Effect of glycyrrhizin, cyclosporin A, and tumor necrosis factor a on infection of U-937 and MRC-5 cells by human cytomegalovi rus. Kei Numazaki, Nobuo ...
Effect of glycyrrhizin, cyclosporin A, and tumor necrosis a on infection of U-937 and MRC-5 cells by human cytomegalovi rus Kei Numazaki, Deparimeni
of
Nobuo Nagata,
Pediatrics,
Sapporo
Toshiya Sato, and Shunzo
Medical
University,
School
Abstract: Reactivation of latent or persistent human cytomegalovirus (HCMV) infection of monocytes or macrophages occurs under immunosuppressive conditions. We investigated the effect of glycyrrhizin (GL), cyclosporin A (CsA), and tumor necrosis factor-a (TNF-a) on the viral DNA synthesis and antigen expression of HCMV in U-937 and MRC-5 cells. Although GL inhibited the viral antigen expression of HCMV in human monocytic cell line U-937 and human embryonic lung cell line MRC-5 in the study, as determined by flow cytometry and immunofluorescence assay, immediate early HCMV DNA was detected by the polymerase chain reaction. CsA or TNF had no inhibitory effect on HCMV in U-937 or MRC-5 cells The HCMV infection model with U-937 or MRC-5 cells is of use for clarifying not only the mechanism of persistent infection but also the anti-HCMV effect of chemical agents. J. Leukoc. Biol. .55: 24-28; 1994.
Key
Words:
cell
polymerase
cytomegalovirus
chain
glycyrrhizin
.
U-937
cell
MRC-5
reaction
INTRODUCTION Although human cytomegalovirus (HCMV) infection occurs at any time during life, the clinical features differ according to the time of infection. Most commonly, HCMV causes congenital and perinatal viral infection, but it contributes significantly to transfusion-acquired infection and frequently to morbidity and mortality in organ transplant recipients, patients receiving immunosuppressive chemotherapy or highdose corticosteroids, or those infected with human immunodeficiency virus type 1 (HIV-1) [1]. Primary infection with CMV follows usually a benign course, but the virus remains latent or persistent in the host cells thereafter. Under immunosuppressive conditions, latent or persistent infection can be reactivated to produce a wide variety of clinical manifestations. pear to be
Cells of the monocyte-macrophage major sites of latent and persistent
lineage apHCMV infec-
tion [2, 3]. However, it is difficult to infect monocytes and macrophages with HCMV in vitro. CMV gene expression cannot be detected reproducibly in peripheral blood cells of asymptomatic carriers [4-6]. We succeeded in infecting the human monocytic cell line U-937 with the AD169 strain and a clinical isolate of HCMV [7]. The effect of anti-CMV agents on HCMV-infected U937 cells must be studied to prevent the reactivation of HCMV. We therefore investigated the effect of glycyrrhizin (GL), cyclosporin A (CsA), and tumor necrosis factor a (TNF-a) on the viral DNA synthesis and antigen and
24
expression the human
Journal
of HCMV embryonic
of Leukocyte
in the monocytic cell lung cell line MRC-5.
Biology
Volume
line
55, January
U-937
1994
of Medicine,
Chiba Sapporo,
MATERIALS
AND
Cell lines
and
The
monocytic
U-937
factor
Hok/caido,
Japan
METHODS
propagation
of HCMV
cell
line
was
obtained
from
the
Amen-
can Type Culture Collection (ATCC) and maintained as a cell suspension (2 x 10 to 2 x 106) in RPMI 1640 medium (Gibco Products) supplemented with 10% heat-inactivated fetal
and
calf
serum,
250 tg/ml
2 mM
L-glutamine,
streptomycin.
The
250
AD169
U/mI
penicillin,
laboratory
strain
of
HCMV (obtained from AThC) was used throughout this study. This strain was propagated in human embryonic lung cells (MRC-5) and harvested as cell-associated virus. HCMV was quantified by plaque assay on MRC-5 cells. For purposes of infection, 5 x 10 U-937 cells were pretreated with polybrene (2 g/ml) for 20 mm in 5 ml of RPMI
medium.
The
cells
were
then
washed
by centrifugation
and
resuspended in 0.5 ml of cell-free AD169 strain at 1.0 multiplicity of infection (MOI) for 3 h. The cells were centrifuged again to remove unbound virus, rinsed three times with fresh medium by centrifugation, and resuspended in fresh medium. At 24-h intervals the cells were pelleted and resuspended in fresh medium. In some experiments, the culture medium contained GL, CsA, and TNF in a variety of concentrations, as explained below. Thus, multiple medium changes, accomplished by washing the cells by means of centrifugation and resuspension in fresh medium, were per-
formed. The AD169 strain at 0.1 onto a 1-dram shell vial containing with monolayers of MRC-5 cells. trifuged
at 700g
at room
3 h, rinsed
three
to remove
unbound
After CMV
incubation, Culture
CA).
The
mined (IFA)
[8].
immediate
temperature
times
with virus,
a fixed
Percentages
early
of
cell
and
and
then
incubated
saline
incubated
for
48
were stained for the Test (Syva Company,
infected
indirect
MRC-5
(lEA)
cells
cells
and
positive
early
for
(PBS)
h at
37#{176}C.
MicroTrak Palo Alto, was
deter-
immunofluorescence
offluorescent
antigen
also inoculated round coverslip vials were cen-
phosphate-buffered
shell vials Identification
percentage
using
MOI was a 12-mm The shell
assay for HCMV
antigen
(EA)
in
Abbreviatiens: A1CC, American Type Culture Collection; CMV, cytomegalovirus; CsA, cyclosporin A; EA, early antigen; GL, glycyrrhizin; HCMV, human cytomegalovirus; HIV-1, human immunodeficiency virus type 1; HLA, human leukocyte antigen; lEA, immediate early antigen; IFA, immunofluorescence
assay;
IgG,
immunoglobulin
G;
LA,
late
antigen;
mAb,
M0I, multiplicity of infection; PBS, phosphatebuffered saline; PCR, polymerase chain reaction; PKC, protein kinase C; TNF-a, tumor necrosis factor a; VZV, varicella-zoster virus. Reprint requests: Kei Numazaki, Department of Pediatrics, Sapporo Medical University, School of Medicine, S.1 W.16 Chuo-ku, Sapporo, 060 Japan. Received June 21, 1993; accepted August 9, 1993. monoclonal
antibody;
cultures control
with
GL,
CsA,
and
(AD169-inoculated
Flow
cytometric
TNF
U-937
analysis
were cells
of surface
compared untreated
with with
the oligonucleotide primers were used in the PCR study to amplify DNA sequences of HCMV that code for regions of a major immediate early (IE) antigen (US3). The following oligonucleotide was designed for the PCR as the primer: PIE-i (5’CAAGAGAAAGA1DGACCCTGAT3’ , nucleotides 980-1001). Their sequences and locations are in AD169 Hindill fragment E. Synthetic oligonucleotides for use as primers were prepared using a Milligan 7500 DNA synthesizer and purified by high-pressure liquid chromatogra-
drugs).
markers
The expression of surface antigens on the cells was assessed by flow cytometry using the following fluorescein-conjugated monoclonal antibodies (mAbs): CD3, CD4, CD8, CD4 5R, Leu-15 (Mac-i), Leu-M1, HLA-DR, and Fc immunoglobulin G (IgG) (Becton Dickinson, Mountain View, CA). Paired isotype-specific control antibodies were run with each sample. Fluorescence was compared with appropriate fluoresceinlabeled isotype controls (IgGi-FITC; Becton Dickinson) using logarithmic-to-linear conversion tables. In the staining procedure, mAbs to HCMV lEA (DAKO Japan, Kyoto) and to late antigen (LA) (Chemicon International, Temecula, CA) were used.
phy. The cDNA, for 6 mm before
Drugs GL was supplied by Minophagen Pharmaceutical, Tokyo. It was dissolved in 0.01 M PBS and adjusted to pH 7.2 with 1 N sodium hydroxide. The CsA used in these studies was a gift from Sandoz, Tokyo. TNF-a was purchased commercially from Sigma Chemical Co., St. Louis, MO. The cytotoxicity of the drugs against U-937 and MRC-5 cells was assessed by inhibition of host cell DNA synthesis as reported previously [9]. The CD for host DNA synthesis was defined as the concentration of drug required to reduce [3H]thymidine incorporation to 50% of that of the control cell culture.
primers, adding
and reagents 2.5 U of Taq
were heated polymerase
at 100#{176}C (Perkin-
Elmer Cetus) to produce a final volume of 100 jl. The protocol of cycles was as follows: cycles 1-30 were performed using a DNA Thermal Cycler (Perkin-Elmer Cetus) at 94#{176}C for 5 mm (denaturation), 55#{176}Cfor 2 mm (annealing) and 72#{176}Cfor 2 mm (extension). Then 10-20 jl of the PCR reaction mixture was analyzed on 3% NuSieve and 1% Seakem agarose (FMC Corp.). The gels were stained with ethidium bromide and examined before blotting onto Nytrand membranes (Schleicher & Schuell, Keene, NH). The membranes were subsequently probed with 32P, 5-end labeled oligonucleotide. The probe was labeled to a specific activity of 108_109 cpm/tg. Hybridization was carried out for 1 h at 50#{176}C.The membranes were washed out at ambient temperature for 15 mm and at 55#{176}Cfor 15 mm before autoradiography. The specificity and sensitivity of these primers for HCMV DNA were established in our laboratory.
RESULTS
Detection reaction
of HCMV
DNA
The PCR was performed phenol-chloroform extraction ethanol precipitation step
by the
polymerase
chain
as
described previously [10]. SSof protein was followed by an for DNA preparation. Synthetic
100’
80 C
a) C) U)
60 0
Cl)
Effect
of GL, CsA,
U-937
cells
When
investigated
was
found
to exert
and
PCR an
inhibi-
20
demonstrate HCMV-infected
0
2.0
4.0
Concentration 1. Inhibitory effects of GL on exposed to various concentration for
48
h,
and
the
HCMV.
numbers
8.0
of GL
(mg/mi)
AD169-infected
of GL
in the of
10.0
6.0
cells
medium. showing
MRC-5
The
cells
positive
cells
were im-
munofluorescence for HCMV lEA and EA were compared with the control (no CL in the medium). The % cells fluorescent is calculated as the number of cells fluorescent with GL divided by the number of cells fluorescent without CL. The squares, circles, and triangles represent three different experiments.
GL
and
tory effect on MRC-5 cells at a concentration of 1.0 mg/mi and completely inhibited infection of MRC-5 cells by AD169 at a concentration of 10.0 mg/ml (Fig. 1). The CD50 of GL in MRC-5 cells was 15.0 mg/mi. Figure 2A shows results of amplification of HCMV IE DNA from MRC-5 cells infected or uninfected with AD169 strain. After 48 h of culture, viral IE DNA was detected in HCMV-inoculated MRC-5 cells at all concentrations ofGL, 0.5 to 12.0 mg/ml, by means of the PCR. Figure 2B shows the results of detection of viral IE DNA from HCMV-inoculated U-937 cells. The CD50 of GL in U-937 cells was 3.0 mg/mi. HCMV IE DNA was detected in HCMV-inoculated U-937 cells by PCR after 3 days in cul-
Flow
incubated
IFA,
of MRC-5
by IFA
40
0
Fig.
by
on infection
as assessed
ture at GL concentrations of 0.5 to 2.5 mg/mI. The CD50 of CsA for MRC-5 cells was 50 ig/mi. TNF-a at concentrations ranging from 10 to 1000 U/ml had no effect on the viability of either uninfected MRC-5 cells or HCMV-infected MRC-5 cells (data not shown). IFA and PCR failed to
a)
were
and TNF
by HCMV
any
cytometric
inhibitory MRC-5 cells.
studies
of
effect
changes
of
CsA
or
in surface
TNF
on
markers
The U-937 cells in this study were divided into two major subgroups (Gi and G2), as reported previously. After 3 days of culture, the expression of U-937 cell surface antigens was analyzed in cultures with and without GL. The cells in one subgroup (Gi) mainly expressed Leu-M1 and HLA-DR. The cells in the other group (G2) mainly expressed CD4, CD4 5R, Leu-15, Leu-Mi, and HLA-DR. The number of CD4-positive cells decreased after GL treatment at a low concentration (0.5 mg/ml) but slightly increased at a high concentration (1.5 mg/mi) (results not shown). HCMV lEA and LA were detected in AD169-inocuiated U-937 cells using
Nurnazaki,
ci al.
Antiviral
effect
on
human
cytomegalovirus
25
dothelial cells the adhesive following vator of secondary tivation
(BPAEC) with CD18 glycoprotein
either stimulus. PKC [33] and to diacylglycerol of
the
PKC
equal
efficacy, mediated
against
indicating that PMN adhesion
The fact that PMA is a direct actithat OZ induces PMN activation generation [25] suggests that ac-
pathway
may
be
an
important
deter-
minant ofCDl8-dependent PMN adhesion. The mechanism by which mAb 1B4 inhibited the OZand PMA-induced PMN adhesion to BPAEC is unknown but may be associated with binding of the mAb to the cell surface CD18, which can prevent
to its endothelial cell “antiadherence effect” of anti-CD18 antibodies may be related to their ability to increase cellular cAMP when PMNs are stimulated. A report by Gresham et al. [34] indicated that IB4 increased cAMP levels in PMNs stimulated with fMLP, suggesting that antiCD18 mAbs may activate cAMP-dependent protein kinase ligand
the
CD11/CDi8
ICAM-i.
from
and
thereby prevent PMN The stimulation of PMNs endothelial cells examined, 6.8-fold
increase
binding
Alternatively,
in
the
adhesion. with PMA and this was
transendothelial
injured associated
albumin
73% with
of a
permeability
over the control value. IB4 significantly reduced the number of injured endothelial cells and the increase in endothelial permeability. Similarly, stimulation of PMNs with OZ resulted in injury of 53% ofendothelial cells and a 3.5-fold increase in endothelial permeability, both of which were inhibited by mAb 1B4. Thus, the inhibition of PMN adherence by IB4 paralleled the ability of the anti-CD18 mAb to protect against endothelial injury induced by either OZ or PMA. This protection occurred despite the inability of mAb IB4 to prevent the PMAand OZ-induced 02 production. Similarly, mAb 1B4 did not inhibit the PMA-induced myeloperoxidase release and only partially inhibited myeloperoxidase release ( 29%) in response to OZ. Thus, the protective effect of IB4 was related to its ability to impair PMN adhesion to endothelial cells and not to inhibition of the respiratory burst or degranulation responses. The results indicate that the release of PMN activation products in the absence of PMN adhesion to the endothelial cells is incapable of producing endothelial injury. This points to the critical role of CD18-mediated PMN adhesion in the induction of endothelial injury. The findings are consistent with the idea that PMN adhesion to the endothelial cells provides the mechanism by which the released oxidants and proteases can be localized and concentrated at the endothelial-PMN interface, and thereby mediate injury ofendothelial cells [4, i4-i7]. The relative amounts of O2 and myeloperoxidase released differed caused
between greater
OZ and production
PMA of O2
stimuli. than
OZ,
PMA typically whereas OZ
released greater amounts of myeloperoxidase than PMA. We observed that PMA-stimulated PMNs tended to produce greater endothelial injury and permeability increases than OZ-stimulated PMNs. However, whether this trend is the result of greater release of O2 than proteases is not clear from this study. The literature regarding the relative toxicity of oxygen radicals versus proteases is controversial. For example, Vanani et al. [35] found that PMNs stimulated with immune complexes, OZ, on i2-O-tetradecanoyl phonbol acetate caused endothelial cell killing that was oxygen radical dependent. Yet Harlan et al. [36] and Diener et al. [15] reported that OZand PMA-stimulated PMNs resulted in endothelial cell detachment dent. This latter observation that superoxide dismutase
62
Journal
of Leukocyte
that
was oxygen is consistent and/or catalase
Biology
Volume
radical indepenwith the finding was not protective
55, January
endothelial
PMNs
1994
injury
in
response
to
PMA-stimulated
[37].
Smith et al. [38] observed that activation of PKC in PMNs (such as with PMA) elicits preferential release of specific granules, rather than azurophilic granules (which contain myeloperoxidase). These workers suggest that PKC activation mediates the release of specific granules but that an increase in [Ca2]1 in PMNs may be necessary for the release of azurophiic granules. This hypothesis is consistent with our observation that OZ (which increased [Ca2]1) caused greater release of myeloperoxidase than PMA (which did not increase [Ca2]1). Activation of PMNs is linked to an increase in PMN [Ca2] [23, 24]. Anti-CD18 and anti-CDlib mAbs have been reported to inhibit Ca2 oscillations in adherent PMNs [22], suggesting that CDiib/CD18 integnin contributes to the regulation ofchanges in [Ca2]. However, our findings mdicate that mAb 1B4 did not block the OZ-induced rise in [Ca2]1 in PMNs; thus, its protective effect was not related to its ability to inhibit the increase in [Ca2] PMNs. The basal adherence of nonstimulated PMNs to endothelial cells was decreased by mAb IB4 pretreatment ol PMNs (from 9.0 ± 1.0 PMN/field to 1.2 ± 0.3 PMN/field). This result can be explained by the constitutive level ol CDii/CD18 expression on the PMN cell membrane [ii]. There may also be a minimal degree of PMN activation duning
the
PMN
CD11/CD18 of
isolation,
expression.
CDi1/CD18
be increased as PMA [11]. CD11/CD18 hanced PMN ingly, mAb degree with
(10,000-20,000
which
Resting
could
PMNs molecules
result
express pen
cell),
in
basa]
a basal
level
which
car
to 10-fold upon activation with agonists such This increase in the quantitative expression ol has been proposed to be important in the enadherence to endothelial cells [39]. Interest1B4 inhibited PMN adherence to a similat either PMA or OZ as the stimulus, suggestin 5-
PMA and OZ caused similar CD18 up-regulation on the plasma membrane. Whether this up-regulation involves a quantitative increase in the surface expression o CDi1/CD18 or a qualitative change in the existing surface CD11/CD18 (e.g., a phosphorylation step) needs further study. In summary, PMAor OZ-stimulated PMNs showed increased adherence to BPAEC, which was associated with morphological evidence of endothelial injury and increased transendothelial permeability of albumin. Pretreatment of PMNs with anti-CD18 mAb 1B4 prevented PMN adherence that
PMN
and endothelial injury. IB4 was also protective against the increase in endothelial permeability in response to layered PMNs stimulated with PMA or OZ, although 1B4 was less effective in the PMA response. However, mAb 1B4 inhibited neither PMA-stimulated PMN activation production and myeloperoxidase release) PMN activation responses (i.e., the rise production). 1B4 only partially inhibited
responses (i.e., nor OZ-stimulated in [Ca2] and the myeloperoxi-
O2 O2
dase release from PMNs stimulated with OZ. Therefore, the protective effect of mAb IB4 was associated with its antiadherence effect. We conclude that PMN adherence to endothelial cells mediated by activation of the CD18 integnin is a critical factor in the development of vascular endothelial injury.
ACKNOWLEDGMENT The authors gratefully thank Ms. Lynn McCarthy careful preparation of the manuscript. Supported grants HL27016, HL45638, HL46350, and T32
for the by NIH HL07289.
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144,
Chollet-Martin,
Babin-Chevage,
three (CR3)
Im-
j
T.,
P.,
Chem.
hydrogen
5K.,
(secondary)
in vitro
Neutrophil
lymphocytes. Lo
specific
activated endothelial cells potentiates leukotriene B4 production. j Cell. Physiol. 153, 187-195. Anderson, D.C., Miller, L.J., Schmalstieg, F.C., Rothlein, R., Springer, TA. (1986) Contributions of the Mac-l glycoprotein family to adherence-dependent granulocyte functions: structure-function assessments employing subunit-specific monoclonal antibodies. J. ImmunoL 137, 15-27. Arnaout, MA., Lanier, L.L., Faller, DV. (1988) Relative contribution of the leukocyte molecules Mol, LFA-l and p150,95 (Leu M5) in adhesion
10.
of adherence
Hun,
bier,
21.
Immunol.
j
Pham
38.
39.
with
Mol
pression
deficiency. in
normal
Effects
of cell
and
deficient
activation
on
Mol/LFA-l
J.
leukocytes.
Clin.
surface
cx-
Invest.
74,
1291-1300.
Lum
et al.
Endothelial
injury
and
CD18
integrin
63
TABLE
1. on
Effect the
of Pretreatment with Trypsin Uptake of Sulfatides by J774
and Metabolic Macrophagesa PC-S PC/mg
(;zg
Pretreatment
azide
+
deoxyglucose
presence ofLDL, inhibition ofuptake ofPC-S liposomes was not observed (Fig. 8). Decreased uptake of PC-S liposomes was not observed in the presence of low concentrations of
uptake cell
acetylated LDL (25 tg tions (100 g pnotein/ml) macrophage uptake of LDL, at all concentrations
protein)
37#{176}C
None Trypsin Sodium
Inhibitors
4#{176}C
7.82
±
1.96
3.32
±
0.79
3.18
± ±
0.92 0.33
1.03 2.69
±
2.15
0.31 0.02
±
inhibiting uptake of PC-S liposomes; uptake somes in the presence of oxidized LDL (100 was
aMacrophages
were
preincubated
at
37#{176}Ceither
with
trypsin
(1
The
liposomes.
Data
each
experiment
cells are
were
then
analyzed
presented
performed
as the
for mean
uptake
of the
of two
experiments
radiolabeled
PC-S ±
SE,
with
in triplicate.
shown); however, the uptake of these PC-S liposomes was still only 4% of that observed with liposomes prepared with PC and sulfatides at a molar ratio of 1.5:1. The nature of the binding and uptake of sulfatidecontaining liposomes was investigated (Table 1). Pretreatment of the cells with trypsin inhibited binding of PC-S liposomes .to macrophages at 4#{176}Cto 31% of control values and inhibited uptake at 37#{176}Cto 41% ofcontnol values. Pretreatment of macrophages with sodium azide and deoxyglucose, inhibitors of ATP generation, produced a profound decrease in the uptake of PC-S liposomes at 37#{176}C, but at 4#{176}Cno significant effect of these inhibitors was observed. These results are consistent with the concept that the uptake of sulfatide-containing liposomes by macrophages at 37#{176}Cis energy dependent and protein (receptor) mediated, whereas association of these liposomes with macnophages at 4#{176}Cis only protein (receptor) mediated. The effect ofdextran sulfate and fucoidin on the uptake of PC-S liposomes by macrophages was examined at 4#{176}C (Fig. 6A) and 37#{176}C(Fig. 6B). These polyanions have been shown to inhibit the macrophage uptake of PS-containing liposomes [5], acetylated LDL [6], and oxidized LDL [181. Both dextran sulfate and fucoidin profoundly inhibited the amount of liposomes that were cell associated at both temperatures. Concentrations of these polyanions as low as 1 tg/ml depressed the binding of the liposomes at 4#{176}Cand the uptake at 37#{176}Cto approximately 20% of control levels, and maximal inhibition was achieved at a concentration of
‘
5 jg/ml
(Fig.
was the of PC-S
most effective inhibitor liposomes, reducing
somes to 35% of control values. The effect of native and modified sulfatide-containing liposomes was
102
Journal
to
of control
40%
values
jg
(Fig.
in
these lipoprotein/ml)
8).
DISCUSSION The uptake recognition
of
PS
system)
by has
macrophages been
(referred extensively
to studied.
as
the The
PS PS
recognition system is thought to be responsible for the cleanance ofaged red blood cells [19] and human tumor cells [20] by macrophages. The appearance of PS on the outer membrane leaflet may well be the signal for the recognition and subsequent phagocytosis of abnormal on damaged cells. Endocytosis of PS has been shown to occur in invertebrates, indicating that this important recognition system has been
A
a)
80
Ce 0.
60 0 C
0
0
40
0
20
0
10
inhibitor
a)
concentration
(pg/mi)
80
(8 0.
6).
Uptake of PC-S liposomes by macrophages was also cxamined in the presence of negatively charged phospholipids (Fig. 7). The effectiveness of PS to inhibit uptake was quite poor even at high concentrations (100 jsg PS/ml). This agrees with our previous findings (Figs. 2 and 3) that sulfatides are very effective inhibitors of PS uptake and suggests that the avidity of binding of sulfatides to the macrophage receptor(s) that mediates the uptake of these polar lipids is much greater than that for PS. When macrophages were incubated with phosphatidylglycerol liposomes (100 jig/ml), uptake of PC-S liposomes was inhibited to approximately 55% of control values (Fig. 7). Ofthe three phospholipids examined, cardiolipin take
decreased
of
mg/ml)
for 10 mm or with the combination of 50 mM deoxyglucose and 10 mM sodium azide for 30 mm at 37#{176}C.The cells were then incubated with sulfatidecontaining liposomes (5 sg PC/mI) for 2 h at either 37#{176} or 4#{176}C.In the case of the cells that were preincubated with the inhibitors of ATP generation, the same inhibitors were also present during the 2-h incubation with the PC-S liposomes.
protcin/ml), but higher concentraof this modified LDL inhibited PC-S liposomes by 30%. Oxidized examined, was more effective
of Leukocyte
Biology
the
of the macrophage uptake of these
LDL also
on the examined.
Volume
uptake In
55, January
uplipoof the
1994
60 0 C
0
0
I”
40
0 ‘-
-
20
0
10
5
Inhibitor
concentration
(pg/mi)
Fig. 6. Effect ofdextran sulfate and fucoidin on the uptake of PC-sulfatides by J774 macrophages measured at either 37#{176}C(A) or 4#{176}C (B). Cells were incubated with sulfatide-containing liposomes (5 sg PC/mI, 2 sg sulfatides/ ml) in the presence of either dextran sulfate () or fucoidin (0) for 2 h at either 37#{176} or 4#{176}C. The cellular content of [3Hcholesteryl hexadecyl ether was determined. Results represent the mean of triplicate determinations.
