Activated lymphocytes increase expression of 5-lipoxygenase
and its activating protein in THP-1 cells
William L.
Ring,
Carl A.
Riddick,
Joseph R.
Baker,
Christopher K.
Glass, and
Timothy D.
Bigby
Department of Medicine, Department of Veterans Affairs Medical
Center, San Diego, 92161; and University of California, San Diego,
California 92037
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ABSTRACT |
The aim of this study was to investigate the regulation of the
5-lipoxygenase pathway of arachidonic acid metabolism by lymphocytes using the monocyte-like cell line, THP-1. When THP-1 cells were incubated over 4-7 days in 10% supernatant from lectin-activated human lymphocytes, their capacity to synthesize 5-lipoxygenase products
was significantly increased. In contrast, the supernatant from
nonactivated lymphocytes had no effect. The increase in capacity to
synthesize 5-lipoxygenase products was mimicked by the addition of
either granulocyte macrophage colony-stimulating factor (GM-CSF) or
interleukin-3. These increases in synthetic capacity reflected increased enzymatic activity. Increased immunoreactive protein and mRNA
for the enzymes 5-lipoxygenase and 5-lipoxygenase-activating protein were also found in cells conditioned with activated lymphocyte supernatants. Furthermore, the increase in mRNA for both enzymes was
not blocked by cycloheximide, suggesting that the effect on steady-state mRNA levels does not require the synthesis of new protein.
The increase in mRNA could be reproduced by GM-CSF. We conclude that
lymphocytes can regulate the expression of 5-lipoxygenase in THP-1
cells over a period of days via the release of soluble factors.
arachidonic acid; leukotrienes; inflammation; cytokines; 5-lipoxygenase-activating protein
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INTRODUCTION |
THE 5-LIPOXYGENASE PATHWAY produces
leukotrienes, which are oxygenated metabolites of arachidonic acid.
Leukotrienes have been implicated in a wide range of inflammatory
diseases (7). The enzyme 5-lipoxygenase, which acts on free arachidonic
acid, is responsible for catalyzing the first two steps in the
synthesis of leukotrienes, namely the conversion of arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic
acid (5-HPETE) and then 5-HPETE to leukotriene
A4
(LTA4). The synthesis of
5-lipoxygenase products in intact cells is also dependent on a nuclear
membrane-associated protein, 5-lipoxygenase-activating protein (FLAP)
(6). The mechanism by which 5-lipoxygenase expression and activity is
controlled is still poorly understood.
THP-1 cells are a well-established line of human tumor cells derived
from a Japanese pediatric patient with acute monocytic leukemia (23).
These cells possess characteristics of mononuclear phagocytes: they are
nonspecific esterase positive, they produce lysozyme, and they are
phagocytic (1). However, THP-1 cells have not been viewed previously as
an adequate in vitro model for examination of 5-lipoxygenase pathway
regulation. Specifically, this cell line has been reported to either
lack 5-lipoxygenase activity or to have 5-lipoxygenase
activity that cannot be modulated (13, 14, 19).
We recently reported that human peripheral blood monocytes, when grown
in coculture with human lymphocytes for 2 days, developed a threefold
increase in their capacity to synthesize 5-lipoxygenase products (16).
Moreover, when the supernatant from lectin-activated lymphocytes, but
not from nonactivated lymphocytes, was added to monocytes at a 10%
concentration (vol/vol) for 2 days, there was a large increase in the
capacity of the monocytes for 5-lipoxygenation. This increase in
capacity was associated with significant increases in 5-lipoxygenase
and FLAP proteins in conditioned cells compared with control cells, as
assessed by immunoblot analysis. Likewise, Northern blot analysis of
total RNA showed significant increases in 5-lipoxygenase and FLAP mRNA
in conditioned monocytes compared with control monocytes. Additional
work revealed that granulocyte macrophage colony-stimulating factor
(GM-CSF) and interleukin-3 (IL-3), present in supernatants from
activated lymphocytes, were responsible for most of the observed
increases in 5-lipoxygenase and FLAP (16). However, further
investigations into the regulation of the 5-lipoxygenase
pathway expression have been limited by the difficulties inherent in
working with primary cell isolates and by practical limits to the
number of human monocytes that can be harvested. These difficulties
preclude further in-depth studies of mRNA levels or of the mechanism(s)
whereby transcription is regulated in primary isolates of human
mononuclear phagocytes.
The broad goal of our investigations was to further study the
mechanism(s) whereby the expression and, in turn, the activity of the
5-lipoxygenase pathway are regulated over the long term. Therefore, the
specific aim was to examine, using a stable cell line, the interaction
we have previously demonstrated between lymphocytes and monocytes and
to utilize the cell line to begin to define the molecular aspects of
regulation of expression of the 5-lipoxygenase pathway in mononuclear
phagocytes.
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METHODS |
Cell culture. THP-1 cells were
cultured in RPMI 1640 medium with 10% fetal calf serum, to which 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml gentamicin, and
50 µM 2-mercaptoethanol were added. Media were replaced every
2-3 days. Studies were performed at cell densities of 2.0 × 106 to 2.5 × 106 cells/ml. All cell
preparations were performed at room temperature using sterile
techniques in a laminar flow hood and endotoxin-free (<10 pg/ml)
solutions.
Preparation of nonactivated and lectin-activated
lymphocyte supernatant. Lymphocyte supernatants were
prepared as previously described (16). Briefly, peripheral blood was
obtained from normal healthy subjects without known
allergies, who had not taken any medication for 2 wk and
any aspirin products for 6 wk before blood donation. All subjects were
volunteers and had given informed consent to a protocol approved by the
Committee on Investigations Involving Human Subjects of the University
of California, San Diego. Lymphocytes were isolated by density gradient
centrifugation over isotonic Percoll with a specific gravity of 1.077 followed by removal of monocytes by their adherence to serum-coated
plastic. Lymphocytes were incubated for 4 days without (nonactivated)
or with (lectin-activated) lectins. For all lectin stimulation studies, cells were incubated with 10 µg/ml concanavalin A and 10 µg/ml phytohemagglutinin. The media were then collected and centrifuged to
remove cells and debris, and the supernatant was stored at 4°C. For
THP-1 conditioning studies, lymphocyte supernatant was added to a final
concentration of 10% (vol/vol).
Studies of disrupted cells. THP-1
cells were collected from tissue culture plates, and any nonadherent
cells were gently released from tissue culture plastic with a sterile
rubber policeman and added to the collected cells. The resulting cell
suspension was centrifuged, and the cell pellet was disrupted by
sonication at 4°C as previously described (15).
Radiolabeling and assessment of arachidonic acid
release. Labeling studies were performed by overnight
incubation of THP-1 cells with 0.05 µCi of
[14C]arachidonic acid,
as described (9).
Assay of 5-lipoxygenase and LTA4
hydrolase activity in whole and disrupted cells.
5-Lipoxygenase activity and LTA4
hydrolase activity were measured in intact THP-1 cells as previously
described (15). Cells were collected in warmed Hanks' balanced salt
solution (HBSS) and stimulated with 1 µM A-23187 at 37°C for 15 min. The supernatant was collected, and lipids were extracted from this
supernatant using methanol and chloroform by previously described
techniques (2, 12). 5-Lipoxygenase and
LTA4 hydrolase activity in
disrupted cells was measured by specific enzymatic assay as previously
described (12). For 5-lipoxygenase activity, samples were incubated in a 37°C shaking water bath with 2 mM
CaCl2, 2 mM ATP, and 100 µM arachidonic acid for 15 min. For
LTA4 hydrolase activity, samples were incubated in a 37°C shaking water bath in assay buffer at a
final concentration of 100 mM tris(hydroxymethyl)aminomethane, 0.35%
bovine serum albumin, and 15 µM
LTA4 for 15 min. In both specific
enzymatic assays, the reaction was then quenched by adding cold
methanol, and precipitated protein was removed by centrifugation. Supernatants were removed and evaporated to dryness, and residues were
dissolved in methanol for storage at
70°C until further analysis. For lipoxygenase and hydrolase assays in both intact and
disrupted cells, lipoxygenase metabolites were resolved and quantitated
by reverse-phase high-performance liquid chromatography using
previously described techniques (12).
Immunoblot analyses. Immunoblotting
was performed on total protein using previously described techniques
(15). Immunoblots were probed with antibodies (generously provided by
Dr. Jilly Evans, Merck-Frosst Centre for Therapeutic Research, Point
Claire-Dorval, Quebec, Canada) that were raised against either
recombinant human leukocyte 5-lipoxygenase or amino acid
residues 41-52 of the human FLAP sequence (1:5,000 dilution). The
specificity of these antibodies has been extensively characterized (8,
17, 24). Bands were quantitated by scanning laser densitometry (LKB
Instruments, Uppsala, Sweden).
Northern blot analyses.
Poly(A)+ RNA was isolated from
total cell extracts on oligo(dT) cellulose as previously described (4). Northern blotting was performed as previously described (18, 22).
Northern blots were probed with cDNA probes for 5-lipoxygenase (a
generous gift from Dr. Richard A. F. Dixon; Merck Sharp & Dohme Research Laboratories, West Point, PA), FLAP (a generous gift from Dr.
Jilly Evans), LTA4 hydrolase (a
generous gift from Dr. Takao Shimizu, University of Tokyo, Japan), and
-actin. In some cases, 10 µg/ml cycloheximide was added to the
cells before conditioning with lymphocyte supernatant, and mRNA was
isolated. Bands were quantitated by scanning laser densitometry (LKB
Instruments) and were normalized to bands observed with the
-actin
probe.
Materials. HBSS and RPMI 1640 were
obtained from BioWhittaker (Wakersville, MD); penicillin and
streptomycin were from the Cell Culture Facility, University of
California, San Diego; gentamicin was from Gemini Bioproducts
(Calabasas, CA); Percoll was from Pharmacia Fine Chemicals (Piscataway,
NJ); A-23187 was from Calbiochem-Behring (La Jolla, CA);
redistilled-in-glass grade chromatography solvents were obtained from
Burdick and Jackson Division, Baxter (Muskegon, MI);
5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic
acid (5-HETE) was from Biomol (Plymouth Meeting, PA);
LTA4,
LTB4, and
LTC4 were generous gifts from
Merck-Frosst Centre for Therapeutic Research, Point Claire-Dorval,
Quebec, Canada; arachidonic acid was from NuChek Prep (Elysian, MN);
and recombinant human GM-CSF and IL-3 were from R & D Systems
(Minneapolis, MN). All other chemicals were from Sigma Chemical (St.
Louis, MO) and were of the finest grade available. All cell lines were
obtained from American Type Culture Collection (Rockville, MD).
Data analysis. Data are expressed as
means ± SE in all circumstances where mean values are compared. The
differences between two means were analyzed with a two-tailed unpaired
t-test. The time course study and the
GM-CSF/IL-3 intact cell study were analyzed with a repeated measures
analysis of variance using a Fisher's post hoc test. Differences were
considered significant when P < 0.05 (20).
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RESULTS |
Effect of lectin-activated lymphocyte supernatants on
5-lipoxygenase activity in intact THP-1 cells. A
variety of inflammatory cell lines were screened for an effect on the
regulation of 5-lipoxygenase activity by activated lymphocyte
supernatants. These cells included HL-60 cells (a promyelocytic
leukemic cell line), KG-1 cells (an acute myelogenous leukemic cell
line), U-937 cells (a histiocytic lymphoma cell line), and THP-1 cells.
Cells were cultured for 7 days either in media alone or with media
containing 10% supernatant from lectin-activated lymphocytes, and then
A-23187-stimulated intact cell 5-lipoxygenase activity was assessed. In
these screening studies, only THP-1 cells, when conditioned with
lectin-activated lymphocyte supernatnant, demonstrated a significant
increase in 5-lipoxygenase activity compared with control cells (Fig.
1). Moreover, there was no
significant difference in 5-lipoxygenase activity between control THP-1
cells, THP-1 cells conditioned directly with lectins, or THP-1 cells
conditioned with the supernatant from nonactivated lymphocytes (Fig.
1). Specifically, over a period of 7 days, the supernatant from
lectin-activated lymphocytes caused a 15-fold increase in the capacity
of THP-1 cells to synthesize the 5-lipoxygenase product, 5-HETE
(P < 0.05). Similiar results were
obtained for leukotriene B4
(LTB4; 0.0 vs. 0.0 vs. 0.0 vs. 2.6 ± 0.5 pmol
LTB4/106
cells; data are for THP-1 cells, THP-1 cells treated with lectins, supernatant from nonactivated lymphocytes, and supernatant from lectin-activated lymphocytes, respectively;
n
4). There was no detectable
leukotriene C4
(LTC4) release from THP-1 cells
under the conditions studied. There was also no detectable 5-HETE or LTB4 in supernatants from either
lectin-activated or control lymphocytes. Over a period of 7 days, the
supernatant from lectin-activated lymphocytes did not significantly
change the number of cells present in culture compared with control
cells (101.2 ± 11.7% vs. 100.0 ± 16.2%, total cells in
culture as a percentage of the mean number of control cells, for
conditioned vs. control cells, respectively).

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Fig. 1.
Effects of lymphocyte supernatants on 5-lipoxygenase activity in THP-1
cells. Lymphocytes were incubated for 4 days with or without lectins,
and conditioned supernatants were collected. THP-1 cells were cultured
either alone (control), with lectins, or with 10% (vol/vol) of
conditioned supernatants from either unactivated or lectin-activated
lymphocytes. THP-1 cells were then washed with Hanks' balanced salt
solution and were stimulated with 1 µM A-23187 for 15 min at
37°C. Supernatants were collected, extracted, and analyzed for
lipoxygenase products by reverse-phase high-performance liquid
chromatography and ultraviolet spectroscopy. Data are expressed as pmol
5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic
acid (5-HETE)/106 cells and
represent means ± SE of at least 5 experiments.
* Significantly different from control cells
(P < 0.05, two-tailed unpaired
t-test).
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Time course of the effect of lectin-activated
lymphocyte supernatant on 5-lipoxygenase activity in THP-1
cells. THP-1 cells were cultured with or without 10%
supernatant from lectin-activated lymphocytes for various lengths of
time, and then A-23187-stimulated intact cell 5-lipoxygenase activity
was assessed. There was no difference between conditioned cells and
control cells at 15 min and 1 day (Fig. 2).
However, at 2 days there was a 3.2-fold increase in the capacity of
conditioned cells to release 5-HETE, compared with controls
(n
7;
P < 0.05). This effect of
lectin-activated lymphocyte supernatant was maximal at 7 days and
persisted for at least 14 days (the longest time period examined).
There was no detectable A-23187-stimulated release of
LTB4 from either control or
conditioned THP-1 cells at 15 min through 4 days. However, after 7 days
of conditioning, the LTB4 released
was enhanced in conditioned vs. control cells as described above, and
this effect again persisted for at least 14 days. As noted above, there
was no detectable LTC4 release
from either conditioned or control cells at any time.

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Fig. 2.
Time course of effect of lectin-activated lymphocyte supernatant on
5-lipoxygenase capacity in THP-1 cells. THP-1 cells were cultured for
up to 7 days in RPMI 1640 with (L-Stim) or without (control) 10%
activated lymphocyte supernatant. At various time points, stimulated
5-lipoxygenase activity was assessed as in Fig. 1. Data are expressed
as pmol 5-HETE/106 cells and
represent means ± SE of at least 3 experiments.
* Significantly different from corresponding time point obtained
with control cells (P < 0.05, analysis of variance using Fisher's post hoc test).
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Effect of GM-CSF and IL-3 on 5-lipoxygenase activity
in THP-1 cells. We have shown that GM-CSF and IL-3 are
the principal substances in the supernatant from lectin-activated
lymphocytes responsible for increasing 5-lipoxygenase activity in
monocytes (16). Therefore, we asked whether these cytokines also
regulate 5-lipoxygenase activity in THP-1 cells. THP-1 cells
were cultured with or without either 10 ng/ml GM-CSF or 10 ng/ml
IL-3 for 7 days, and then A-23187-stimulated intact cell 5-lipoxygenase
activity was assessed. Both GM-CSF and IL-3 caused an ~12-fold
increase in the capacity of THP-1 cells to synthesize the
5-lipoxygenase product 5-HETE (P < 0.05; Fig. 3). Parallel results were
obtained for LTB4 (0.0 vs. 1.5 ± 0.2 vs. 1.1 ± 0.8 pmol
LTB4/106
cells, control vs. GM-CSF vs. IL-3, respectively;
n
3). The magnitude of this
increase in capacity to synthesize 5-lipoxygenase products by each
cytokine was comparable with that exerted by 10% supernatant from
lectin-activated lymphocytes (Fig. 3). Neither cytokine was able to
stimulate detectable LTC4 release.

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Fig. 3.
Effects of granulocyte macrophage colony-stimulating factor (GM-CSF) or
interleukin-3 (IL-3) on 5-lipoxygenase activity in THP-1 cells. THP-1
cells were cultured for up to 7 days in RPMI 1640 alone (control) or
with either 10 ng/ml GM-CSF, 10 ng/ml IL-3, or 10% (vol/vol) activated
lymphocyte supernatant (L-Stim). Stimulated 5-lipoxygenase activity was
assessed as in Fig. 1. Data are expressed as pmol
5-HETE/106 cells and represent
means ± SE of at least 3 experiments. 5-HETE release was
significantly increased in all conditioning studies compared with
control. * P < 0.05, analysis
of variance using Fisher's post hoc test.
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Effect of lectin-activated lymphocyte supernatant on 5-lipoxygenase
activity and LTA4 hydrolase activity in
disrupted THP-1 cells.
To determine whether an increase in 5-lipoxygenase activity
and/or LTA4 hydrolase
activity was responsible for the increased release of 5-HETE and
LTB4 observed in intact
conditioned THP-1 cells, cells were cultured with or without 10%
supernatant from lectin-activated lymphocytes for 7 days and then
disrupted by sonication. Over this period of 7 days, the supernatant
from lectin-activated lymphocytes did not significantly change the
total protein per cell compared with control cells (3.1 ± 0.4 vs.
3.3 ± 0.4 mg/106 cells of
total protein, for conditioned vs. control cells, respectively). However, disrupted fractions from conditioned cells had 2.5 more 5-lipoxygenase activity than that found in disrupted fractions from
control cells, as assessed by 5-HETE production
(P < 0.05; Fig.
4). There was no difference in
LTA4 hydrolase activity between samples prepared from conditioned vs. control cells (117.4 ± 34.3%, LTB4 production as percent
of control; n = 5;
P = not significant).

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Fig. 4.
5-Lipoxygenase activity in disrupted THP-1 cells previously conditioned
with lectin-activated lymphocyte supernatant. THP-1 cells were cultured
for 7 days without (control) or with 10% activated lymphocyte
supernatant (L-Stim) as in Fig. 1. Cells were then disrupted by
sonication and assayed for 5-lipoxygenase activity using a specific
enzymatic assay as described in
METHODS. Data expressed as pmol
5-HETE/mg protein are means ± SE of at least 5 experiments.
* 5-HETE release was significantly increased in disrupted
conditioned cells compared with control cells
(P < 0.05, two-tailed unpaired
t-test).
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Effect of lymphocyte supernatant on arachidonic acid
release by THP-1 cells. Supernatants from
lectin-activated lymphocytes caused a greater increase in
5-lipoxygenase product release from intact cells than the increase in
enzymatic activity measured in disrupted cells. Therefore, to determine
whether a change in arachidonic acid release contributed to the
increased release of 5-lipoxygenase products by intact conditioned
THP-1 cells, cells were cultured for 7 days with media alone or with
media and 10% supernatant from lectin-activated lymphocytes. The cells were also radiolabeled with
[14C]arachidonic acid
during the last day of conditioning. The cells were then stimulated
with 1 µM A-23187 for 15 min, and release of label was assessed as a
percent of the total label incorporated. Similar quantities of label
were released from conditioned vs. control cells (Fig.
5). Thus enhanced release of the substrate, arachidonic acid, is unlikely to account for the increased release of
5-lipoxygenase products from conditioned cells.

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Fig. 5.
Effect of lectin-activated lymphocyte supernatant on THP-1 cell
arachidonic acid release. THP-1 cells were cultured without (control)
or with 10% lectin-activated lymphocyte supernatants (L-Stim) for 7 days. During last day of culture period, cells were labeled with
[14C]arachidonic acid.
Release of radiolabel into the media after stimulation with A-23187 was
assessed as described in METHODS. Data
are expressed as label released as a percent of total label
incorporated and represent means ± SE of at least 3 experiments.
There was no difference between conditioned and control cells
(P = NS).
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Effect of lectin-activated lymphocyte supernatant on
immunoreactive 5-lipoxygenase and FLAP in THP-1 cells.
To determine whether lectin-activated lymphocyte supernatant increases
immunoreactive 5-lipoxygenase and FLAP, THP-1 cells were cultured for 7 days with or without 10% supernatant from lectin-activated lymphocytes and were then disrupted by sonication. Immunoblotting of these sonicates revealed a reproducible increase in 5-lipoxygenase and FLAP
protein when immunoblots from conditioned cells were compared with
those from control cells (Fig. 6).
Densitometric analysis of autoradiographs was preformed. There was a
3.3 ± 1.2- and 2.3 ± 0.3-fold increase in 5-lipoxygenase and
FLAP protein, respectively, when immunoblots from conditioned cells
were compared with those from control cells (means ± SE of at least
4 experiments).

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Fig. 6.
Effect of lectin-activated lymphocyte supernatant of
5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein
(FLAP) levels in THP-1 cells, as assessed by immunoblot. THP-1 cells
were cultured without (control) or with 10% lectin-activated
lymphocyte supernatants (L-Stim) for 7 days. Immunoblots were performed
on disrupted cell fractions. Immunoblot was probed for 5-lipoxygenase
(A) and FLAP
(B), comparing control
(left) and conditioned
(right).
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Effect of lectin-activated lymphocyte supernatant on
5-lipoxygenase and FLAP mRNA in THP-1 cells. To
determine the effect of lectin-activated lymphocyte supernatant on
steady-state mRNA encoding for 5-lipoxygenase, FLAP, and
LTA4 hydrolase, THP-1 cells were
cultured with or without 10% supernatant from lectin-activated lymphocytes for various lengths of time. Northern blot analysis of
poly(A)+ RNA from these cells
revealed no effect on mRNA for either 5-lipoxygenase or FLAP after 4 h
of conditioning, compared with control cells (Fig.
7). However, there was an approximately
threefold increase in mRNA for both 5-lipoxygenase and FLAP at 1 day of
conditioning, and this increase persisted without change through 2, 4, and 7 days. In contrast, supernatants from lectin-activated lymphocytes had no effect on levels of LTA4
hydrolase mRNA (110%, 80%, 120%, and 90% of control at 4 h, 1 day,
2 days, and 7 days, respectively).

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Fig. 7.
Effect of lectin-activated lymphocyte supernatants on
5-lipoxygenase and FLAP mRNA levels in THP-1 cells, as
assessed by Northern blot analysis. THP-1 cells were cultured without
(control) or with 10% lectin-activated lymphocyte supernatants
(L-Stim) for up to 7 days.
Poly(A)+ RNA was extracted and
subjected to Northern blot analysis. Autoradiographs of a
representative blot probed for 5-lipoxygenase and FLAP, comparing
control and conditioned for each pair of rows at various time points,
as indicated, are shown.
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Effect of GM-CSF on 5-lipoxygenase and FLAP mRNA
levels in THP-1 cells. To determine the effect of
GM-CSF on steady-state mRNA encoding for 5-lipoxygenase and FLAP, THP-1
cells were cultured with or without 10 ng/ml of GM-CSF for 2 days.
Northern blot analysis of total RNA from these cells revealed an
increase in mRNA for both 5-lipoxygenase and FLAP (Fig.
8). There was no effect on mRNA for
LTA4 hydrolase (90% of control).

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Fig. 8.
Effect of GM-CSF on 5-lipoxygenase and FLAP mRNA levels in THP-1 cells,
as assessed by Northern blot analysis. THP-1 cells were cultured
without (control) or with 10 ng/ml GM-CSF for 7 days. Total RNA was
extracted and subjected to Northern blot analysis. Autoradiographs of a
representative blot probed for 5-lipoxygenase and FLAP, comparing
control and GM-CSF-treated, are shown.
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Effect of cycloheximide on changes in mRNA encoding
for 5-lipoxygenase and FLAP induced by conditioning with
lectin-activated lymphocyte supernatant. To determine
whether new protein synthesis was involved in the effect of
lectin-activated lymphocyte supernatant on levels of mRNA for
5-lipoxygenase and FLAP, THP-1 cells were treated for 2 days with or
without 10% supernatant from lectin-activated lymphocytes, in the
presence or absence of 10 µg/ml of a protein synthesis inhibitor,
cycloheximide. Cycloheximide had no significant effect on the ability
of lectin-activated lymphocyte supernatant to cause an increase in
steady-state mRNA levels for either 5-lipoxygenase or FLAP (Fig.
9). There was no effect of cycloheximide-
and/or lectin-activated lymphocyte supernatant on
LTA4 hydrolase mRNA levels (117%
and 96% of control cells, for cells conditioned with lectin-activated
lymphocyte supernatant, with or without cycloheximide, respectively).
Thus lectin-activated lymphocyte supernatant increased mRNA levels for
5-lipoxygenase by 6.0-fold in the presence of cycloheximide vs.
4.4-fold in the absence of cycloheximide. Parallel results were
obtained for FLAP mRNA levels (4.6- vs. 3.4-fold increase in the
presence and absence of cycloheximide, respectively).

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Fig. 9.
Effects of cycloheximide (CHX) on ability of lectin-activated
lymphocyte supernatants to alter 5-lipoxygenase and FLAP mRNA
expression in THP-1. THP-1 cells were prepared as in Fig. 6. In some
cultures, 10 µg/ml cycloheximide was added before 10%
lectin-activated lymphocyte supernatant.
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DISCUSSION |
We report that the human monocytic leukemia cell line, THP-1, has the
capacity to synthesize 5-lipoxygenase products, has detectable levels
of 5-lipoxygenase and FLAP, and expresses mRNA encoding for
5-lipoxygenase and FLAP. Furthermore, we report that the
supernatant from lectin-activated human lymphocytes causes a large
increase in the capacity of THP-1 cells to synthesize 5-lipoxygenase
products, whereas supernatants from nonactivated lymphocytes have no
effect. The increase in 5-lipoxygenase capacity is significant within
4-7 days, as measured by the release of 5-HETE and
LTB4, and persists for at least 14 days of conditioning. The increase is also associated with significant
increases in the expression of protein and mRNA for both 5-lipoxygenase
and FLAP but is not associated with changes in levels of mRNA for LTA4 hydrolase. Similar increases
in the capacity of THP-1 cells to synthesize 5-lipoxygenase products
occur after 7 days of conditioning with either GM-CSF or IL-3, and
GM-CSF also caused an increase in mRNA for both
5-lipoxygenase and FLAP. Thus THP-1 cells have a
5-lipoxygenase pathway that can be upregulated by activated lymphocytes
and by GM-CSF or IL-3 via increases in expression of 5-lipoxygenase and
FLAP.
The increased capacity for 5-lipoxygenation of arachidonic acid in
THP-1 cells when conditioned with lectin-activated lymphocyte supernatants appears to be primarily associated with changes in 5-lipoxygenase and FLAP. Changes in availability of arachidonic acid do
not appear to largely contribute to this increased capacity, as
reflected by radiolabeling studies. Interestingly, in the conditioned THP-1 cells, there is a delay in the detection of increased
LTB4 production compared with
5-HETE production. Although this might suggest a delayed effect of
conditioning on expression of LTA4 hydrolase, this seems less likely, as there is neither a change with
conditioning in LTA4 hydrolase
activity by specific enzymatic assay nor a change in the level of mRNA
for the enzyme. In fact, the delay in detecting
LTB4 production may simply be due
to differences in sensitivity of the intact cell assay. There is a
greater increase in the capacity of conditioned THP-1 cells to generate
5-lipoxygenase products in the intact cell assay, which reflects
changes anywhere in the pathway, compared with the specific enzymatic
assays for 5-lipoxygenase and FLAP. Although this may suggest
additional regulation at other levels of the pathway, it may also
reflect differences in assay conditions.
Other investigators using THP-1 cells have suggested that they were a
poor system for studying the 5-lipoxygenase pathway. Previous work has
not demonstrated the presence of FLAP in unconditioned cells (14).
Earlier studies have reported that, when THP-1 cells were conditioned
with interferon-
or with phorbol myristate acetate for 2 days, there
was no detectable synthesis of 5-lipoxygenase products (13). Likewise,
more recently, investigators have reported that THP-1 cells conditioned
for 4 days with retinoic acid, interferon-
, 1,25-dihydroxyvitamin
D3, or combinations of these have
no detectable synthesis of leukotrienes or HETEs (19). In contrast, we
detected both 5-lipoxygenase and FLAP in immunoblots of THP-1 cells
that had not been conditioned. In addition, we measured low levels of
both 5-HETE and LTB4 synthesis in
nonconditioned THP-1 cells stimulated with A-23187. This finding may be
due to differences between sublines of this same cell line or
differences in cell culture conditions. We also report a large increase
in the capacity of THP-1 cells to synthesize 5-lipoxygenase products
after 4 days of conditioning with supernatant from lectin-activated
lymphocyte, GM-CSF, or IL-3. The differences between our findings and
those published previously may be due to a number of factors, including an increased length of conditioning time and conditioning with presumably a wide range of soluble products produced by
lectin-activated lymphocytes. However, previous work in the area did
not specifically examine the effect of GM-CSF or IL-3 on 5-lipoxygenase
capacity in THP-1 cells.
Previous work in monocytes or the cell line Mono-Mac 6 have suggested a
role for transforming growth factor-
in the regulation of the
5-lipoxygenase pathway (5, 21). Transforming growth factor-
is known
to promote the maturation of THP-1 cells into macrophage-like cells (3)
and may influence the 5-lipoxygenase pathway in these cells. However,
this was not the focus of this investigation. We have reported that, in
monocytes conditioned with lectin-activated lymphocyte supernatants,
the increase in the capacity of monocytes to synthesize 5-lipoxygenase
products is largely mediated by GM-CSF and IL-3 (16). In THP-1 cells, GM-CSF and IL-3 appear to play a similar role.
The protein synthesis inhibitor cycloheximide had no effect on the
increased expression of both 5-lipoxygenase and FLAP mRNA seen in THP-1
cells after conditioning with lectin-activated lymphocyte supernatant.
This suggests a direct effect on 5-lipoxygenase and FLAP expression by
the conditioning agent(s) and argues against the stimulation of
expression of an intermediary protein (transcription factor) modulating
mRNA levels. This would be in keeping with recent work on GM-CSF and
IL-3 signal transduction, which seems primarily to involve stimulated
tyrosine phosphorylation of proteins already present within resting
cells, including JAK2, STAT5a, and STAT5b (10, 11).
We have previously reported that the response to lectin-activated
lymphocyte supernatant observed in THP-1 cells is also seen in human
monocytes obtained from peripheral blood (16). However, in monocytes,
there is an initial increase of 5-lipoxygenase synthesis capacity at 15 min that then returns to baseline values at 1 h. This rapid effect was
not seen in THP-1 cells. In both monocytes and THP-1 cells, in
contrast, there is a more prolonged effect on 5-lipoxygenase capacity.
This begins at 1 day and persists at least through 4 days in monocytes.
This failure to observe an initial increase in 5-lipoxygenase synthetic
capacity in conditioned THP-1 cells compared with conditioned monocytes
is as yet unexplained. It may be due to that fact that THP-1 cells are
relatively immature macrophage precursors and that, therefore, this
cell line responds somewhat differently than monocytes to the
conditioning stimulus. In fact, the time course of increased capacity
for 5-lipoxygenation of arachidonic acid in THP-1 cells is
somewhat slower than for monocytes, requiring approximately three times
as long before both the first detectable increase and the maximum
effect. This again would suggest the possibility that THP-1 cells,
before conditioning, are less mature than cultured monocytes. Moreover,
the maximal effect of lectin-activated lymphocyte supernatant on THP-1
cells corresponds to a larger times increase in their
capacity to synthesize 5-lipoxygenase products compared with
the effect seen in monocytes. However, despite the minor quantitative
and kinetic differences observed between THP-1 cells and monocytes,
and, with the exception of the very early effect of lectin-activated
lymphocyte supernatant seen in the latter cells, THP-1 cells appear to
have broadly similar regulation of their 5-lipoxygenase pathway
compared with monocytes.
In summary, THP-1 cells have the capacity to synthesize 5-lipoxygenase
products, particularly after exposure of the cells to the supernatant
from lectin-activated lymphocytes. The increased capacity of
conditioned cells for 5-lipoxygenation is associated with increases in
both protein and mRNA for both 5-lipoxygenase and FLAP and
may be mediated by GM-CSF and/or IL-3. This effect of
lectin-activated lymphocyte supernatant on the 5-lipoxygenase pathway
of THP-1 cells is broadly comparable to effects observed with primary
isolates of human peripheral blood monocytes. Thus lymphocytes can
regulate the expression of 5-lipoxygenase and FLAP in THP-1 cells over
a period of days via the release of soluble factors. The current
observations advance the prior work in monocytes determining the time
course of increases in mRNA and determining that increases in mRNA do
not require intermediate synthesis of transcription factors that
modulate gene expression. Moreover, THP-1 cells may be used for an
in-depth analysis of the mechanisms whereby the expression of the
5-lipoxygenase pathway in inflammatory cells is regulated, providing
additional insights into the mechanism by which
5-lipoxygenase expression and activity are controlled in
mononuclear phagocytes.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Heart, Lung, and Blood
Institute Grant HL-01888, by a grant from the Merit Review Board of the
Department of Veterans Affairs, and by a grant from Glaxo Wellcome. W. L. Ring was supported by a training fellowship award from the American
Lung Association of California and by a Physician Scientist Award from
the National Institutes of Health. C. A. Riddick was
supported by a Faculty Development grant from the Robert Wood Johnson
Foundation. T. D. Bigby was supported in part by a Career Investigator
Award from the American Lung Association.
 |
FOOTNOTES |
Address for reprint requests: T. D. Bigby, Division of Pulmonary and
Critical Care Medicine, Dept. of Medicine, Univ. of California, San
Diego, 111-J, Dept. of Veterans Affairs Medical Center, 3350 La Jolla
Village Dr., San Diego, CA 92161.
Received 3 December 1996; accepted in final form 29 August 1997.
 |
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