Advances in Bioscience and Biotechnology, 2012, 3, 770-781 ABB
http://dx.doi.org/10.4236/abb.2012.326097 Published Online October 2012 (http://www.SciRP.org/journal/abb/)
Characterization of macrophage mutants established by
their resistance to LPS and cycloheximide-induced
apopotic cell death
Fumio Amano1,2*, Shoko Tsukabe1, Reiko Teshima1, Keiko Waku1, Kiyoko Kohama1
1Laboratory of Biodefense & Regulation, Osaka University of Pharmaceutical Sciences, Osaka, Japan
2Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan
Email: *amano@gly.oups.ac.jp
Received 24 August 2012; revised 29 September 2012; accepted 5 October 2012
ABSTRACT
Macrophages are activated by bacterial lipopolysac-
charide (LPS) to produce inflammatory cytokines
such as TNF-α or reactive oxygen species such as ni-
tric oxide or superoxide anion. However, in the pres-
ence of an inhibitor of protein synthesis, cyclo-
heximide (CHX), at 10 μg/mL, LPS at 100 μg/mL in-
duced macrophage apoptosis rapidly without pro-
ducing phenotypes of activated macrophages. In or-
der to understand the mechanism underlying LPS-
induced cytotoxicity toward macrophages, we isolated
mutant cells from a macrophage-like cell line, J774.1,
as clones resistant against the cytotoxic effects of LPS
+ CHX by using a somatic cell genetics protocol. All
of the mutant clones, designated as LCR mutants,
showed resistance to the cell death induced by LPS +
CHX as well as to that induced by higher doses of
LPS alone, as did the LPS1916 mutant cell line, which
had been previously established by its resistance to
100 μg/mL LPS. Characterization of the activated
macrophage phenotypes revealed that these mutants
showed reduced production of TNF-α and nitric oxide
in response to LPS. Further analysis showed a much
reduced amount of [125I]LPS-binding and lower CD14
expression on the cell surface, in spite of an adequate
intracellular expression of CD14 molecules. Besides,
the molecular weight of CD14 on these mutants was
around 40 - 48 kDa, smaller than that of the wild-type
JA-4 cells (around 50 - 55 kDa), suggesting impaired
CD14 maturation in these mutants. However, expres-
sion of Toll-like receptor 4 (TLR4) and Myd 88 on the
cell surface was not different between the wild type
and the mutant cells. These results suggest that LCR
mutants have common phenotypes of mal-expression
of CD14 molecules on the macrophage cell surface,
leading to not only reduced responses to LPS-medi-
ated macrophage activation but acquisition of resis-
tance to LPS-induced apoptotic cell death in the pre-
sence of CHX.
Keywords: Macrophage; J774.1 Cell Line; LPS;
Apoptosis; CD14
1. INTRODUCTION
Macrophages are known to be activated by bacterial
lipopolysaccharides (LPS; [1,2]), and the activated
macrophages produce reactive oxygen species (ROS)
such as superoxide anion (2), hydrogen peroxide
(H2O2), and nitric oxide (NO), or prostaglandins and
inflammatory cytokines such as TNF-α, IL-1β, and so on
[3,4]. It is also known that high concentrations of LPS
lead to macrophage dysfunction and death [5,6] probably
through certain products from such activated macro-
phages [6,7]. In macrophages, expression of LPS binding
due to lymphokines or gamma interferon (IFN-γ) seems
to be closely correlated with the LPS-induced cytotoxic
effects of macrophages toward tumor cells [8]. However,
little is known about the precise mechanisms underlying
these cytotoxic effects of LPS on macrophages. To elu-
cidate the mechanisms of the LPS action on macro-
phages and to investigate the correlation between LPS-
induced activation of macrophages and LPS-induced
cytotoxicity toward macrophages, we isolated LPS-re-
sistant mutants from the J774.1 macrophage-like cell line
with altered activated macrophage phenotypes [9,10]. As
shown at the right side of Figure 1, one of the typical
mutants, the LPS1916 cell line [9], shows very high re-
sistance to LPS with a much reduced ability to produce
ROS in response to LPS [9,11]. Later, as shown at the
left side of Figure 1, we found a novel cytotoxic effect
of LPS toward macrophages in the presence of a protein
synthesis inhibitor, cycloheximide (CHX), which was
proved to be induced without activated macrophage
O
*Corresponding author.
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781 771
Figure 1. A model of the macrophage cell death induced by
LPS in the presence or absence of CHX. In the right half of the
figure, macrophages are activated by LPS; and several hours
later, the activated macrophages produce ROS such as 2
O
and NO as well as inflammatory cytokines such as TNF-α or
IL-1β, or prostaglandins. These products impair macrophage
functions and cause cytotoxicity, leading to apoptotic or non-
apoptotic cell death of macrophages. The left half of the figure
shows that if CHX is present with LPS, macrophages respond
to LPS but rapidly, within a few hours, proceed to apoptotic
cell death without showing activated-macrophage phenotypes.
Resistant mutant cell lines were isolated either in the presence
of a very high concentration (100 μg/mL) of LPS alone (right
side at bottom) or in the presence of 100 μg/mL LPS and 10
μg/mL CHX (left side at bottom), and are designated as the
LPS1916 mutant [9] and LCR mutants (present study), respec-
tively.
phenotypes [12] but with altered LPS-signaling, espe-
cially with sustained phosphorylation of p38 MAP ki-
nease (p38MAPK; [13]) and subsequent apoptosis [13-16].
In this study, we isolated macrophage mutant, resistant to
LPS and CHX-induced macrophage cell death, and in-
vestigated these mutant cell lines with respect to the rela-
tionships between LPS-resistance and not only activated
macrophage phenotypes but also LPS-binding properties
including expression of CD14, Toll-like receptor 4
(TLR4), and Myd88 molecules.
2. MATERIALS AND METHODS
2.1. Materials
Escherichia coli 055:B5 LPS, chromatographically puri-
fied, was obtained from Sigma (St. Louis, MO.) Ham’s
F12 medium was purchased from Flow Laboratories
(McLean, VA); and fetal bovine serum (FBS) containing
less than 60 pg LPS per mL, from GIBCO (Grand Island,
NY). Both were used throughout these experiments.
Na125I was purchased from ICN Biochemicals (Irvine,
CA). Recombinant murine IFN-γ was a generous gift
from TORAY (Tokyo, Japan). All other reagents and
chemicals were of the purest commercial grade available.
2.2. Cell Culture and Isolation of LPS-Resistant
Mutants
JA-4, a subline of a murine macrophage-like cell line
J774.1 [9], was used as the parental, wild-type cell line
throughout this study. JA-4 cells were cultured in Ham’s
F-12 medium containing 50 U of penicillin and 50 μg of
streptomycin per mL, and 10% (v/v) heat-inactivated
FBS (GIBCO, Grand Island, NY) in a CO2 incubator
(5% CO2-95% humidified air) at 37˚C. Isolation of
clones resistant against LPS + CHX-induced cell death
was performed essentially as described previously [9]. In
brief, mutagenesis of the parental JA-4 cells was done by
incubation of the cells (5 × 105 cells/10 mL) at 37˚C for
18 h in T-75 flasks (Corning/Iwaki Glass, Tokyo, Japan)
containing 10 mL of fresh medium and 400 μg of ethyl
methane sulfonate (EMS; Sigma). Then the cells were
rinsed 3 times with medium lacking EMS and incubated
at 37˚C for 6 h in 10 mL of fresh medium containing 100
ng/mL LPS + 10 μg/mL CHX, under which conditions
almost all of the non-treated JA-4 cells died. For selec-
tion of LPS + CHX-resistant mutants, the surviving cells
were detached by trypsinization, reseeded at 1 × 106
cells/100-mm-diameter dish per 10 mL of medium, and
then incubated at 37˚C for several days to allow the cells
to grow and form stable colonies on the dishes. At this
stage, the frequency of LPS + CHX-resistance was about
8.1 × 10–4. Fourteen clones were picked up from these
stable colonies by trypsinization in steel rings, and
maintained in the medium. Finally, the 14 clones were
individually seeded onto 100 mm-dishes and stable sub-
lines were isolated from each dish and designated as
LCR-1 to LCR-14. After estimation of the LPS dose-
dependent resistance in the presence of 10 μg/mL CHX,
LCR-1, 3, 4, 10 and 11 clones were used as LCR mutants
(Figure 1, left side, bottom) throughout this study. All of
these cell clones were stored frozen in a liquid nitrogen
tank before use, and were thawed and maintained in non-
selective medium for at least 1 week before assays.
2.3. Assays for LPS + CHX-Resistance and
LPS-Resistance
Resistance to LPS + CHX-induced cell death was quan-
titatively estimated as described before [12]. In brief, 2 ×
105 cells were seeded into the wells of a 24-well clus-
tered microplate (Costar, Cambridge, MA) and incubated
at 37˚C for 4 h. Then the medium was replaced with
fresh medium containing various concentrations of LPS
in the presence or absence of 10 μg/mL CHX, and the
cells were incubated at 37˚C for 4 h. The reaction was
stopped by sudden chilling of the microplates on ice,
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781
772
after the culture supernatants were collected into micro-
fuge tubes and centrifuged at 11,000 × g for 1 min at 4˚C.
The resultant supernatants were collected and assayed for
lactate dehydrogenase (LDH) by using an LDH-Cyto-
toxic Test Wako (Wako Pure Chemicals, Osaka, Japan)
according to the manufacturer’s protocol. Culture super-
natants collected at zero-time incubation were used to
determine the background release of LDH. To determine
the total LDH activity, cultures of non-treated cells were
mixed with a final concentration of 0.1% Triton X-100 at
the end of the incubation at 37˚C for 4 h, and then incu-
bated for an additional 30 min at 37˚C for complete lysis
of the cells. Cytotoxicity was expressed as % of the total
activity according to the following formula:


% of totalexperimental release
b
ackground releasetotal activity100

We also examined LPS-resistance of the mutant cells
by performing a cell growth assay [9]. In brief, 5 × 103
cells were seeded into the wells of a 24-well clustered
microplate in 0.5 mL of medium and incubated at 37˚C
for 4 h. Then the medium was exchanged for fresh me-
dium containing 0.001 - 100 μg/mL LPS, and the cells
were incubated at 37˚C for 4 days in a CO2 incubator.
Finally, the medium was removed; and the adherent cells
were then detached with 0.5 mL of 0.025% trypsin and 1
mM EDTA in PBS. The cell numbers were counted with
a Coulter Counter (Coulter Electronics, Hialeah, FL) as a
suspension in Isoton II, and the results were expressed as
values (%) relative to the control without LPS addition.
2.4. Assays for TNF-α and NO Production
To estimate the activated-macrophage phenotypes of the
mutants, we used ELISA [17] and Griess reagents [18] to
assess TNF-α release from and NO production by the
LPS-treated cells, respectively. In brief, the cells were
seeded at 2 × 105 cells/0.5 mL/well of a 24-well clustered
microplate and incubated at 37˚C for 4 h, after which the
medium was replaced with fresh medium containing
various concentrations of LPS. The cells were further
incubated at 37˚C for 4 h, and the culture supernatants
were then collected and centrifuged at 11,000 × g for 1
min at 4˚C. The resultant supernatants were examined for
TNF-α content by using an ELISA kit (R & D Systems,
Minneapolis, MN), according to the manufacture’s pro-
tocol. NO production was examined by similarly using
the culture supernatants of LPS-treated cells that had
been incubated in the presence or absence of 10 U/mL
IFN-γ at 37˚C for 24 h, and the amounts of 2
NO
were
determined with Griess reagents for the nitrite assay
(Wako Pure Chemicals). Following mixing of the reac-
tion mixture in a 96-well microplate, the A550 was
monitored with a microplate reader (Titertek Multiscan
Plus, model MKII), with background subtraction at A630.
Quantitative analysis was performed with known stan-
dard solutions of NaNO2.
2.5. FACScan Analysis
The cells were seeded at 4 × 106 cells/10 mL/dish (Iwaki
#3000-035, Tokyo, Japan), and then incubated at 37˚C
overnight. Then the cells were rinsed with ice-cold
FACScan buffer comprising 0.1% BSA and 0.1% so-
dium azide in PBS (19), scraped off from the dish, col-
lected by centrifugation, and washed with FACScan
buffer. For the staining of cell-surface antigens, aliquots
of 1 × 106 cells were incubated on ice with a polyclonal
rabbit anti-mouse CD14 antibody (Santa Cruz Biotech-
nology, Santa Cruz, CA) or FITC-conjugated anti-TLR4
antibody (e-Bioscience, San Diego, CA) for 30 min and
washed once with 0.5 mL of FACScan buffer by cen-
trifugation at 670 × g for 3 min. Then the cells that had
been reacted with anti-mouse CD14 antibody were
mixed with FITC-conjugated anti-rabbit antibody and
incubated on ice for 30 min. Then the cells were washed
again with 0.5 mL of FACScan buffer, finally suspended
in 1 mL of the FACScan buffer, and filtered through a
400-mesh nylon cloth. For the staining of the cell interior,
aliquots of 1 × 106 cells were incubated for 30 min on ice
in culture medium (pH 7.5) containing 10% FBS and
3.7% formaldehyde, washed by centrifugation with ice-
cold PBS as described above. The resultant cell pellets
were treated with 0.1% Triton X-100 in PBS for 30 min
on ice. Next, the cells were washed with PBS by cen-
trifugation and then incubated with either polyclonal
rabbit anti-mouse CD14 antibody or polyclonal rabbit
anti-Myd88 antibody (Santa Cruz Biotechnology) for 30
min on ice. The subsequent processes of washing, reac-
tion with FITC-conjugated anti-rabbit antibody, and fil-
tration were performed as described above. Every sample
for cell surface staining was reacted with 7-amino ac-
tinomycin D (7-AAD; e-Bioscience) to eliminate the
damaged cells with a leaky membrane barrier, before
sorting with a FACScan (Beckton Dickinson Immuno-
cytometry Systems; San Jose, CA). The results were
analyzed with the Cell QuestTM Program (BD TEchnolo-
gies).
2.6. SDS-PAGE/Western Blotting
As described previously [19], the cells were seeded at 4
× 106 cells/10 mL/dish ((Iwaki #3000-035) and incubated
at 37˚C overnight. The cells were then chilled on ice and
washed twice with PBS, scraped off the dishes, and pel-
leted by brief centrifugation at 4˚C at 11,000 × g. The
cell pellets were suspended in 100 μL of lysis buffer
comprising 0.1 mM EDTA, 10 mM NaF, 1 mM Na3VO4,
1% aprotinin and 0.1% Triton X-100 in 20 mM Tris-HCl,
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781 773
pH 7.5. After having been stood on ice for 30 min, the
cells were centrifuged at 11,000 × g for 1 min at 4˚C, and
the resultant supernatant was used as the cell extract. For
SDS-PAGE/Western blotting, 50 μg aliquots of the cell
extracts were treated with the SDS-sample buffer and
then boiled at 95˚C for 5 min. Samples were loaded onto
a 5% - 20% gradient Slab gel (ATTO, Tokyo, Japan),
electrophoresed in a discontinuous buffer system of
Laemli [20], and then electro-transferred to an Immo-
bilon PVDF membrane (Millipore, Billerica, MA) at 30
V overnight and then at 100 V for 30 min. After blocking
of the filter with 30 mg/mL milk casein (Snow Brand,
Sapporo) in a rinse buffer comprising 0.1% Triton X-100,
0.1 mM EDTA, and 0.8% NaCl in 10 mMTris-HCl
buffer, pH7.5, the proteins on the filter were reacted with
polyclonal rabbit antibody against mouse CD14 (Santa
Cruz) at 37˚C for 2 h, and then the membrane filter was
rinsed 3 times with the rinse buffer. Next, the membrane
was reacted with an HRP-conjugated anti-rabbit IgG
(Cell Signaling Technology, Danvers, MA) at room tem-
perature for 1 h. The immune complexes on the mem-
brane were detected by the addition of luminol (Cell Sig-
naling Technology) and H2O2, according to the manu-
facturer’s protocol. The intensity of the reaction products
was determined by use of an LAS2000 image analyzer
(Fuji Film, Tokyo, Japan), and the results were analyzed
with Multi Gauge v2.2 (Fuji Film, Tokyo, Japan).
2.7. Preparation of [125I]-LPS and Binding of
[125I]-LPS to Cell Monolayers
[125I]-LPS (E. coli O55:B5) was prepared as described
previously [10] by using a variation of the procedure of
Ulevitch [21]. The specific activity of the labeled LPS
varied from preparation to preparation, ranging from 0.1
to 1.2 μCi/μg LPS; and the approximate mean value was
0.3 μCi/μg LPS. The LPS binding assay was performed
according to the method described before [10] with slight
modifications. In brief, cells were seeded at 2 × l05
cells/well of a 24-well microplate containing 0.5 ml of
the culture medium. After preculturing overnight, the
medium was changed to that containing 10%FBS, and
then the cells were cooled on ice at 4˚C for 15 min. Next,
the cells were incubated with 100 ng/mL [125I]-LPS in
the presence or absence of 5 μg/mL of non-labeled LPS
at 4˚C for 6 h. The binding incubations were terminated
by rapid and repeated washing 3 times of each well with
0.5 mL of the buffers, composed of 0.02% (w/v) BSA in
PBS. Finally, the cells were lysed with 0.5 mL of 0.1 N
NaOH and transferred to a counting tube, and then the
radioactivity was measured with an auto-well γ-counter
(Aloka, Tokyo, Japan). The results were expressed as
specific binding activity of LPS (ng/well/2 × l05 cells)
after calculation by subtraction of non-specific activity
(radioactivity bound in the presence of 5 μg/mL non-
labeled LPS) from the total activity (radioactivity bound
in the absence of 5 μg/mL of non-labeled LPS).
2.8. Morphologic Observation
Cells were observed under a phase-contrast microscope
(Diamat; Nikon, Tokyo, Japan), and photographs were
taken with black and white film (Neopan F; Fuji Film,
Tokyo, Japan) with a Nikon FE camera.
3. RESULTS
3.1. Resistance of Mutant Cells against the LPS
and CHX-Induced Cytotoxicity
We examined the resistance of the mutant cell lines
against the LPS and CHX-induced cytotoxicity. LCR-1
and LCR-3 cell lines, representative LCR mutant cell
lines isolated by using a somatic cell genetics protocol to
select for cells resistant to LPS and CHX-induced cyto-
toxicity (Figure 1, left side at bottom), showed little
change after incubation with 100 ng/mL LPS + 10 μg/mL
CHX (Figures 2(f) and (h)). However, the JA-4 cell line,
i.e., the parental, wild-type cell line, exhibited severe cell
damage including evidence of apoptosis as well as a
swollen, necrotic morphology under the same conditions
(Figure 2(b)). The LPS1916 cell line, isolated previously
[9] by its resistance to high-dose LPS (Figure 1, right
side at bottom) showed little cell damage after LPS +
CHX-treatment (Figure 2(d)). However, all of these cell
lines showed intact cell morphology with adherent and a
round shape, if the cells were incubated without LPS +
CHX-treatment (Figures 2(a), (c), (e) and (g)). Similar
results were obtained with other LCR mutants, i.e., LCR-
4, 10 and 11 in the presence or absence of LPS + CHX
(data not shown). These results show that the LCR mu-
tant cell lines, as well as the LPS1916 cell line, were re-
sistant to the cytotoxic effects of LPS + CHX on macro-
phages [12].
To examine the extent of the resistance of LCR mu-
tants quantitatively, we next examined the LPS dose-
dependent release of LDH in the presence or absence of
CHX (Figure 3). JA-4 cells released LDH dependent on
the LPS dose in the presence of CHX. They released
significant amounts of LDH at and more than 1 ng/mL
LPS, and exhibited maximal release of around 66% at
and more than 10 ng/mL (Figure 3(a)). However, none
of the LCR mutants or LPS1916 cells showed LDH re-
lease in the presence of CHX, even at 1000 ng/mL LPS.
In the absence of CHX, JA-4, LCR, and LPS1916 cell
lines showed no LDH release in response to LPS (Figure
3(b)), indicating that LCR mutant cell lines had acquired
high resistance to LPS + CHX-induced macrophage cell
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781
774
Figure 2. Morphological changes in the mutant cells treated
with LPS and CHX. Macrophage cell damage observed under a
phase-contrast microscope after incubation of JA-4 (a), (b),
LPS1916 (c), (d), LCR-1 (e), (f), or LCR-3 (g), (h) cells in the
absence (a), (c), (e), (g) or presence (b), (d), (f), (h) of both 100
ng/mL LPS and 10 μg/mL CHX at 37˚C for 4 h. Note the dif-
ference in the extent of LPS + CHX-induced cell damage be-
tween the wild-type, JA-4 cells (b) and the LCR-mutants (f), (h)
or LPS1916 cells (d). Original magnification, ×400.
death.
Because the resistance of the LCR mutants seemed to
be primarily LPS-resistance based on the results of the
LPS dose-response study shown in Figure 3, we next
examined the LPS-resistance of these mutants not by the
short-term incubation in the presence of CHX (Figure
3(a)), but by the long-term incubation with LPS alone at
higher doses (Figure 4)). LPS showed growth inhibition
of the macrophage cell lines, and the JA-4 cell line was
the most sensitive to LPS, whereas the LPS1916 cell line
showed the highest resistance to LPS among the cell
lines. All of the LCR mutants showed resistance to LPS
intermediate between that of JA-4 and LPS1916 cell
lines. The IC50 value of LPS, meaning inhibition of cell
growth to 50% of the non-treated control without LPS,
0
10
20
30
40
50
60
70
80
01101001000
LDH release (% of total)
LPS (ng/mL)
(+CHX)
(a)
0
10
20
30
40
50
60
70
80
0110100 1000
LDH release (% of total)
LPS
(
n
g
/mL
)
(-CHX)
(b)
Figure 3. Lack of LDH release from the macrophage
cell lines treated with LPS + CHX. LDH release was
examined as a quantitative indicator of the cell death
induced by LPS + CHX. The cells were treated with
various concentrations of LPS shown on the abscissa
in the presence (a) or absence (b) of 10 μg/mL CHX at
37˚C for 4 h, and LDH release into the culture super-
natants was assayed as described in the text. The fig-
ures show JA-4 (), LPS1916 (), LCR-1 (), LCR-3
(), LCR-4 (), LCR-
10 (), and LCR-
11 () cells.
The results are the means ± S.E. of independent 3 ex-
periments, each performed in duplicate.
was 0.057 μg/mL for JA-4, 14.4 μg/mL for LPS1916,
and 0.49, 0.32, 2.28, 0.36, and 0.35 μg/mL for LCR-1,
LCR-3, LCR-4, LCR-10, and LCR-11 cell lines, respect-
tively. These results show that the LCR mutants had
property of LPS-resistance several fold higher than that
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781 775
0
10
20
30
40
50
60
70
80
90
100
110
00.001 0.010.1110100
Cell number
(% of contro l)
LPS (g/mL)
JA4
LPS1916
LCR1
LCR3
LCR4
LCR10
LCR11
Figure 4. LPS-resistant growth of the macrophage mutant cell
lines during long-term culture. Attenuation of cell growth by
high doses of LPS was examined by using JA-4 (), LPS1916
(), LCR-1 (), LCR-3 (), LCR-4 (), LCR-
10 (), and
LCR-
11 () cells. The cells were incubated at 37˚C for 4 days
in medium supplemented with various concentrations of LPS,
as shown on abscissa, but without CHX. The cell numbers were
counted with a Coulter Counter after detachment of the cells
from the microplates, as described in the text. The results are
the means from a typical experiment out of repeated experi-
ments, each performed in duplicate.
of the JA-4 parental cell line but that the extent of their
resistance was less than that of the LPS1916 cell line,
which was isolated not by resistance to the LPS + CHX-
induced cytotoxicity but by that to high-dose LPS, sug-
gesting the enhancing effects of CHX on the susceptibil-
ity of macrophages to the LPS-induced cell death during
the short-term culture.
3.2. Secretion of TNF-α
We next examined the activated macrophage phenotypes
that appeared in response to LPS. Secretion of TNF-α is
one of the most remarkable phenotypes of activated
macrophages [1]. As shown in Figure 5, JA-4 cells se-
creted TNF-α dependent on the LPS dose, and 1 ng/mL
LPS induced significant amounts of TNF-α. On the con-
trary, all of the LCR mutants and LPS1916 cells secreted
a lesser amount of TNF-α, especially at the low doses of
LPS at 1 and 10 ng/mL. These results suggest that LCR
mutants were able to be activated by LPS but that the
extent of the response and the threshold of it were less
and higher, respectively, than those of the parental cell
line, JA-4.
Next, the LCR mutants were examined for another
phenotype of activated macrophages, i.e., NO production.
As shown in Figure 6(a), in the absence of 10 U/mL
IFN-γ, only JA-4 cells produced scarce but significant
amounts of 2 during incubation with 0.1 - 10
μg/mL LPS, where none of the LCR mutants or LPS1916
NO
0
2
4
6
8
10
12
14
16
18
0110100 1000
TNF-(ng/mL)
LPS ( ng/mL)
JA4
LPS1916
LCR1
LCR3
LCR4
LCR10
LCR11
Figure 5. TNF-α production from the macrophage mutant cell
lines treated with LPS. TNF-α production from macrophage
mutant cell lines was examined as an activated-macrophage
phenotype. JA-4 (), LPS1916 (), LCR-1 (), LCR-3 (),
LCR-4 (), LCR-
10 (), and LCR-
11 () cells were treated
with various concentrations of LPS, as shown on the abscissa,
at 37˚C for 4 hours. Thereafter, the release of TNF-α into the
culture supernatant was assayed, as described in the text. The
results are the means ± S.E. of 3 independent experiments, each
performed in duplicate.
cells did so. Because JA-4 cells produce 2
NO
but not
3
NO
after induction of iNOS by LPS [11], the results in
Figure 6(a) may be thought to correspond to NO pro-
duction by these macrophages. In the presence of 10
U/mL IFN-γ, not only JA-4 but also the LCR mutants as
well as the LPS1916 cells produced 2, in an LPS
dose-dependent manner. However, the extent of 2
NO
NO
production was less in the LCR mutants than in the JA-4
cells, especially at 0.1 μg/mL LPS. These results suggest
that the LCR mutants had reduced ability for LPS-re-
sponsiveness in terms of NO production.
3.3. Binding of [125I]LPS to the Mutant Cells
Because another LPS-resistant mutant, LR-9, shows re-
duced binding of LPS to the cell surface [10], we exam-
ined the binding of [125I]LPS to the LCR mutants as well
as to the LPS1916 and JA-4 cells. As shown in Figure 7,
specific binding of LPS was much lower in the LCR
mutants and LPS1916 cell line than in the JA-4 parental
cell line. These results suggest that the resistance to LPS
both in the presence of CHX during 4 hours’ incubation
(Figure 3(a)) and in the absence of CHX during 4 days’
incubation (Figure 4), as well as the reduced responses
of the activated-macrophage phenotypes (Figures 5 and
6) might have been due to this reduced LPS-binding of
the LCR mutants.
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781
776
Figure 6. NO production from the macrophage mutant cell
lines treated with LPS. As another activated macrophage phe-
notype, NO production was examined. JA-4 (), LPS1916 (),
LCR-1 (), LCR-3 (), LCR-4 (), LCR-
10 (), and LCR-11
() cells were treated with various concentrations of LPS, as
shown on the abscissa, at 37˚C for 24 hours in the absence (a)
or presence (b) of 10 U/mL IFN-γ and then in the cul-
ture supernatant was determined, as described in the text. The
results are the means from a typical experiment out of repeated
experiments performed in duplicate.
2
NO
3.4. Expression of CD14 in the Mutant Cells
CD14 is known to be involved in the binding of LPS to
macrophages, leading to transduction of the LPS-binding
signal to the TLR4 receptor systems [1]. Because the
LCR mutants showed reduced LPS-binding (Figure 7),
CD14 expression was examined in JA-4, LPS1916,
LCR-1, and LCR-3 cells. As shown in Figure 8(a), the
expression of CD14 was remarkable on the surface of
JA-4 cells, whereas it was scarcely seen on that of
LCR-1 cells (Figure 8(c)), and significantly reduced in
LPS1916 (Figure 8(b)) and LCR-3 (Figure 3(d)) cells.
These results would seem to explain in part the reason
why these mutants showed reduced LPS-binding.
Because CD14 is known to be processed intracellu-
larly from a molecule with high mannose-type oligosac-
charide to that of the mature-type one [22], we next ex-
amined the distribution of CD14 inside of the cells. As
shown in Figures 8(e)-(h), all of the cells examined ex-
hibited a similar intracellular expression of CD14 mole-
cules, in contrast to the difference in the cell-surface dis-
tribution of CD14.
3.5. Expression of TLR4 and Myd88 in the
Mutant Cells
As LPS signals are transduced via TLR4 and Myd88 [1,
22], the reduced LPS-responses in terms of both LPS-
resistance (Figures 3 and 4) and activated-macrophage
phe- notypes (Figures 5 and 6) might be attributed to the
altered expression of TLR4 and/or Myd88. FACScan
analysis of cell-surface TLR4 revealed significant ex-
pression with very little difference between JA-4cell line
and the LCR mutant or LPS1916 cell lines (Figure 9).
Besides TLR4, intracellular Myd88 expression in JA-4,
LPS1916, LCR-1 and LCR-3 cell lines was also exam-
ined. As shown in Figure 10, Myd88 was present sig-
nificantly and similarly in all 4 of these cell lines. These
results suggest that neither TLR4 nor Myd88 was pri-
marily involved in the acquisition of the LPS-resistance
or in the reduced LPS responses concerning activated-
macrophage phenotypes.
3.6. Analysis of Molecular Sizes of CD14
Expressed in the Mutant Cells
The results in Figure 8 suggest that CD14 in the LCR
mutants and LPS1916 cells was poorly expressed on the
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
JA-4LPS1916LCR-1 LCR-3 LCR-4LCR-10LCR-11
Specific bindi ng
(ng/well)
Cell lines
Figure 7. [
125I]LPS binding to the macrophage mutant cell
lines. Binding of LPS to JA-4, LPS1916, and LCR mutant cell
lines was examined with [125I]LPS. The ordinate shows the
specific binding of LPS, calculated by subtraction of non-spe-
cific binding from the total binding, as described in the text.
The results are the means ± S.E. of independent 3 experiments,
each performed in duplicate.
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Copyright © 2012 SciRes.
777
Figure 8. Expression of CD14 in the mutant cells. Expression of CD14 on the cell surface (a)-(d) and inside of the
cells (e)-(h) was examined by FACScan analysis. JA-4 (a), (e), LPS1916 (b), (f), LCR-1 (c), (g), and LCR-3 (d), (h)
cells were reacted with rabbit antibody against mouse CD14 (open area of plot) or not (closed area of plot), and then
reacted with FITC-conjugated anti-rabbit antibody. In order to stain the intact cell surface, the cells were reacted with
7-AAD before cell sorting; and the cells stained with 7-AAD were excluded from the analysis as damaged cells. For
staining of the cell interior, the cells were sequentially fixed with formaldehyde, rendered permeable with 0.1% Triton
X-100, and stained with rabbit antibody against mouse CD14, followed by FITC-conjugated antibody against rabbit
IgG, as described in the text. The results shown were obtained from a typical experiment out of repeated experiments
that gave similar results.
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781
778
Figure 9. Expression of TLR4 on the mutant cells. Expression of TLR-4 on the cell surface of JA-4 (a); LPS1916 (b);
LCR-1 (c); and LCR-3 (d) cells was analyzed by FACScan. The cells were reacted with FITC-conjugated anti-mouse
TLR-4 antibody (open area plot) or not (closed area plot), as well as treated with 7-AAD; and the cell populations
without 7-AAD staining were analyzed as intact cells for TLR4 expression on their cell surface. The results were ob-
tained from a typical experiment out of repeated experiments that gave similar results.
Figure 10. Expression of Myd88 in the mutant cells. Expression of Myd88 inside of JA-4 (a); LPS1916 (b); LCR-1 (c);
and LCR-3 (d) cells was analyzed by FACScan. The cells were fixed with formaldehyde, made permeable with 0.1%
Triton X-100, and then stained with rabbit antibody against mouse Myd88 (open area of plot) or not (closed area of
plot), followed by FITC-conjugated antibody against rabbit IgG, as described in the text. The results were obtained
from a typical experiment out of the repeated experiments that gave similar results.
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781
Copyright © 2012 SciRes.
779
cell surface but was abundant inside of the cells. There-
fore, there might be a possibility that the process of
CD14 maturation was somehow defective in these LCR
mutant cells and LPS1916 cells. Analysis of these cell
extracts and subsequent immune-detection of CD14 by
SDS-PAGE/Western blotting revealed that JA-4 cells
expressed CD14 molecules with a molecular weight
higher than 50 kDa, whereas the LCR mutants and
LPS1916 cells showed a smaller molecule (Figure 11).
These results suggest that processing of CD14 molecules
was normal in the JA-4 cells but that that of the LCR
mutants and LPS1916 cells might have been suspended
at or before transition steps from the immature type with
high-mannose oligosaccharides to the mature type with
complex oligosaccharides [22]. These results seem to be
in close relationship with the results on CD14 expression
(Figure 8), also suggesting impaired processing of CD14
and inadequate distribution on the cell surface of the
LCR mutants and LPS1916 cells.
lished by resistance to 100 μg/mL LPS alone [9], these
LCR mutants showed significantly less resistance to
LPS-induced cell growth inhibition than LPS1916 cells
(Figure 4) in the absence of CHX. These results suggest
that the stringency of the selection force of the mutant
clones after mutagenesis of JA-4 cells with EMS was
different between 100 μg/mL LPS alone in the previous
study [9] and 100 μg/mL LPS + 10 μg/mL CHX in the
present study. This is because the frequency of LPS re-
sistance in the previous study was about 1.5 × 10–5,
whereas that of LPS + CHX-resistance in the present
study was 8.1 × 10–4.
However, LPS-activated macrophage phenotypes of
the LCR mutants and LPS1916 cells were similarly re-
duced as compared with those of the wild-type, JA-4 cell
line, concerning LPS-induced TNF-α release (Figure 5)
as well as LPS-induced NO production in the presence or
absence of IFN-γ (Figure 6). These results suggest that
the LCR mutants had the common properties of declined
or reduced LPS-responses in terms of either LPS-resis-
tance or LPS-induced macrophage activation. Although
we found 2 distinct pathways of LPS-induced macro-
phage cytotoxicity, either through macrophage activation
(Figure 1, right) or through apoptotic cell death proc-
esses without macrophage activation in the presence of
CHX (Figure 1, left), the resultant resistant mutants in
these two pathways, either the LPS1916 cell line (Figure
1, right), or the LCR mutants (Figure 1, left) seem to
have exhibited altered reduced LPS responses in com-
mon.
4. DISCUSSION
In this report, we described the isolation of macrophage
mutants, resistant to LPS + CHX-induced cell death,
from JA-4 as the parental cell line, which had originated
from a macrophage-like cell line, J774.1 [9] by using
somatic cell genetics, and designated them as LCR mu-
tants. The mutants showed little damage to their mor-
phology after treatment with LPS + CHX at 37˚C for 4 h
(Figure 2), and released little LDH in response to LPS
even at a dose of 1000 ng/mL in the presence of 10
μg/mL CHX (Figure 3(a)). Although these phenotypes
indicating resistance to LPS were also observed in the
LPS1916 cell line, which had been isolated and estab-
In order to understand the mechanisms underlying the
resistance of these mutants to LPS, we examined charac-
teristics of both the LCR mutants and LPS1916 cells in
70
60
50
40
MW (kDa)
JA-4
LCR-1
LCR-3
LCR-4
LCR-10
LCR-11
LPS1916
Figure 11. Analysis of molecular sizes of CD14 expressed in the mutant cells. JA-4,
LPS1916, and LCR mutant cells were collected, washed, and extracted as described in
the text. Fifty-microgram aliquots of the cell extracts were analyzed by SDS-PAGE/
Western blotting using a polyclonal anti-mouse CD14 antibody, as described in the text.
The results shown are images of chemiluminescence detected with the LAS2000 im-
age analyzer, as described in the text. Note that none of the extracts of LCR mutants or
LPS1916 cells yielded CD14 bands corresponding to a molecular weight larger than
50 kDa, which was evident in the JA-4 parental cell line.
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F. Amano et al. / Advances in Bioscience and Biotechnology 3 (2012) 770-781
780
this study. The results in Figure 7 showed reduced bind-
ing of LPS to these mutants as compared with the bind-
ing to the parental cell line, JA-4. Besides, the expression
of CD14 molecules on the cell surface was significantly
reduced in both the LCR mutants and LPS1916 cell line,
despite the fact that the intracellular distribution of CD14
molecules in these mutants was similar to that of the
parental cell line (Figure 8), supporting the idea that
CD14 participated in the accumulation of LPS-LPS
binding protein complexes on the cell surface, and in the
efficient subsequent transfer of these complexes to TLR4
receptors [24].
Contrary to the difference in distribution of CD14 on
the cell surface of the mutants and JA-4 cells, TLR4 and
Myd88 were similarly expressed in these mutants and
JA-4 cells (Figures 9 and 10). These results suggest that
the LPS-resistance of the macrophage cell lines might
have been acquired through altered CD14 expression or
malfunction of CD14, resulting in reduced cell surface
expression and a decrease in LPS binding, as was shown
in Figure 7. It should be noted that TLR4 and Myd88 in
the mutants showed the same distribution as seen in the
pa- rental cells, resulting in the retention of such LPS-re-
sponses as TNF-α release and NO production at high
doses of LPS at and more than 100 μg/mL (Figures 5
and 6).
In addition to the altered cell-surface distribution of
CD14 in these mutants compared with that in the JA-4
cells (Figure 8), the size of the CD14 molecule was quite
different between these mutants and JA-4 cells (Figure
11). Whereas the molecular weight of the CD14 of JA-4
cells ranged from 44 to 55 kDa, the range in the LCR
mutants was lower, i.e., 40 - 48 kDa; and the molecular
weight in the LPS1916 cells was, about 48 kDa. There
were no bands higher than 50 kDa in the case of the LCR
mutants and LPS1916 cells. It is known that processing
of CD14 is accompanied by carbohydrate modifications
and that immature CD14 molecules have high mannose-
type oligosaccharides with lower molecular weights,
whereas mature CD14 has complex-type oligosaccha-
rides of high molecular weight [22].
Taken together, the results in the present study suggest
that macrophages would acquire LPS-resistance, selected
by either a very high dose of LPS or LPS + CHX, due to
interrupted processing of CD14 molecules with high
mannose-type oligosaccharides at the immature stage,
thus preventing maturation to CD14 with complex-type
oligosaccharides. Such interruption would result in de-
creased transport to and expression of mature CD14
molecules on the cell surface, with the consequence be-
ing decreased sensitivity of macrophages to LPS. Al-
though the LPS-signaling cascades and molecular mecha-
nisms underlying the signal transduction through TLR-4
have been extensively studied and reported, the role of
CD14 molecules in the acquisition of LPS-resistance by
macrophages has not been studied so far. Our present
results provide novel insights into LPS-macrophage in-
teractions with respect to LPS-resistance and biodefense
against massive infections by Gram-negative bacteria
and endotoxin shock [23,25,26].
5. ACKNOWLEDGEMENTS
This work was supported by a Grant-in-aid for the Promotion of Sci-
ences and a Grant-in-aid for High Technology Research from the Min-
istry of Education, Science and Culture of Japan and by funding from
the Human Science Foundation. We thank Dr. Hisae Karahashi for
technical assistance, and Dr. Ko Fujimori for useful comments.
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