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Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, WI 53706
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Diphosphoryl lipid A from Rhodobacter sphaeroides (RsDPLA)
is the first potent antagonist of toxic ReLPS in both human and murine
cells and also prevents LPS-induced shock in mice (8).
Qureshi et al. (6, 9) have purified RsDPLA and completed
the structural analysis of this lipid A. The structures of both agonist
(ReLPS) and antagonist (RsDPLA) are presented in Fig. 1
. RsDPLA blocks both the
CD14-dependent and the CD14-independent pathway for the activation of
immune cells by LPS. RsDPLA effectively competes with LPS for binding
to LBP and soluble CD14 (10), and RsDPLA also
blocks the binding of iodinated ReLPS to the cell (11).
However, others have shown that LPS from R. sphaeroides
(RsLPS) does not prevent the binding of LPS-BODIPY to the
neutrophils (12). Furthermore, RsLPS blocks the entry of
toxic ReLPS through the plasma membrane into the intracellular
organelles. However, the details of the interaction of RsLPS with the
cell and its internalization pathway have not been defined
(12).
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In the present study, we have developed the method for direct labeling of both ReLPS (agonist) and RsDPLA (antagonist) with colloidal gold of different particle sizes. Binding and localization of ReLPS and RsDPLA were compared within the cell by transmission and scanning electron microscopy because internalization pathway and intracellular distribution of RsDPLA has not been established yet. A side-by-side quantitative comparison of both ReLPS (agonist) and RsDPLA (antagonist) fate in the cell is extremely important for understanding of mechanisms involved in antagonism exhibited by RsDPLA.
Our data indicate that binding and internalization of ReLPS-gold conjugates occurs within seconds and that an excess of RsDPLA blocks both of these processes in RAW 264.7 cells. ReLPS internalization proceeds not only via all three proposed pathways, such as clathrin-coated pits, micropinocytosis, and macropinocytosis, but also by nonspecific endocytosis into the cytoplasm. However, RsDPLA is internalized predominantly by macropinocytosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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"Spectrally pure" ReLPS and RsDPLA were prepared and structurally characterized as described by Qureshi et al. (26, 27). Without this level of spectral purity, contaminating phospholipids and proteins in LPS preparations may confound the results (7). DMEM containing glucose and glutamine, FBS, and gentamicin sulfate were purchased from BioWhittaker (Walkersville, MD). Triethylamine (TEA), MES, HEPES, and trisodium citrate were obtained from Sigma (St. Louis, MO). Electron microscopy-grade acetone and a Durcopan ACM embedding kit were purchased from Polysciences (Warrington, PA), and 25% glutaraldehyde and osmium tetroxide were obtained from Electron Microscopy Sciences (Fort Washington, PA). Siliconized-flat top microtubes were obtained from Fisher Scientific (Pittsburgh, PA). All glassware and plasticware were rendered pyrogen-free by heating at 120°C overnight.
Cells
Murine RAW 264.7 cells (ATCC TIB 71) were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM, 90%; FBS, 10%; and 100 µg/ml gentamicin (BioWhittaker).
Preparation of the ReLPS-gold (30 nm) conjugates and RsDPLA-gold (30 nm) conjugates
A novel method for direct 30-nm gold labeling of both ReLPS and RsDPLA has been developed. For preparation of ReLPS-gold conjugates, ReLPS, (120 µl; 2.7 mg/ml) suspended in 43 mM TEA in water was mixed rapidly with 30-nm colloidal gold (0.6 ml) in 20 mM MES, pH 6.25, and sonicated for 10 min. For preparation of RsDPLA-gold conjugates, RsDPLA (120 µl; 3.8 mg/ml) suspended in 24 mM TEA in water, was rapidly mixed with 0.6 ml of 30-nm colloidal gold in 20 mM MES, pH 6.67, and sonicated for 10 min. The gold aggregation test (28) was performed to detect salt-induced colloidal gold aggregation, and no free gold was present in the labeling suspensions after ReLPS- or RsDPLA-gold conjugates were prepared.
Purification of the ReLPS-gold (30 nm) conjugates
ReLPS-gold conjugates (0.5 ml) were centrifuged at 2000 rpm for 1 h in siliconized microtubes (Fisher Scientific) to remove the excess of ReLPS. ReLPS-gold conjugates were precipitated, and ReLPS not associated with gold particles remained in the supernatant. The pellet was resuspended in the 20 mM MES/20 mM TEA, pH 6.5, and conjugates proved to be stable by the gold aggregation test. The concentration of ReLPS in purified gold conjugates was determined by the phosphorus assay (29).
Preparation of the ReLPS-gold (5 nm) conjugates and RsDPLA-gold (5 nm) conjugates
For preparation of ReLPS-gold conjugates, ReLPS (90 µl; 4.0 mg/ml) suspended in 43 mM TEA in water was mixed rapidly with 5-nm colloidal gold (1.8 ml) in 20 mM HEPES, pH 7.3, and sonicated for 10 min. To prepare RsDPLA-gold conjugates, RsDPLA (170 µl; 1.5 mg/ml) suspended in 18 mM TEA in water was mixed rapidly with 2.0 ml of 5-nm colloidal gold in 20 mM HEPES, pH 7.3, and sonicated for 10 min.
Purification of the ReLPS-gold (5 nm) conjugates and RsDPLA-gold (5 nm) conjugates
ReLPS- and RsDPLA-gold conjugates (1.8 ml each) were centrifuged at 20,000 rpm for 1 h in a Beckman LE-80 Ultracentrifuge (Beckman Instruments, Fullerton, CA) to remove excess ReLPS/RsDPLA present in the supernatant. Precipitated gold conjugates were resuspended in 20 mM HEPES, pH 7.3. Purified ReLPS- and RsDPLA-gold conjugates were stable as determined by gold aggregation test. The concentration of ReLPS and RsDPLA in purified gold conjugates was determined by the phosphorus assay (29).
Larger-size gold (30 nm) conjugates with toxic ReLPS (ReLPS-gc) and its antagonist RsDPLA (RsDPLA-gc) were used to quantify their binding and internalization in RAW 264.7 cells. For a qualitative and quantitative (200–250 cells) comparison of the major differences in the pathway and distribution of ReLPS and RsDPLA within the RAW 264.7 cell, purified 5-nm gold conjugates of ReLPS and RsDPLA were used.
TNF-
bioassay
The assay was performed with murine RAW 264.7 cells treated with
equivalent concentrations of unconjugated ReLPS or gold conjugated
ReLPS. Briefly, 0.5 ml of RAW 264.7 cell culture
(106 cell/ml) in DMEM supplemented with 10% FBS
and 100 µg/ml gentamicin were seeded in 12-well flat-bottom plates
and incubated for 1 h at 37°C in a humidified 5%
CO2 environment. Medium then was replaced with
0.5 ml of medium containing serial dilutions of unconjugated ReLPS or
gold-conjugated ReLPS in a range from 0.1 pg/ml to 10 ng/ml and
incubated for 4 h at 37°C in a 5% CO2
humidified incubator. Supernatants then were removed and analyzed for
quantity of TNF- according to the directions provided with the
Quantikine M mouse TNF- immunoassay kit (R&D Systems, Minneapolis,
MN) with a CERES UV900HDi (Bio-Tek Instruments, Winooski, VT) plate
reader.
Field-emission scanning electron microscopy
RAW 264.7 macrophages were grown at 37°C in DMEM medium supplemented with 10% FBS and 100 µg/ml gentamicin on 200 mesh formvar-coated nickel grids (Ted Pella, Redding, CA) for 48 h. Cells were either incubated for 10 min with 5 µg/ml ReLPS-gold conjugates, or pretreated with a 100-fold excess of RsDPLA, before ReLPS-gold conjugates, and incubated at 37°C for 10 min. Unbound ReLPS was removed by washing cells with DMEM and PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4). Cells were fixed with 1% glutaraldehyde in PBS for 30 min, and dehydrated in a graded series of alcohol for 5 min each, followed by drying according to the critical point drying method using a Samdri-780 (Tousimis Research, Rockville, MD) as described by Albrecht and MacKenzie (30). Grids with the attached cells were platinum coated on IBS/TM200 Ion-beam sputter-coating device (VCR Group, South San Francisco, CA). Samples were viewed on a Field-Emission Scanning Electron Microscope Hitachi S-900 (Hitachi Instruments, Santa Clara, CA). Micrographs were recorded on Polaroid type 55 P/N films (Polaroid, Cambridge, MA) at direct magnifications. Gold particles associated with the cells were counted on each of the 10 cells and an average amount calculated for each sample.
Transmission electron microscopy
RAW 264.7 macrophages were grown at 37°C in DMEM supplemented
with 10% FBS on 4-mm mica discs for 72 h. ReLPS or RsDPLA-gold
conjugates at various concentrations were mixed with the warmed medium,
added to the cells, and incubated for the indicated time intervals at
37°C or 4°C. Unbound ReLPS or RsDPLA-gold conjugates were removed
by washing cells with DMEM and PBS and cultures then were plunge-frozen
in liquid ethane. Cryosubstitution was done with 1% osmium tetroxide
in acetone according to the method of Rebhun (31). Discs
with the cells were embedded into resin Durcopan ACM (Polysciences).
Thin sections of 
80 nm were cut with a diamond knife on a
Reichert-Jung Ultracut E (Reichert-Jung, Vienna, Austria), transferred
to 200 mesh copper grids, and stained with the lead citrate. Samples
were viewed on a transmission electron microscope (CM120
STEM; Philips Electronics, Eindhoven, The Netherlands) at 60–80 kV
accelerating voltage. The number of ReLPS- or RsDPLA-gold
conjugates per 10 µm (presumed size of the average cell) was
calculated by counting the number of cell-associated gold particles in
different organelles in each of the 130 randomly selected cells’
sections.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ReLPS and RsDPLA were directly labeled with 5-nm and 30-nm colloidal gold particles for a comparative study of their interaction and localization within the cell. ReLPS- and RsDPLA-gold conjugates were purified by centrifugation, and excess nonconjugated LPS or RsDPLA was removed. With a phosphorus assay (29), the concentration of ReLPS and RsDPLA in purified gold conjugates was estimated to be 30 and 80 molecules of ReLPS and RsDPLA, respectively, bound to one 5-nm gold particle. The binding to one 30-nm gold particle was two orders of magnitude higher (3000–8000 molecules), which is roughly equivalent to the ratio of surface areas for 30- and 5-nm gold particles.
To determine whether the association of ReLPS with the colloidal gold
would inhibit the stimulatory activity of ReLPS, the activities of
gold-labeled and unlabeled ReLPS were compared. We have observed a
very similar TNF- response in RAW 264.7 cells (Table I) treated with equivalent concentrations
of either nonconjugated or gold-conjugated ReLPS (5- or 30-nm gold
conjugates did not show any difference in activity). Therefore, we used
colloidal gold as the label, because in previous studies it was shown
that picogram concentrations of LPS coated on latex beads generated
less than 50% of soluble LPS induction of procoagulant activity in
cultured monocytes (32). The effect was probably
attributable to the relatively large size of latex beads (0.114 µm)
used in that study. As seen from Table I, the ratio of TNF-
stimulating activity for gold-conjugated ReLPS vs nonconjugated ReLPS
varied from 0.89 to 0.99 as the concentration of ReLPS increased from
0.2 pg/ml up to 220 pg/ml (Table I).
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Localization of ReLPS-30-nm gold conjugates
We used transmission electron microscopy for the morphological
evaluation of the distribution of ReLPS-gold conjugates in the RAW
264.7 cells. It previously was shown that ReLPS activates kinases in
macrophages within seconds (33). We found that by 30
s, 78% of the cell-associated ReLPS-gold conjugates were on the cell
surface, and 22% were internalized as shown in Fig. 2A. The majority (73%) of the
internalized ReLPS-gold conjugates were present within early
endosomes or caveolae (nonclathrin-coated; Fig. 2B) by a
process termed micropinocytosis or were found in the cytoplasmic
matrix. Some gold particles also were observed in coated pits. The
remaining 27% of internalized ReLPS-gold were found in large (200–800
nm) macropinosomes or phagosome-like vacuoles, with all gold particles
adhering to the limiting membrane, as shown in Fig. 2C.
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A). In contrast, cells
that were incubated with purified ReLPS-gold conjugates at 4°C (to
eliminate phagocytosis of ReLPS that occurs at 37°C), had 100% (230
gold particles) of conjugates on the membrane surface, and there was no
internalization of ReLPS.
When the incubation of cells with ReLPS-gold conjugates was continued
for 10 min at 37°C, the amount of cell surface-associated ReLPS-gold
conjugates remained the same; however, we observed the ReLPS-gold
conjugates not only in the organelles mentioned above, but also in
dense lysosome-like structures in the cytoplasm. The entry of a single
ReLPS-gold conjugate and the formation of caveolae and endosomes as
observed by transmission electron microscopy are shown in Fig. 3. This figure shows that the LPS binds
to the cell surface followed by membrane invagination and the formation
of caveolae and endosomes by a process called micropinocytosis.
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In contrast to ReLPS, no internalization was observed during the
first 30 s of incubation of RsDPLA-gold conjugates with cells. The
binding and internalization of RsDPLA-gold conjugates were slower
compared with ReLPS-gold conjugates as could be seen from comparison of
their kinetics of binding and internalization (data not shown).
RsDPLA-gold conjugates were not observed in clathrin-coated vesicles,
and >90% of internalized conjugates were localized in large
macropinosomes or vacuoles with all gold particles adhering to the
limiting membrane after 5 min as shown in Fig. 2C for
ReLPS-gold conjugates. The remaining 8% of conjugates were observed in
endosomes and caveolae, suggesting that RsDPLA internalization probably
proceeds primarily by macropinocytosis. After 20 min of incubation,
RsDPLA-gold conjugates were present inside the secondary lysosomes and
the residual bodies of the cell (Fig. 4).
After 40 min of incubation, the number of RsDPLA-gold conjugates
increased in the secondary lysosomes, and 3% were evident in the Golgi
apparatus (Fig. 4).
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The lack of clathrin-coated vesicle formation during ReLPS and
RsDPLA internalization described above could be attributed to
large-sized conjugates made with 30-nm gold. To determine whether the
size of ReLPS and RsDPLA gold conjugates can alter the pathway of their
binding and internalization, purified ReLPS-5-nm gold conjugates and
RsDPLA-5-nm gold conjugates were compared for their interaction with
cells after 2 min of incubation (Fig. 5).
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). By using transmission electron microscopy, we were able to distinguish ReLPS-5-nm gold conjugates associated with plasma membrane as located on the top of plasma membrane (11%), in coated pits (10%), or that which seemed to be located inside the plasma membrane or right under it (30%). At the same time, 52% of all plasma membrane-associated RsDPLA-5-nm gold conjugates were seen on the top of plasma membrane, 2.2% were found in coated pits, and 23% were located inside the plasma membrane or immediately under it.
Thirty-seven percent of all ReLPS-5-nm gold conjugates were
found in the cytoplasmic matrix of the cell, and some of the conjugates
were located in noncoated vesicles and vacuoles (Fig. 5). Fewer
ReLPS-5-nm gold conjugates were found in caveolae; however, no
conjugates were present in clathrin-coated vesicles. The total number
of ReLPS-5-nm gold conjugates associated with the cell was 476. When we
treated cells with 10x lower concentration (26 ng/ml) of ReLPS-5-nm
gold conjugates, we found the same distribution of conjugates within
the cell as above, and the total number of ReLPS-5-nm gold conjugates
associated with the cell was 52.
After a 2-min treatment of cells with 260 ng/ml purified
RsDPLA-5-nm gold conjugates we found a total of 1543 purified
RsDPLA-5-nm gold conjugates associated with each cell, which is three
times more than those observed with ReLPS-5-nm gold conjugates. Only
5% of conjugates were found in the cytoplasmic matrix, and only a
small percentage of conjugates were found in noncoated vesicles and
phagosomes as observed with ReLPS-5-nm gold conjugates (Fig. 5). We did
not find any RsDPLA-5-nm gold conjugates in clathrin-coated
vesicles.
Therefore, the major difference in distribution of the ReLPS- vs RsDPLA-5-nm gold conjugates within the cell after a 2-min incubation was that ReLPS-gold conjugates were present in greater numbers in the cytosol (7-fold) and in coated pits (5-fold) as compared with RsDPLA, whereas the distribution among other organelles appeared similar.
RsDPLA blocks the binding and internalization of ReLPS-gold conjugates
Previously, an excess of RsDPLA has been shown to block ReLPS
cell-stimulating activity (8). We found 21 ReLPS-gold
conjugates on the membrane surface and 27 inside the cell without
RsDPLA treatment. When RAW 264.7 cells were pretreated with an excess
of RsDPLA (not labeled with gold) and then incubated with 5 µg/ml
ReLPS-gold conjugates and examined by thin-section transmission
electron microscopy, no gold particles were present on the cell surface
or inside the cell. When purified ReLPS-gold conjugates for the same
experiment were used, 37 purified ReLPS-gold conjugates were present on
the membrane surface and 81 were inside the cell. In the presence of
RsDPLA, only one ReLPS-gold conjugate per cell was located at the cell
plasma membrane surface (Table II). These
experiments illustrate that RsDPLA completely blocks the binding of LPS
and thus its entry into the cell.
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and 7).
In Fig. 6B, one ReLPS-gold conjugate was on the surface and
one entered the cell. In Fig. 7A, ReLPS-gold conjugates
(white dots) were observed on the cell surface. However, in the
presence of excess RsDPLA, ReLPS-gold conjugates failed to bind the
cell. These results indicate that RsDPLA blocks the ReLPS
activation of cells by blocking access to the binding sites on the cell
surface and thus preventing its internalization into the cell.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-1
(8). RsDPLA also blocks the LPS-induced tyrosine
phosphorylation in macrophages and protects mice against the lethal
effects of toxic LPS (8). However, the blockade of the LPS
binding sites on the cell surface by LPS antagonists has never been
demonstrated by electron microscopy. There have been several technical
problems in the past. Previously, gold-labeled Abs to LPS have been
used to track the LPS in the cell, and these results are not reliable.
LPS is often contaminated with proteins (7) that can
easily bind to gold particles, and conjugation of gold particles to
spectrally pure aggregated LPS has been difficult. We have now
developed a method for complexing different-sized gold particles with
purified and protein-free ReLPS and RsDPLA.
In the present study, spectrally pure ReLPS from E. coli was
used (27). Spectrally pure ReLPS allowed colloidal gold to
bind the LPS and not contaminating proteins, which would have been the
case had commercial preparations of ReLPS been used. Stable ReLPS and
RsDPLA-gold conjugates with 5-nm and 30-nm colloidal gold particles
were prepared, and the activities of both gold-labeled and unlabeled
ReLPS were compared. Both gold labeled and unlabeled samples induced
similar TNF- responses in RAW 264.7 cells (Table I), thus indicating
that gold does not affect the biological activity of the LPS. This is
not surprising because in nearly all instances, the conjugation of
colloidal gold particles with Abs, ligands, or enzymes does not change
their binding or biological activity (25).
Binding of LPS to the plasma membrane is an essential initial step for
interaction between LPS and the cell. LPS enters the cell via three
different mechanisms as proposed recently: formation of the
clathrin-coated vesicles (14); macropinocytosis similar to
bacterial phagocytosis (18); and via noncoated plasma
membrane invaginations involving caveolae and micropinocytosis leading
to the formation of micropinocytic vesicles (19). Our data
indicate that after a 2-min incubation of ReLPS with the cell, the
majority of internalized ReLPS was found in the cytoplasmic matrix
(Fig. 5), but as the incubation time increased ReLPS was found
primarily in macropinosomes and phagosomes (data not shown). This
observation suggests that ReLPS internalization proceeds not only via
all three earlier proposed pathways, but it also can penetrate through
plasma membrane of the cell by an unknown mechanism.
We have shown previously that RsDPLA has 105
times lower TNF-stimulating activity as compared with ReLPS
(9) and is the potent LPS antagonist in both human and
murine cells (8). But internalization pathway and
intracellular distribution of RsDPLA within the cell has not been
established yet. A side-by-side quantitative comparison of both ReLPS
and RsDPLA fate in the cell is extremely important for understanding
the mechanisms involved in antagonism exhibited by RsDPLA. RsDPLA was
internalized predominantly via macropinocytosis and phagocytosis as
evident from our data. The internalized RsDPLA-gold conjugates were
found mostly in large vacuoles as described by Yoshida et al.
(34) with all gold particles adhering to the limiting
membrane (Fig. 4C). We did not observe any clathrin-coated
vesicles carrying RsDPLA-gold conjugates.
ReLPS (agonist) and RsDPLA (antagonist) bind to the cell plasma
membrane, and both can be internalized via macropinocytosis. The main
difference is that ReLPS also can be internalized via other pathways;
in addition, the majority of ReLPS was found in the cytoplasmic matrix,
whereas RsDPLA was very rarely observed in the cytoplasm (Fig. 5).
The mechanism by which RsDPLA and ReLPS are internalized may be
different: ReLPS internalization proceeds by both micropinocytosis and
macropinocytosis (18) and endocytosis into the cytoplasm,
whereas RsDPLA internalization proceeds predominantly by
macropinocytosis. These results are consistent with Kitchens et al.
(19), who showed that LPS aggregation promotes accelerated
monocyte entry via a noncoated pathway. We did not detect ReLPS- or
RsDPLA-gold conjugates in the mitochondria, nucleus, or peroxisomes,
which is consistent with the data of Kriegsmann et al.
(15).
Our data revealed that an excess of RsDPLA blocks both the binding and
internalization of ReLPS (Table II and Fig. 7). The fact that RsDPLA
binds to the cell and prevents ReLPS binding suggests that they are
competing for the same binding sites. These findings support the
mechanism for RsDPLA antagonism suggested by Qureshi et al.
(8): RsDPLA binds to LBP, CD14, and possibly other
signaling protein(s) on cells. TLR-4, TLR-2, and MD-2 recently have
been suggested to be the signaling proteins (2, 3, 4). TLR-4
has been suggested to be the central lipid A-recognition protein in the
LPS receptor complex (34). However, LPS has not been shown
to bind strongly to these proteins. LBPs other than CD14 present on
cells remain to be determined. RsDPLA competes for the LPS binding site
on LBP (10). The RsDPLA-LBP complex then competes with
LPS-LBP complex at the second level of binding with membrane CD14.
RsDPLA also may compete at the third level of binding at
the LPS signaling protein (TLR-4) level (35). Therefore,
RsDPLA prevents the binding of ReLPS to CD14 or other proteins on the
cell surface. However, RsDPLA, with fewer and shorter fatty acyl
chains, binds to the cell but is unable to initiate cell activation,
and thus acts as an LPS antagonist. The importance of LPS
internalization and the fate of LPS inside the cell are presently not
known. Future studies are required to clarify LPS interaction with host
organelles.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nilofer Qureshi at the current address: Department of Basic Science, School of Medicine, University of Missouri, 2411 Holmes Street, Kansas City, MO 64108. E-mail address: qureshin{at}umkc.edu
3 Abbreviations used in this paper: LBP, LPS-binding protein; TLR, Toll-like receptor; ReLPS, deep rough chemotype LPS from E. coli D31 m4; RsLPS, LPS from R. sphaeroides; RsDPLA, diphosphoryl lipid A from R. sphaeroides; TEA, triethylamine.
Received for publication November 17, 2000. Accepted for publication April 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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