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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 4011-4018
Lipopolysaccharide Regulates Macrophage Fluid Phase Pinocytosis Via
CD14-Dependent and CD14-Independent Pathways
By
Maikel P. Peppelenbosch,
Marjory DeSmedt,
Tessa ten Hove,
Sander
J.H. van Deventer, and
Johan Grooten
From Laboratory for Experimental Internal Medicine, G2-130 Academic
Medical Centre, Amsterdam, The Netherlands; and Laboratory for
Molecular Biology, Flemish Institute for Biotechnology, G, Belgium.
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ABSTRACT |
Lipopolysaccharide (LPS) is a mediator of inflammation and septic
shock during bacterial infection. Although monocytes and macrophages
are highly responsive to LPS, the biological effects of LPS in these
cell types are only partially understood. We decided, therefore, to
investigate the influence of LPS on macrophage pinocytosis and Fc
receptor-mediated endocytosis, two prominent and related macrophage
effector functions. We observed that LPS did not greatly influence
endocytosis in either macrophages or monocytes, but did exert a dual
action on pinocytosis: at lower concentrations (0.1 to 100 ng/mL), LPS
caused a decrease in pinocytosis in both macrophages and monocytes,
whereas at higher LPS concentrations, enhanced pinocytosis in
macrophages was observed. Detoxified LPS was two orders of magnitude
less potent in producing these effects. After inhibition of the LPS
receptor CD14, the LPS-induced decrease in pinocytosis was absent, and
stimulation of pinocytosis at lower LPS concentrations was unmasked. We
conclude that LPS can influence pinocytosis via CD14-dependent and
CD14-independent signaling pathways. Furthermore, as
addition of LPS to macrophages effected pinocytosis but not Fc
receptor-mediated endocytosis, these two processes are independently
regulated in macrophages.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
LIPOPOLYSACCHARIDE (LPS) is composed of
O-specific polysaccharide side chains attached to a basal
core oligosaccharide, which in turn is covalently bound to a lipid
moiety known as lipid A.1 LPS is a component of the outer
lipid bilayer of gram-negative bacteria and is an important inducer of
host defense during bacterial infection. After its release from
bacteria, LPS binds to a specific binding protein, known as LPS-Binding
Protein (LBP).2,3 LBP transfers monomeric LPS to CD14, a
protein from which both a soluble and membrane-bound form
exist.4 The membrane-bound form of CD14, which is linked to
the membrane by a glycosylphosphatidylinositol anchor, is highly
expressed in phagocytes. This protein serves as a cellular receptor for
LPS and mediates the LPS-induced cellular changes important for
enhanced host defense, although the means by which binding of the
LPS/LBP complex to CD14 results in the transfer of a signal across the
cell membrane is still unclear. Further signaling includes activation
of NF- B, p38 MAP kinase, and stress-activated protein kinases,
finally resulting in increased production of inflammatory cytokines
(eg, tumor necrosis factor [TNF], interleukin-1 , interleukin-6,
granulocyte-macrophage colony stimulating factor) and chemokines (eg,
interleukin-8, macrophage inflammatory peptide-1 ), generation of
nitric oxide, and upregulation of surface antigen
presentation.5 LPS is, therefore, an important regulator of
monocyte and macrophage function in pathophysiology.
Among the most characteristic properties of monocytes and macrophages
is the capacity of these cells to take up large volumes by fluid-phase
pinocytosis and to ingest microbes and other particles by phagocytosis.
Phagocytosis and pinocytosis require similar changes in the actin
cytoskeleton, and both processes are sensitive to certain dominant
negative mutants of the small ras-like GTPases of the Rho
family6-8 and are blocked by inhibitors of phosphoinositide 3-kinase activity.9 It is assumed, therefore, that both
processes are closely related with respect to the underlying molecular
mechanism. Fluid phase pinocytosis by macrophages is a constitutive
process, but may be greatly enhanced by phorbol esters or macrophage
colony stimulating factor.10 Recently, we showed that TNF
has a similar activity also.10a Phagocytosis can be
initiated directly by direct binding of gram-negative bacteria to
monocyte CD14,11 but in general, phagocytosis is initiated
by binding of C3b or IgG to the appropriate receptors on the phagocyte
surface. Some insight into the signal transduction mechanism leading
from the activated Fc receptor to stimulation of phagocytosis was
recently obtained by the observation that mouse macrophages lacking a
functional copy of the gene coding for the Syk tyrosine kinase are
deficient in Fc receptor-dependent phagocytosis.12
Nevertheless, interaction between the endocytotic machinery and
receptor signal transduction is poorly understood. Also, the possible
modulation by LPS of TNF- and phorbol ester-stimulated pinocytosis or
Fc receptor-stimulated endocytosis is unknown.
The above-mentioned considerations, as well as the general importance
of LPS-dependent signaling in infection and immunity, prompted us to
study the effects of LPS on pinocytosis and endocytosis in mouse
macrophages and human peripheral blood monocytes. Use was made of the
4-4 clone of VN11 retrovirus-immortalized macrophages, recently
generated in our laboratory from the spleen of a C57BI/6 mouse.13 We showed earlier that these cells display
expression of the mature macrophage markers Mac-1 (CD11b), Mac-2, BM-8,
F4-80, the transferrin receptor CD71, and the adhesion molecule CD18, whereas the immature macrophage marker ER-MP58 is not
expressed.13 Furthermore, the cells show constitutive
expression of the costimulatory ligands B7-1 (CD80) and B7-2 (CD86),
and treatment of these cells with interferon  readily induced strong
expression of major histocompatibility class II (I-A)
molecules.13 Functionally, these cells adhered strongly to
plastic or glass surfaces, and antigen loading of these macrophages
induced a proliferative T-cell response in vitro. Importantly,
reintroduction of antigen-loaded 4-4 cells in syngeneic mice induced a
primary T-cell response to the antigen and was not accompanied by
myeloid leukemia.13 Apparently, apart from the capacity of
the 4-4 clone to be maintained in vitro, these cells are phenotypically
and functionally not different from primary isolated mature
macrophages. Further analysis showed that these cells display high
expression of the Fc receptor II (CD32) and that this receptor is
coupled to the endocytotic machinery as 4-4 cells efficiently
phagocytosed IgG-coated fluorescent microspheres and sheep
erythrocytes.13 Finally, the cells expressed high levels of
CD14, and addition of LPS to these cells induces secretion of
interleukin-1, interleukin-6, interleukin-12, and TNF.13 We
concluded that the 4-4 cells form a suitable model system to study the
effects of LPS and decided to use these cells for investigating LPS
action on macrophage phagocytosis and pinocytosis. In addition, we
employed peripheral blood monocytes, isolated from healthy volunteers,
as we have recently shown that these cells are highly responsive to LPS.
In this study, we show that LPS inhibits fluid phase pinocytosis at
lower concentrations, but at higher concentrations stimulates pinocytosis. Interestingly, inhibition of CD14 with a specific antibody
blocked the inhibitory influence of LPS on pinocytosis, and under these
conditions, LPS-dependent stimulation of pinocytosis at lower
concentrations became apparent. Finally, LPS did not effect Fc
receptor-mediated phagocytosis. Therefore, these results not only
identify CD14-dependent and CD14-independent effects on macrophage
fluid phase pinocytosis, but also show that pinocytosis and Fc
receptor-mediated endocytosis can be dissociated.
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MATERIALS AND METHODS |
Cell culture.
Isolation of the 4-4 clone of mouse macrophages has been described
earlier in detail.13 For routine culture, cells were grown
in RPMI 1640 (Life Technologies, Paisley, UK) and were supplemented with 7.5% fetal calf serum, 2 mmol/L L-glutamine, 100 U/mL penicilin, 100 µg/mL streptomycin, 1 mmol/L sodium pyruvate, and 40 µmol/L -mercaptoethanol. Cells were seeded on six well dishes 48 hours before experimentation. Peripheral blood mononuclear cells were isolated from heparinized blood of healthy volunteers using
Ficoll-Hypaque (Sigma Chemical Co, St Louis, MO) density
gradient centrifugation (400g, 20 minutes). Subsequently, the
cells residing at the interface were washed three times and resuspended
at a concentration of 106 cells/mL in RPMI 1640 supplemented with 2% pooled human serum. After washing, cells were
allowed to recover 30 minutes at 37°C, 5% CO2, before
the onset of experimentation. The healthy status of the volunteers was
confirmed by analysis of blood sedimentation.
[3H]Sucrose uptake.
If appropriate, cells were preincubated with a CD14-blocking antibody
(Pharmingen, San Diego, CA) or irrelevant antibody (SUK5 antibody,
directed against the tail domain of kinesin heavy chain; a kind gift of
Dr K. de Vos, Flanders Institute for Biotechnology, Ghent, Belgium) for
15 minutes. For determining fluid phase uptake, the culture medium was
supplemented with 1 µCi [3H]-sucrose
(Amersham, Arlington Heights, IL) and the appropriate stimulus. Unless
stated otherwise, the uptake of [3H]-sucrose was allowed
to continue for 45 minutes at 37°C and 5% CO2.
Subsequently, the cells were placed on ice, and the cells were washed
six times with ice-cold phosphate-buffered saline. Afterwards, the
cells were dissolved in 1% sodium dodecyl sulfate (SDS),
and accumulated radioactivity was determined by scintillation counting.
Parallel wells were incubated with [3H]-sucrose at 4°C
to determine the contribution of nonendocytosed [3H]sucrose to total radioactivity. Other parallel wells
were used to determine cell number per well. Using the specific
activity of the [3H]-sucrose, fluid phase uptake was
calculated and was always approximately 2 × 10 14
L/min/cell, which is in agreement with the values reported by others
and represents approximately 1% of the total cell volume. Solvent
controls were without effect.
Measurement of pinocytosis and Fc receptor-mediated endocytosis
using fluorescent human catalase.
Human catalase (Sigma) was fluorescently labeled using Fluorlink
(Amersham). Subsequently, cells were incubated with the appropriate stimulus and 6.6 mg/mL fluorescent catalase. For determining Fc receptor-mediated endocytosis, human catalase was preincubated with a
mix of the culture supernatants of three different hybridomas producing
antihuman catalase antibodies (a description of antibody generation and
their characteristics will be published elsewhere). Incubation of
macrophages with human catalase was allowed to continue for 30' minutes
at 37°C and 5% CO2, after which the cells were placed on
ice, washed four times with ice cold phosphate-buffered saline, and
subsequently harvested using 2 mmol/L EDTA. Uptake of fluorescence was
then determined using flow cytometry. To distinguish uptake from
binding, parallel experiments were performed at 4°C. Determination of
the pinocytotic activity in human peripheral blood monocytes was
performed by incubating 106 freshly isolated mononuclear
cells with 1 mg/mL fluorescently labeled (using Fluorlink from
Amersham) human serum protein (Ig for intravenous injections; obtained
from the Central Laboratory for Bloodtransfusion, Amsterdam; lot number
950315H60) for 45 minutes at 37°C and 5% CO2.
Subsequently, cells were put on ice, washed twice, and analyzed by
fluorescence-activated cell sorting (FACS). The scatter profile was
used to identify monocytes, and average fluorescence per cell was
determined. Cell types not identified as monocytes by the scatter
profile displayed negligible accumulation of fluorescence.
LPS detoxification.
LPS was dissolved in a 0.1 mol/L NaOH solution in 95% ethanol and
incubated for a prolonged time period (approximately 2 months) at room
temperature. Subsequently, the solution was adjusted to pH 7.0 with
acetic acid, and the LPS was spinned down and washed with ethanol.
Subsequently, LPS was reconstituted in phosphate-buffered saline.
Injection of 100 ng of thus-treated LPS into actinomycin D-sensitized
mice was inactive, whereas untreated LPS was lethal at this concentration.
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RESULTS |
LPS inhibits or stimulates fluid phase pinocytosis depending on the LPS
concentration involved.
To study fluid phase pinocytosis, [3H]-sucrose was used
as a probe. It seemed that addition of [3H]-sucrose to
the extracellular medium of 4-4 macrophages resulted in rapid
accumulation of radioactivity in the cells and that this uptake
remained in the semilinear phase for at least 60 minutes (Fig
1). For further experiments, an assay time
of 45 minutes was used. The effects of LPS on
[3H]-sucrose uptake were twofold: at lower
concentrations, LPS impaired pinocytosis, maximal inhibition being
reached at 10 ng/mL (Fig 2A). At
LPS concentrations in excess of 10 ng/mL, pinocytosis increased again,
and in cells challenged with an LPS concentration of 10 µg/mL (the
highest concentration used in this study), a clear stimulation of
[3H]-sucrose uptake, as compared with unstimulated cells,
was noted (Fig 2A). LPS exerts, therefore, different effects
on macrophage fluid phase pinocytosis, depending on the LPS
concentration involved.
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| Fig 1.
Pinocytosis in macrophages. Murine clone 4-4 macrophages
were incubated for various time intervals with
[3H]-sucrose at 37°C. Subsequently, the cells were
placed on ice and washed, and accumulated radioactivity was determined
by scintillation counting. Using the specific activity of the
[3H]sucrose, volume uptake was calculated and expressed
as femtoliter per cell.
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| Fig 2.
Effects of LPS on macrophage fluid phase uptake. Murine
clone 4-4 macrophages were stimulated with vehicle or different
concentration of LPS for 45 minutes at 37°C and 5% CO2
in the presence of [3H]sucrose. Subsequently, the cells
were placed on ice and washed, and accumulated radioactivity was
determined by scintillation counting. Using the specific activity of
the [3H]sucrose, fluid phase uptake was expressed as
femtoliter per cells per minute. The results show the average and
standard error of three different experiments, each experiment being
the average of three independent determinations. The statistical
significance of the difference between the unstimulated and stimulated
value is indicated by stars, one star indicating a P value
<0.05 and two stars indicating a P value <0.01 (P
values were obtained using an unpaired two-tailed heteroscedastic
Student's t-test on the original nine values on which the
graph is based.). (A) Effects of different LPS concentrations on
macrophage pinocytosis. (B) Effect of different LPS concentrations on
TNF (500 U/mL)-stimulated pinocytosis. (C) Effects of LPS in the
presence of the CD14-blocking antibody rmC5-3. (D) Effects of LPS in
the presence of an irrelevant antibody (SUK5).
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TNF-stimulated pinocytosis is inhibited by LPS.
To obtain further insight into LPS action on pinocytosis, we
investigated the effect of LPS under conditions that result in stimulated pinocytosis. To this end, cells were exposed to 500 U/mL
TNF. As expected, this treatment resulted in a substantial increase in
pinocytosis (Fig 2B). Subsequently, the effects of LPS on this
TNF-stimulated [3H]-sucrose uptake were studied. It
seemed that low concentrations of LPS strongly impaired TNF-stimulated
fluid phase pinocytosis, a clear effect already being noted at an LPS
concentration of 100 pg/mL (Fig 2B). At an LPS concentration of 10 ng/mL LPS, uptake of radioactivity was reduced to levels below those
observed in unstimulated cells, similar to those observed after
stimulation of macrophages with LPS in the absence of TNF. At LPS
concentrations in excess of 10 ng/mL, fluid phase pinocytosis increased
again, the absolute levels of [3H]-sucrose uptake closely
resembling those observed in the absence of TNF (compare Fig 2A and B).
We concluded that the LPS not only inhibited constitutive pinocytosis,
but also TNF-stimulated pinocytosis, and thus, that LPS effects on
pinocytosis are dominant to TNF effects with respect to this process.
CD14 mediates the LPS-dependent inhibition of macrophage pinocytosis.
Most, though not all, effects of LPS on cellular function are mediated
by the LPS receptor CD14. We asked, therefore, to which extent the
dualistic effects of LPS on macrophage pinocytosis involve CD14. To
this end, 4-4 macrophages were incubated with a saturating amount of a
monoclonal antibody directed against murine CD14 and subsequently
challenged with different concentrations of LPS. Under these
conditions, low concentrations of LPS stimulated, rather than reduced,
pinocytosis (Fig 2C). Furthermore, at high concentrations of LPS (1 to
10 µg/mL), which already substantially enhance
[3H]-sucrose uptake in the absence of the antibody, the
presence of the anti-CD14 antibody even further increased pinocytosis
(compare Fig 2A and C). As a control experiment, we incubated the cells with an irrelevant antibody (SUK5), but this antibody did influence neither the LPS-induced inhibition of pinocytosis at lower
concentrations, nor the LPS-dependent stimulation at higher
concentrations (Fig 2D). Interaction of LPS with CD14 requires LBP, a
protein present in serum. Therefore, we washed cells with serum-free
medium and stimulated cells in such medium. Under these conditions, an
LPS-induced inhibition of fluid phase uptake was not detected, and
stimulation of pinocytosis was enhanced (Fig
3). We concluded that the LPS-induced inhibition of pinocytosis is mediated by a CD14-dependent mechanism, whereas the LPS-induced inhibition of pinocytosis is not.
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| Fig 3.
Effects of LPS on macrophage fluid phase uptake in the
absence of serum. Murine clone 4-4 macrophages were washed four times
with serum-free medium and stimulated with LPS for 45 minutes at 37°C
in the presence of [3H]sucrose. Subsequently, the cells
were placed on ice and washed, and accumulated radioactivity was
determined by scintillation counting. Using the specific activity of
the [3H]sucrose, fluid phase uptake was expressed as
femtoliter per cells per minute. The results show the average of three
independent determinations.
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LPS is known to activate members of the mitogen-activated protein (MAP)
kinase family of proteins. Therefore, we investigated the roles of
p42/44 MAP kinase and p38 MAP kinase in LPS effects on pinocytosis
using the mitogen-activated protein kinase (MEK) inhibitor PD098059 and
SB203580, a p38 MAP kinase inhibitor. PD098059 or SB203580, however,
did not alter LPS effects (Fig 4), and LPS effects on fluid phase uptake are apparently mediated via alternative signaling events.
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| Fig 4.
Effects of LPS on macrophage fluid phase uptake in the
presence of (A) 1 µmol/L SB203580 or (B) 2 µmol/L PD098059. Murine
clone 4-4 macrophages were preincubated with the inhibitors for 1 hour
and stimulated with LPS for 45 minutes at 37°C in the presence of
[3H]sucrose and the appropriate inhibitor. Subsequently,
the cells were placed on ice and washed, and accumulated radioactivity
was determined by scintillation counting. Using the specific activity
of the [3H]sucrose, fluid phase uptake was expressed as
femtoliter per cells per minute. The results show the average of three
independent determinations.
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LPS acts specifically on fluid phase pinocytosis.
An important question is whether the effects of LPS on
[3H]-sucrose reflect a general LPS action on macrophage
endocytosis or whether these effects are associated specifically with
the pinocytotic machinery. To this end, human catalase was
fluorescently labeled. As mouse macrophages are unlikely to express
specific receptors for human catalase, fluorescent catalase may only
enter the cell by nonspecific mechanisms. If catalase is preincubated, however, with a mix of three mouse monoclonal antibodies directed against different epitopes of human catalase, aggregates of catalase and antibody will form, which may enter the cell by phagocytosis and/or
receptor-mediated endocytosis after interaction with the macrophage Fc
receptor. Indeed, cells incubated with antibody-catalase complexes
accumulated approximately five times as much fluorescence as compared
with cells that were incubated with fluorescent catalase alone
(Fig 5A), showing the efficiency
of antibody-directed uptake over nonspecific endocytosis. Low
concentrations of LPS decreased uptake of nonantibody-bound catalase,
maximal inhibition being reached at an LPS concentration of 10 ng/mL
(Fig 5A). At higher concentrations of LPS, uptake of fluorescent
catalase increased again, and at an LPS concentration of 10 µg/mL, a
clear stimulation of cellular fluorescence, as compared with
unstimulated cells, was apparent (Fig 5A). These results show,
therefore, that the effects of LPS on fluid phase pinocytosis are not
only observed using [3H]-sucrose as a probe, but are also
observed with probes having a larger molecular size. No effect of LPS,
however, was noted on the endocytosis of antibody-bound fluorescent
catalase, except at the highest LPS concentration, at which a small
decrease in uptake was noted (Fig 5A). Apparently, LPS has differential
effects on antibody-directed endocytosis and fluid phase pinocytosis.
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| Fig 5.
Influence of LPS on uptake of fluorescently labeled
catalase. Murine clone 4-4 macrophages were stimulated with vehicle or
different concentration of LPS for 45 minutes at 37°C and 5%
CO2 in the presence of 6.6 mg/mL fluorescent catalase to
assay pinocytosis (open circles) or fluorescent catalase and a mixture
of three different supernatants of hybridomas producing antihuman
catalase antibodies to assay Fc receptor-mediated uptake (filled
circles). Uptake of fluorescence was then determined using FACS
analysis. To distinguish uptake from binding, parallel experiments were
performed at 4°C. The results show the average and standard error of
four different experiments, each experiment being the average of two
independent determinations. (A) Effects of different LPS
concentrations. (B) Effects of different LPS concentrations in TNF (500 U/mL)-stimulated macrophages. (C) Effects of LPS on TPA (100 ng/mL)-stimulated macrophages.
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Similar results were obtained when the LPS action was investigated
under conditions that result in enhanced fluid phase pinocytosis. Stimulation of the cells with TNF resulted in increased uptake of
fluorescent catalase (Fig 5B), although uptake of antibody-bound catalase was somewhat lower as compared with cells that had not been
treated with TNF (compare Fig 5A and B). Addition of LPS to TNF cells
decreased uptake of fluorescence at lower LPS concentrations, and at
higher concentrations of LPS, uptake of fluorescence increased again,
in agreement with results using [3H]-sucrose as a probe.
LPS, however, did not have a marked effect on the uptake of
antibody-bound catalase. Addition of the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) to the macrophages resulted
in a very large increase in the intracellular accumulation of
fluorescent catalase (Fig 5C). If TPA, however, was added to the cells
together with low concentration of LPS, this TPA-induced uptake of
catalase was severely impaired (Fig 5C). At 10 to 100 ng/mL LPS,
accumulation of fluorescence was lower than that observed in
unstimulated cells and similar to the level observed in cells that had
been stimulated with LPS in the absence of TPA (compare Fig 5A and C).
Effects of LPS on the uptake of antibody-bound catalase were much less
pronounced (Fig 5C). Furthermore, TPA-treated cells were less efficient
in the uptake of antibody-bound catalase in comparison with untreated
cells (compare Fig 5A and C). We concluded that LPS
specifically influences fluid phase pinocytosis and is not a general
regulator of endocytosis in macrophages.
LPS-dependent stimulation of pinocytosis is macrophage-specific.
Apart from macrophages, monocytes are also capable of measurable
pinocytosis and phagocytosis. Furthermore, monocytes are highly
responsive to LPS. Significant differences exist,
however, between macrophages and monocytes with respect
to the regulation of pinocytosis.14 We decided, therefore,
to investigate the effects of LPS on phagocytosis and pinocytosis in
freshly isolated peripheral blood monocytes. It seemed that LPS had
little influence on phagocytosis of fluorescently labeled S
pneumoniae (not shown). In contrast, LPS significantly inhibited
pinocytosis of fluorescently labeled human serum protein by monocytes
isolated from four different healthy volunteers (Fig
6). The stimulation of pinocytosis at higher LPS concentrations, however, was not detected (Fig 6). We
concluded that the CD14-mediated inhibition of fluid phase uptake is a
general feature of LPS-induced signaling, but that the LPS-independent
stimulation of pinocytosis at higher LPS concentrations is a
macrophage-specific phenomenon.
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| Fig 6.
Effects of LPS on fluid phase uptake of peripheral blood
monocytes. Peripheral blood mononuclear cells were isolated from
heparinized blood of healthy volunteers using Ficoll-Hypaque density
gradient centrifugation. Subsequently, the cells residing at the
interface were washed, allowed to recover from isolation, stimulated
for 45 minutes at 37°C and 5% CO2 in the presence of 1 mg/mL fluorescently labeled human serum protein, and analyzed by FACS.
The scatter profile was used to identify monocytes, and average
fluorescence per monocyte was determined. Nonmonocyte cell populations
did not accumulate significant amounts of fluorescence. The graph shows
the curves from four different healthy volunteers.
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Effects of detoxified LPS on pinocytosis.
The stimulatory effects of LPS on pinocytosis at higher concentrations
(1 to 10 µg/mL) are somewhat surprising as lower
concentrations of LPS are generally considered to maximally stimulate
cells with respect to oxidative burst and cytokine production. It is
therefore important to investigate if LPS acts on pinocytosis via a
nonsignaling mechanism. We decided to test, therefore, the effect of
detoxified LPS on macrophage pinocytosis. As evident from Fig
7, detoxified LPS was much less potent in
inducing changes in pinocytosis, inducing a rightward shift in the
dose-response curve of two orders of magnitude. The inhibitory effects
of LPS on pinocytosis seem, therefore, to be mediated by a bona fide
LPS-induced signaling system. Although these data would also seem to
support the notion that the stimulation of pinocytosis by LPS is signal
transduction-dependent, it should be kept in mind that the residual
activity of LPS (approximately 1%) may be sufficient to inhibit LPS
stimulation of pinocytosis via a nonsignal transduction mechanism.
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| Fig 7.
Effects of detoxified LPS on macrophage fluid phase
uptake. Murine clone 4-4 macrophages were stimulated with LPS (open
circles) or detoxified LPS (closed circles) for 45 minutes at 37°C
and 5% CO2 in the presence of [3H]sucrose.
Subsequently, the cells were placed on ice and washed, and accumulated
radioactivity was determined by scintillation counting. Using the
specific activity of the [3H]sucrose, fluid phase uptake
was expressed as femtoliter per cells per minute.
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DISCUSSION |
Monocytes and macrophages mediate many of the physiological effects of
LPS. One of the most characteristic features of these cells is their
capacity to endocytose extracellular material, either by pinocytosis,
receptor-mediated endocytosis, or phagocytosis. Although a
CD14-dependent pathway for phagocytosis of gram-negative bacteria has
been characterized,11 the effects of LPS on fluid phase
pinocytosis and Fc receptor-mediated endocytosis had not been studied
in great detail. In the present study, we observed that LPS did not
have a great effect on Fc receptor-mediated endocytosis, but did have
a dual action on fluid phase pinocytosis: at lower LPS concentrations
(0.1 to 10 ng/mL), pinocytosis was diminished in both macrophages and
monocytes, whereas in macrophages at high LPS concentrations (10 µg/mL), this process was stimulated relative to untreated controls.
In monocytes, stimulation of pinocytosis by LPS was not observed, and
only inhibition of fluid phase uptake by LPS was noted. Interestingly,
after treatment with a CD14-blocking antibody, the LPS-induced
inhibition of fluid phase uptake was no longer discernible, and under
these conditions, LPS-dependent stimulation of pinocytosis at lower
concentrations was unmasked, suggesting that CD14 mediates inhibition
of pinocytosis, whereas an LPS-induced CD14-independent mechanism acts
stimulatory on this process. In agreement with this notion is our
observation that in the presence of the CD14-blocking antibody, 10 µg/mL LPS produced even more fluid phase uptake as was observed at
this concentration of LPS without the antibody. This shows that even at
LPS concentrations facilitating pinocytosis, a CD14-dependent antipinocytotic influence is still present.
The molecular nature of the LPS-induced stimulation of fluid phase
pinocytosis is unclear. Several groups have reported CD14-independent actions after stimulation with high concentrations of
LPS,4,15-17 and recently, we showed in our own laboratory
that a fraction of the CD14-negative population of mononuclear cells,
isolated from the blood of healthy volunteers, displays nuclear
translocation of NF-B upon stimulation with 10 µg/mL
LPS.17a In agreement, macrophages isolated from CD14
knockout mice display cytokine production after treatment with similar
concentrations of LPS,18 and several molecules, including
CD11b/CD18,19-21 CD11c/CD18,22,23 and
L-selectin,24 have been proposed to act as low-affinity receptors for LPS, mediating its effects at higher concentrations. Uniquely, from our studies it emerged that the CD14-independent stimulation of fluid phase uptake may be detected at LPS concentrations as low as 1 to 10 ng/mL when a blocking antibody abrogates the CD14-dependent inhibitory influence of LPS on pinocytosis. We feel,
however, that this observation per se does not indicate the existence
of an alternative high-affinity LPS receptor, as such a receptor is not
expected to yield the roughly linear stimulation of fluid phase uptake
we observed over concentrations ranging from 1 ng/mL to 10 µg/mL.
These data are probably more readily explained by a physical effect
like, eg, altered membrane fluidity, although obviously further
experimental work is needed to confirm this notion.
The effects of LPS on Fc receptor-mediated endocytosis were much less
pronounced when compared with its effects on fluid phase uptake.
Nevertheless, we did not fail to notice that at high concentrations of
LPS, increased pinocytosis was associated with a reduction in uptake of
antibody-bound material. Furthermore, the higher level of fluid phase
pinocytosis in TNF-treated macrophages was also accompanied with lower
Fc receptor-mediated endocytosis, whereas the strong stimulation of
pinocytosis induced by the phorbol ester TPA resulted in a large
reduction in the uptake of antibody-bound material. Together, these
observations indicate that pinocytosis and Fc receptor-mediated
endocytosis may compete for the availability of the endocytotic
machinery. In agreement, both fluid phase pinocytosis and phagocytosis
of IgG-opsonised erythrocytes are impaired by inhibitors of
phosphatidylinositol-3-kinase,9 and also, dominant negative
mutants of the p21ras-related small GTPase p21rac are reported to
inhibit pinocytosis as well as phagocytosis,7,8 showing
that similar signaling elements underlie both processes and that
competition for these elements may exist. The molecular mechanism by
which LPS binding to CD14 inhibits pinocytosis remains unclear. Among
the signaling elements reported to be activated by LPS are p38 MAP
kinase,22,25,26 p42/p44 MAP kinase,27,28 stress-activated protein kinases,29,30 and
NF-B.31 These signaling elements, however, are also
activated by TNF, which stimulates rather than inhibits
pinocytosis, suggesting the involvement of an alternative LPS-specific
molecular mechanism for inhibiting fluid phase pinocytosis. In
agreement, we failed to detect an influence of p38 MAP kinase or
p42/p44 MAP kinase inhibition on the LPS-induced changes in
pinocytosis. Recently, it was reported that direct interaction of CD14
with LPS present in the outer membrane of gram-negative bacteria may
directly induce bacterial phagocytosis.11 It is possible,
therefore, that the induction of phagocytosis and the associated
recruitment of signaling elements required for this phagocytosis may
cause a diminished availability of such signaling elements for
pinocytosis, and that this may explain the LPS-induced CD14-dependent
inhibition fluid of phase uptake.
The present study has shown that LPS may have specific influence on
pinocytosis, with a much less pronounced effect on Fc receptor-mediated endocytosis. It is often suggested that macrophage pinocytosis serves as mechanism for taking up extracellular antigen, which can subsequently be presented to CD4+ T
cells.32 Phagocytosis, apart from its role in pathogen
destruction, is well known to function in antigen presentation.
Interestingly, we recently found that material taken up by pinocytosis
and material taken up by Fc receptor-mediated endocytosis are routed
to different intracellular compartments, suggesting that these
processes have a specific function in antigen presentation and that LPS
may influence antigen presentation via CD14-mediated inhibition of
fluid phase uptake. Experiments exploring this possibility are
currently under progress.
| |
FOOTNOTES |
Submitted June 1, 1998; accepted February 1, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Maikel P. Peppelenbosch, Laboratory for
Experimental Internal Medicine, G2-130 Academic Medical Centre,
Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands; e-mail:
M.P.Peppelenbosch{at}AMC.UVA.NL.
| |
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