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PHAGOCYTES
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
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.
r 1999 by The American Society of Hematology.
L
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
IPOPOLYSACCHARIDE (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 membranebound 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
Blood, Vol 93, No 11 (June 1), 1999: pp 4011-4018
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.
Submitted June 1, 1998; accepted February 1, 1999.
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:
[email protected].
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.
r 1999 by The American Society of Hematology.
0006-4971/99/9311-0042$3.00/0
4011
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4012
PEPPELENBOSCH ET AL
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.
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 FicollHypaque (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 308 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.
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
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LPS REGULATES PINOCYTOSIS
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.
fluid phase pinocytosis, depending on the LPS concentration
involved.
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 TNFstimulated 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
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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 LPSinduced inhibition of pinocytosis is not.
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.
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 antibodybound 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.
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
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4014
PEPPELENBOSCH ET AL
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 F0.05 and two stars indicating a P value F0.01 (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).
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 macrophagespecific. 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
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LPS REGULATES PINOCYTOSIS
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.
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
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signaling, but that the LPS-independent stimulation of pinocytosis at higher LPS concentrations is a macrophage-specific
phenomenon.
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.
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 concentra-
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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PEPPELENBOSCH ET AL
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.
tions (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 CD14blocking 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 associ-
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LPS REGULATES PINOCYTOSIS
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.
ated 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.
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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 LPSspecific 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.
REFERENCES
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.
1. Ulevitch RJ, Tobias PS: Receptor-dependent mechanisms of cell
stimulation by bacterial endotoxin. Annu Rev Immunol 13:437, 1995
2. Schumann RR, Leong SR, Flaggs GW, Gray PW, Wright SD,
Mathison JC, Tobias PS, Ulevitch RJ: Structure and function of
lipopolysaccharide binding protein. Science 249:1429, 1990
3. Tobias PS, Soldau K, Ulevitch RJ: Isolation of a lipopolysaccharide-binding acute phase reactant from rabbit serum. J Exp Med
164:777, 1986
4. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC:
CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS
binding protein. Science 249:1431, 1990
5. Sweet MJ, Hume DA: Endotoxin signal transduction in macrophages. J Leukoc Biol 60:8, 1996
6. Allen WE, Jones GE, Pollard JW, Ridley AJ: Rho, Rac and Cdc42
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
4018
regulate actin organization and cell adhesion in macrophages. J Cell Sci
110:707, 1997
7. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A: The
small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70:401, 1992
8. Cox D, Chang P, Zhang Q, Reddy PG, Bokoch GM, Greenberg S:
Requirements for both Rac1 and Cdc42 in membrane ruffling and
phagocytosis in leukocytes. J Exp Med 186:1487, 1997
9. Araki N, Johnson MT, Swanson JA: A role for phosphoinositide
3-kinase in the completion of macropinocytosis and phagocytosis by
macrophages. J Cell Biol 135:1249, 1996
10. Knight KR, Vairo G, Hamilton JA: Regulation of pinocytosis in
murine macrophages by colony-stimulating factors and other agents. J
Leukoc Biol 51:350, 1992
10a. Peppelenbosch MP, Boone E, Jones GE, van Deventer SJH,
Haegeman G, Fiers W, Grooten J, Ridley AJ: Multiple signal transduction pathways regulate TNF-induced actin reorganisation in macrophages. J Immunol 162:837, 1999
11. Grunwald U, Fan X, Jack RS, Workalemahu G, Kallies A, Stelter
F, Schutt C: Monocytes can phagocytose Gram-negative bacteria by a
CD14-dependent mechanism. J Immunol 157:4119, 1996
12. Crowley MT, Costello PS, Fitzer-Attas CJ, Turner M, Meng F,
Lowell C, Tybulewicz VL, DeFranco AL: A critical role for Syk in
signal transduction and phagocytosis mediated by Fcgamma receptors
on macrophages. J Exp Med 186:1027, 1997
13. DeSmedt M, Rottiers P, Dooms H, Fiers W, Grooten J: Macrophages induce cellular immunity by activating Th1 cell responses and
suppressing Th2 cell responses. J Immunol 160:1116, 1998
14. Knight BL, Soutar AK: Changes in the metabolism of modified
and unmodified low-density lipoproteins during the maturation of
cultured blood monocyte-macrophages from normal and homozygous
familial hypercholesterolaemic subjects. Eur J Biochem 125:407, 1982
15. Lynn WA, Liu Y, Golenbock DT: Neither CD14 nor serum is
absolutely necessary for activation of mononuclear phagocytes by
bacterial lipopolysaccharide. Infect Immun 61:4452, 1993
16. Kitchens RL, Ulevitch RJ, Munford RS: Lipopolysaccharide
(LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated
pathway. J Exp Med 176:485, 1992
17. Bellezzo JM, Britton RS, Bacon BR, Fox ES: LPS-mediated
NF-kappa beta activation in rat Kupffer cells can be induced independently of CD14. Am J Physiol 270:G956, 1996
17a. ten Hove T, Vervoordddonk MJM, Dekkers PEP, Reitsma PH,
van Deventer SJH: LPS induced translocation of NF-␬ B occurs only in
a subpopulation of CD14 positive mononuclear cells. J Endotoxin Res
(in press)
18. Perera PY, Vogel SN, Detore GR, Haziot A, Goyert SM:
PEPPELENBOSCH ET AL
CD14-dependent and CD14-independent signaling pathways in murine
macrophages from normal and CD14 knockout mice stimulated with
lipopolysaccharide or taxol. J Immunol 158:4422, 1997
19. Zarewych DM, Kindzelskii AL, Todd RF 3rd, Petty HR: LPS
induces CD14 association with complement receptor type 3, which is
reversed by neutrophil adhesion. J Immunol 156:430, 1996
20. Petty HR, Todd RF 3rd: Integrins as promiscuous signal
transduction devices. Immunol Today 17:209, 1996
21. Wright SD, Ramos RA, Hermanowski-Vosatka A, Rockwell P,
Detmers PA: Activation of the adhesive capacity of CR3 on neutrophils
by endotoxin: Dependence on lipopolysaccharide binding protein and
CD14. J Exp Med 173:1281, 1991
22. Ingalls RR, Arnaout MA, Golenbock DT: Outside-in signaling
by lipopolysaccharide through a tailless integrin. J Immunol 159:433,
1997
23. Ingalls RR, Golenbock DT: CD11c/CD18, a transmembrane
signaling receptor for lipopolysaccharide. J Exp Med 181:1473, 1995
24. Malhotra R, Bird MI: L-selectin: A novel receptor for lipopolysaccharide and its potential role in bacterial sepsis. Bioessays 19:919,
1997
25. Han J, Lee JD, Bibbs L, Ulevitch RJ: A MAP kinase targeted by
endotoxin and hyperosmolarity in mammalian cells. Science 265:808,
1994
26. Han J, Lee JD, Tobias PS, Ulevitch RJ: Endotoxin induces rapid
protein tyrosine phosphorylation in 70Z/3 cells expressing CD14. J Biol
Chem 268:25009, 1993
27. Hambleton J, McMahon M, DeFranco AL: Activation of Raf-1
and mitogen-activated protein kinase in murine macrophages partially
mimics lipopolysaccharide-induced signaling events. J Exp Med 182:
147, 1995
28. Liu MK, Herrera-Velit P, Brownsey RW, Reiner NE: CD14dependent activation of protein kinase C and mitogen-activated protein
kinases (p42 and p44) in human monocytes treated with bacterial
lipopolysaccharide. J Immunol 153:2642, 1994
29. Sanghera JS, Weinstein SL, Aluwalia M, Girn J, Pelech SL:
Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages. J Immunol 156:4457, 1996
30. Hambleton J, Weinstein SL, Lem L, DeFranco AL: Activation of
c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated
macrophages. Proc Natl Acad Sci USA 93:2774, 1996
31. Lee JD, Kato K, Tobias PS, Kirkland TN, Ulevitch RJ: Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to
complexes of lipopolysaccharide (LPS) and LPS binding protein. J Exp
Med 175:1697, 1992
32. Swanson JA, Watts C: Macropinocytosis. Trends Cell Biol
5:424, 1995
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1999 93: 4011-4018
Lipopolysaccharide Regulates Macrophage Fluid Phase Pinocytosis Via
CD14-Dependent and CD14-Independent Pathways
Maikel P. Peppelenbosch, Marjory DeSmedt, Tessa ten Hove, Sander J.H. van Deventer and Johan
Grooten
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