resident macrophages expressed 12/15-LO. In
resident macrophages not exposed to apoptotic cells (Fig.
2a, a volume view, Fig.
2b, a 2-fold magnified optical section), 12/15-LO protein
(red) was evenly distributed throughout the cytosol and did
not colocalize with F-actin (green) at the cell surface. The three-dimensional intensity graphs below Fig. 2b document
the very different distributions of 12/15-LO and F-actin in the
highlighted area. In contrast, in resident macrophages exposed to
apoptotic thymocytes, 12/15-LO concentrated on the cell surfaces in
general, and this was greatly enhanced at the sites where apoptotic
cells were bound (Fig. 2, c and d). To show that
the translocation of 12/15-LO toward the bound apoptotic cell at the
periphery of the macrophage was specific and not just following general
movement of cytosol, we labeled cytosol green with CellTracker, a dye
that evenly binds to thiol groups in the cell (20). The presence of
12/15-LO on the cell surface (red color) close to attached apoptotic thymocytes but the absence of the yellow color
(that would have represented colocalization with CellTracker) suggest the specificity of the 12/15-LO translocation (Fig. 2, c and
d). Accordingly, the intensity graphs below Fig.
2d show a different distribution of 12/15-LO and CellTracker
in the highlighted area.

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Fig. 2.
Translocation of 12/15-LO in phagocytosing
resident macrophages. Images in a and c
represent volume views generated by combining areas of maximal
intensity of each optical section, whereas those in b and
d are representative focal planes from the middle of the
cells (zoom × 2). a and b, resident
macrophage without any treatment. c and d,
resident macrophages incubated for 15 min with apoptotic thymocytes.
Cells were fixed and stained to visualize 12/15-LO (red),
nuclei (blue), and either F-actin (green)
(a and b) or overall cytosol with the CellTracker
label (green) (c and d).
Bar, 5 µm. The image areas enclosed in white
rectangular frames were quantitatively analyzed separately for
12/15-LO and either F-actin or CellTracker staining and shown
below the images. The color spectrum chart from
blue to red shows change in the intensity of each
fluorophore from low to high. Note an order of magnitude difference in
the intensity of 12/15-LO staining in the cytosol of the resting
macrophage (below b) and on the surface of the macrophage
with bound apoptotic cells (below d).
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Fig. 3 presents another example in which
a resident macrophage is in close or partial contact with four
different apoptotic cells simultaneously. Fig. 3b
demonstrates staining for 12/15-LO (red) and Fig.
3c staining for F-actin (green). Fig.
3a is a merged view where the yellow color
demonstrates 12/15-LO colocalization with the sites of actin
polymerization. The yellow color is clearly more heavily
concentrated in the vicinity of bound apoptotic cells. Quantification
of the effect is provided by intensity maps of the whole cell (derived
from the volume view) for each color. In general, the concentrations of
12/15-LO and F-actin on the surface of macrophages phagocytosing
apoptotic cells were 3-6-fold higher than in resting macrophages
(compare intensity scales in Figs. 2 and 3). This supports the
generalized movement of both to the cell periphery. However, the
intensity graph for 12/15-LO (below Fig. 3b) also
demonstrates increased 12/15-LO concentration at the sites of apoptotic
cell binding, as compared with either cytosol or even other sites of
the periphery of the cell. Specific 12/15-LO intensity in the areas of
cell-cell contact was 3.11 ± 0.54/voxel as compared with
1.49 ± 0.73/voxel in all the rest of the cell perimeter
(p < 0.001). The intensity maps derived from the
highlighted area of a specific focal plane (below Fig. 3,
e and f) show nearly identical patterns of
12/15-LO and F-actin distribution in the area of contact with an
apoptotic cell, confirming their specific colocalization.

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Fig. 3.
Colocalization of 12/15-LO and F-actin in
phagocytosing resident macrophages. Resident macrophages were
incubated for 15 min with apoptotic thymocytes. The upper
row of images (a-c) represents a volume view, and the
lower row (d-f) shows a focal plane, as
explained in the legend to Fig. 2. Merged red (12/15-LO),
green (F-actin), and blue (nuclei) colors are
shown in a and d. The spots of red and
green voxels colocalizing in the volume view and pixels in
the focal plane appear as yellow on the images. Separated
red (b and e) and green
(c and f) colors show specific concentration of
12/15-LO and F-actin, respectively, in the vicinity of bound apoptotic
cells. Three-dimensional intensity maps below the upper row
of images quantify overall distribution of nucleic acid, 12/15-LO, and
F-actin in the cell. The color spectrum charts from blue to
red show change in the intensity of each fluorophore from
low to high. Intensity maps at the bottom of the figure show
intensities of nucleic acid, 12/15-LO,and F-actin staining in the
highlighted area of the focal plane. Similarity in the
intensity maps under e and f for 12/15-LO and
F-actin, respectively, represents their colocalization. Note also a
3-4-fold difference in the intensity of F-actin staining in the
resting macrophage (Fig. 2, below b) and the macrophage with
bound apoptotic cells (Fig. 3, below c and
f).
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12/15-LO Translocation in Macrophages with Disrupted
F-actin--
We next examined the relationship between activity of
12/15-LO and F-actin function. To approach this question, we inhibited either the activity of 12/15-LO or the process of actin polymerization. Formation of F-actin was inhibited by treating macrophages with either
cytochalasin D or latrunculin A, toxins that bind monomeric G-actin,
thereby preventing formation of filamentous F-actin (21, 22). The
images in Fig. 4, a-c show a
non-treated resident macrophage caught in the process of phagocytosis
of an apoptotic thymocyte. 12/15-LO has translocated to the surface
where it appears to be interacting with the apoptotic cell. This same
site has also been greatly enriched by F-actin (see
black-white images in b and c, and
intensity graphs below). Again, although there is clear translocation of the 12/15-LO to the periphery of the cell, a formal analysis of the
intensity maps shows 12/15-LO-specific intensity of 5.40 ± 0.73/voxel at the site of apoptotic cell binding versus
2.40 ± 1.16/voxel in the rest of cell perimeter
(p < 0.001) providing additional evidence that
12/15-LO translocation is concentrated at sites of contact with
apoptotic cells.

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Fig. 4.
Cellular localization of 12/15-LO in
macrophages with disrupted actin polymerization. Resident
macrophages incubated with apoptotic thymocytes for 15 min without
prior treatment (a-c) or with pretreatment with either 150 nM cytochalasin D for 1 h (d-f) or 10 µM latrunculin A for 15 min (g-i). Cells were
fixed and stained for 12/15-LO (red), nuclei
(blue), and F-actin (green). Merged three-colored
volume view images are shown in a, d, and g.
Black and white images in b,
e, and h represent staining for 12/15-LO only,
and the images in c, f, and i show staining for
F-actin only. Bar, 5 µm. Intensity maps below
the 1st row of images quantify cell distribution of nucleic
acid, 12/15-LO, and F-actin. The color spectrum charts from
blue to red show change in the intensity of each
fluorophore from low to high. Note that an intense nucleic acid
staining of an apoptotic cell on the left-hand side of the
intensity map ends at the 7-8th mesh quadrangle, the place where an
intense macrophage staining for 12/15-LO and F-actin begins.
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The cytochalasin D treatment partially disrupted the actin
polymerization (Fig. 4, d and f). Nevertheless,
12/15-LO translocation toward the bound apoptotic thymocyte did not
seem to be impaired (Fig. 4, d and e).
Latrunculin A treatment almost completely disrupted F-actin formation
(Fig. 4, g and i), and even under these severe conditions 12/15-LO translocation to sites of apoptotic cell binding occurred (Fig. 4, g and h). These data suggest
that actin polymerization is not a prerequisite for 12/15-LO translocation.
Actin Polymerization in Macrophages with Inhibited or Disrupted
12/15-LO--
Can 12/15-LO activity in turn affect the process of
actin polymerization? In order to assess the level of polymerized actin in individual cells, we used a flow cytometry assay as described under
"Experimental Procedures." A shift of the cell distribution histogram to the area of higher fluorescence intensity (e.g.
to the right) reflects an increase in the level of F-actin. Such a
shift was observed in resident macrophages in response to incubation with apoptotic thymocytes (Fig. 5,
a and c). It was not due to the F-actin of
internalized apoptotic thymocytes because the latter did not show any
F-actin signal by flow cytometry (green histograms barely
seen in left bottom corners of Fig. 5, a and
c), and no F-actin staining was observed microscopically in
the apoptotic cells (Figs. 1-4). The F-actin response usually reached
its peak in 10-20 min and then disappeared 40-60 min after the start
of incubation (data not shown). Macrophages harvested from Swiss Webster mice (Fig. 5a) generally responded with a higher
level of polymerized actin than macrophages from C57BL/6 mice (compare Fig. 5, a versus c). Treatment of the
resident macrophages with the 12/15-LO inhibitor PD 146176 prior to
addition of the apoptotic cells blocked actin polymerization in the
macrophages (Fig. 5b). Finally, in macrophages harvested
from 12/15-LO knockout mice (12/15-LO
/
), no
change in the F-actin content in response to addition of apoptotic
cells was observed (Fig. 5d).

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Fig. 5.
Actin polymerization in phagocytosing
resident macrophages. F-actin level was assessed by flow cytometry
of the FITC-phalloidin-stained cells as described under "Experimental
Procedures." Black and red
histograms show the F-actin level in the cells without and
with addition of apoptotic thymocytes for 10 min, respectively.
Right-hand shift represents an increase in the level of
F-actin. Green histograms represent apoptotic thymocytes
alone. a, macrophages from Swiss Webster mice. b,
macrophages from Swiss Webster mice pretreated for 1 h with 20 µM PD 146176. c, macrophages from wild type
C57BL/6. d, macrophages from the C57BL/6 mice with disrupted
12/15-LO gene.
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Effect of Macrophage Lysates and 12/15-LO Products on Actin
Polymerization in Vitro--
The next set of experiments directly
examined the effect of the products of 12/15-LO on actin
polymerization, using an in vitro polymerization assay in
the presence or absence of cell lysates. In the first set of
experiments, actin polymerization was initiated by addition of cell
lysates (Fig. 6a). Whole cell lysates prepared from the 12/15-LO knockout macrophages had a limited
ability to promote in vitro polymerization of G-actin (Fig.
6a, dotted line). In contrast, lysates from wild type
macrophage had much higher nucleating and elongating activities as seen
by a shorter lag phase before the start of elongation and an increased rate of elongation (a 130 ± 27% increase, p < 0.001; Fig. 6a, solid line). Remarkably, addition of
13(S)-HODE (the oxidation product of linoleic acid) to the
12/15-LO
/
lysates significantly increased
the elongation rate but did not affect the lag phase, indicating that
13(S)-HODE does not have a nucleating activity (Fig.
6a, dashed line). The same positive effect of
13(S)-HODE on actin elongation but not nucleation was also
observed in an in vitro assay conducted in the absence of cell lysates, when actin polymerization was initiated by addition of
KMEI instead of cell lysate (a 50 ± 18% increase,
p < 0.01; Fig. 6b, solid line).
Preincubation of G-actin with non-oxidized linoleic did not show any
sizable difference from the ethanol vehicle (Fig. 6b, dashed
and dotted lines). Because light scattering from cell
lysates could have interfered with fluorescence measurements, emission
spectra were recorded at the beginning and the end of the time courses.
A multipeak analysis of a difference spectrum (Fig. 6a,
inset) shows a peak of 405.5 nm, which is fairly close to the peak
of pyrene emission (406.8 nm) from F-actin in lysate-free samples (Fig.
6b, inset). Similar results were also observed in experiments with the products of 12/15-LO oxidation of arachidonic acid, 15(S)-HETE and 12(S)-HETE, although the
effects were not as pronounced (data not shown).

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Fig. 6.
Effect of macrophage lysates and
13(S)-HODE on in vitro actin
polymerization. Actin polymerization was registered as an increase
in fluorescence intensity of pyrene-actin at excitation/emission
wavelengths of 365/407 nm. a, lysates of either wild type
(WT) or 12/15-LO knockout (KO) macrophages were
added at the final protein concentration of 50 µg/ml to 2.5 µM of G-actin (5% pyrene-actin). In some experiments, KO
lysates were preincubated with 6 µM 13(S)-HODE
on ice for 30 min and then mixed with G-actin. Lysis buffer replacing
cell lysates served as a control. Inset to a
shows a difference emission spectrum (excitation at 365 nm) of F-actin
at the end of the time course relative to G-actin at its beginning in
the KO lysate supplemented with (13S)-HODE. Multipeak
analysis (dashed lines) reveals a longer wavelength emission
maximum of 405.6 nm. b, polymerization of 3 µM
actin (5% pyrene-actin) preincubated with either 0.7% ethanol
(vehicle), 6 µM (13S)-HODE or 6 µM linoleic acid was initiated by addition of KMEI (see
"Experimental Procedures"). Control shows a reaction without
addition of KMEI. Inset to b shows emission
spectra of the samples with 13(S)-HODE (trace 1)
and ethanol (trace 2) at the end of the time course and the
spectrum of G-actin (trace 3) prior to addition of KMEI.
Representative spectra and time courses from three experiments are
shown.
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Spreading of 12/15-LO Overexpressing Fibroblasts--
To determine
if the relationship between LO activity and F-actin formation could
also be observed in another cell type, we also examined spreading of
fibroblasts, another function dependent on actin polymerization. For
these studies we used murine cell lines stably overexpressing either
human 15-LO (clone 12) or
-galactosidase (LacZ, control
cells) (12). Plated clone 12 cells spread much faster than LacZ
cells, and the presence of the 15-LO inhibitor PD 146176 in the medium
delayed spreading of the clone 12 cells (Fig.
7). These observations complement the
hypothesis that 12/15-LO stimulates actin polymerization and that the
functions of 12/15-LO may be more pleiotropic than only assisting
macrophage phagocytic function.

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Fig. 7.
Spreading of clone 12 and LacZ
fibroblasts in culture. a and b, in
this example 15-LO-overexpressing clone 12 (a) and
-galactosidase-overexpressing LacZ (b) fibroblasts were
fixed and stained for F-actin 90 min after plating. Bar, 30 µm. c, a 10-h time course of cells spreading. , clone
12; , LacZ; , clone 12 in the presence of 1 µM of
the 15-LO inhibitor PD 146176 (PD). Data are mean ± S.D. of three independent experiments.
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DISCUSSION |
Many lines of evidence suggest an important role for 15-LO (and
its 12/15-LO homologue in mice) in atherogenesis (7, 8, 12, 18, 19,
23-26). Inhibitors of 12/15-LO decrease the ability of murine
macrophages to oxidize LDL (14, 23), and murine fibroblasts transfected
with human 15-LO have a greatly enhanced ability to initiate oxidation
of LDL (12). Both human and rabbit atherosclerotic lesions express
15-LO mRNA, protein, and enzymatic activity (7, 24-26).
Importantly, inhibitors of 15-LO decreased atherogenesis in rabbits
(18, 19), and deleting 12/15-LO activity in apo
E
/
mice dramatically decreased
atherogenesis (8). These data support an important pathophysiological
role for 12/15-LO in atherogenesis. However, the physiological role of
15-LO remains unclear. In reticulocytes it appears to play an important
role in degradation of organelles as the cell matures, although no
abnormality in red blood cell development was noted in the
12/15-LO
/
mice (27). Its physiological role
in macrophages has remained equally obscure. Recently, our laboratory
demonstrated an important role for 12/15-LO in providing activating
ligands for nuclear receptor PPAR-
(3), which appears to be an
anti-inflammatory and anti-atherogenic factor (28, 29).
The present work demonstrates another novel function of 12/15-LO in
macrophages, namely its ability to regulate actin polymerization and
presumably phagocytosis of apoptotic cells. In the heterogeneous population of elicited macrophages, only 12/15-LO-expressing cells were
efficient in binding and engulfment of apoptotic cells (Fig. 1), and
this was associated with translocation of 12/15-LO from the cytosol to
the periphery of the cell where it concentrated at sites of binding of
apoptotic cells (Figs. 2-4). These data suggest that receptor-mediated
signaling leads to 12/15-LO translocation and involvement in actin
polymerization, in preparation for phagocytosis. It is conceivable that
the presence of 12/15-LO in the filopodia could contribute to the
oxidation of unsaturated lipids in particles that are being ingested,
leading to the engagement of various scavenger receptors and a more
efficient phagocytosis. As well, initial steps of the degradation of
the ingested particle may be initiated. Because phagocytosis of
apoptotic cells and debris (as well as oxidized LDL) is very likely to
be a normal homeostatic mechanism to dampen an inflammatory response
(e.g. to remove pro-inflammatory oxidized lipids and
cellular debris from the extracellular space), one might speculate that
normally 12/15-LO acts as anti-inflammatory enzyme, both by supplying
activating ligands for PPAR-
and by promoting actin polymerization
and phagocytosis.
We assume that the 12/15-LO involvement in phagocytosis requires the
enzyme translocation. Binding of apoptotic cells to macrophages induces
translocation of 12/15-LO from cytosol to the cell membrane and an
enhanced concentration at sites where apoptotic cells are bound (Figs.
2 and 3). 12/15-LO is generally considered to be a cytosolic enzyme
(30). It lacks obvious membrane binding domains. At the same time, a
substrate for 12/15-LO is fatty acids and phospholipids, the components
of lipoproteins and membranes. A recently presented three-dimensional
structure of rabbit 15-LO (31) revealed the presence of an N-terminal
-barrel domain that has a high homology with that of lipases. A
function of this domain in both enzymes is probably binding to their
lipophilic substrates. A similar N-terminal
-barrel domain in 5-LO
has been reported to bind calcium and mediate calcium stimulation of
enzyme activity (32, 33). The translocation of 15-LO from cytosol to
intracellular and plasma membranes has been also described in
reticulocytes and human monocytes and also shown to be dependent on
Ca2+ concentration (34). Moreover, the membrane-bound 15-LO
was more active than the cytosolic enzyme. Indeed, a Ca2+
influx has been observed during the process of macrophage phagocytosis (35). It is possible that one mechanism by which Ca2+ flux
affects phagocytosis is by mediating 12/15-LO translocation and its
effect on actin polymerization.
The 12/15-LO translocation was independent of actin polymerization in
our experiments. Disruption of F-actin by either cytochalasin D or
latrunculin A did not prevent 12/15-LO translocation to the periphery
of the cell and concentration at sites of apoptotic cell binding (Fig.
4). This result is in agreement with a previous report suggesting that
protein translocation in mammalian cell is not affected by disruption
of F-actin network (36). Although we do not yet have any evidence as to
mechanism, it would be of interest to know if the enrichment of these
membrane areas in 12/15-LO occurs via changes in membrane composition
and/or biophysical properties at sites of contact with apoptotic cells
or by some other specific interaction.
On the other hand, inhibition of 12/15-LO activity or disruption of its
gene in macrophages considerably reduced the level of F-actin that was
increased in response to interaction with apoptotic cells (Fig. 5). We
therefore assume that the process of actin polymerization can be
regulated by 12/15-LO activity. In initial experiments to determine the
mechanism(s) by which this might occur, we showed that the products of
12/15-LO oxidation of unsaturated fatty acids, such as
13(S)-HODE, can accelerate the rate of actin filament
elongation (Fig. 6). Although the in vitro effect of
13(S)-HODE appears to be considerably weaker than the
effects reported for certain actin-binding proteins (Ref. 17 for
example), it is hard to predict what would be its role in the cellular
environment. A recent report showing a direct binding of HETEs and
HODEs to the actin molecule (37), combined with the data shown in this
paper, suggests that 12/15-LO products may play a significant role in
promoting F-actin formation in macrophages. Although our data report
for the first time that 12/15-LO products directly affect F-actin
elongation, we also note that 12/15-LO expression in macrophages
affects nucleation as well. This can be concluded from the shortened
lag phase of actin polymerization induced by the lysate of
12/15-LO-expressing macrophage relative to that of 12/15-LO-deficient
macrophages (Fig. 6). Of note is that addition of 13(S)-HODE
to the lysate of 12/15-LO
/
macrophages did
not affect the actin nucleation but did increase the filament
elongation rate (Fig. 6a), in the same manner as it was
observed in lysate-free polymerization experiments (Fig. 6b).
It has been reported that spreading of HeLa cells depends on
lipoxygenase products of arachidonic acid (38). Our data showing a
higher rate of spreading of 15-LO-overexpressing fibroblasts as
compared with control
-galactosidase-expressing fibroblasts (Fig. 7)
support that report and also suggest that 12/15-LO plays a more general
function in cytoskeleton regulation in many cell types rather than only
in activated macrophages. Indeed cytoskeletal rearrangement of B16a
melanoma cells also has been suggested to be dependent on
12(S)-HETE (11). Addition of exogenous 12(S)-HETE induced actin polymerization in B16a melanoma cells responding to
chemoattractants (10). The authors believe that the effect is mediated
by protein kinases. Indeed, preliminary data from our laboratory show
that the addition of exogenous 12(S)-HETE can activate
endogenous lipoxygenase in murine macrophages and further promote actin
polymerization (data not shown). These data would be compatible with
the known ability of fatty acid hydroperoxides to activate 12/15-LO
in vitro (39). Thus, the present studies support a possible
signaling role for endogenous 12/15-LO. The concentration of the
12/15-LO itself at the sites that are targets for actin polymerization
suggests that the enzyme or its fatty acid hydroperoxide products might
be involved in actin signaling. A contemporary view on the filopodia
formation highlights Rho family GTPase Cdc42 and phosphatidylinositol
4,5-biphosphate as important signaling molecules (40) and
Wiskott-Aldrich syndrome protein as a major integrator of signals
activating actin polymerization (41). Whether 12/15-LO products
influence function of these molecules will be an important subject of
further studies. In addition, the recently demonstrated interaction of
a platelet-type 12-LO in epidermoid carcinoma cells with integrin
4 and microtubular proteins lamin A and type II keratin
5 (42) suggests that in addition to its enzymatic products the 12/15-LO
protein itself may have a role in cytoskeleton regulation. It is also
possible of course that by its translocation 12/15-LO is strategically positioned to deliver its products to actin and associated proteins to
promote phagocytosis. Future studies will be needed to elucidate the
exact mechanisms involved in the 12/15-LO-dependent actin polymerization.