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Low density lipoprotein inhibits the physical interaction of phlogistic crystals and inflammatory cells.

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Low density lipoprotein (LDL) is the major
plasma inhibitor of neutrophil oxidative and lytic responses to monosodium urate crystals and may function
to modulate acute gouty inflammation. LDL inhibited
apparent phagocytosis of urate crystals by human
neutrophils, suggesting it may interfere with early
events in the crystal-induced stimulation of neutrophils.
The effects were not specific for neutrophils, since
human platelet secretory and membranolytic responses
to urate crystals were also inhibited by purified LDL in
doses as low as 10 pg/ml. As with neutrophils, the
inhibitory activity of LDL was stimulus-specific. Since
LDL binds to urate crystals and specifically inhibits a
range of cellular responses to them, including phagocytosis, we hypothesized that LDL interferes with the
initial crystal-cell interaction. We measured platelet
interaction with urate crystals and found that LDL was
a potent inhibitor of crystal-induced platelet sedimentation and was 10-100-fold more active than lipoproteindepleted plasma or purified high density lipoprotein. In
summary, LDL inhibits a broad range of responses of a
Publication number IMM 3731 from Scripps Clinic and
Research Foundation.
From the Departments of Immunology and Rheumatology,
Scripps Clinic and Research Foundation, La Jolla, California; the
Division of Rheumatology, Veterans Administration Medical Center; and the Department of Medicine, University of California, San
Diego School of Medicine, San Diego, California.
Supported by the Veterans Administration Research Service and NIH grants AM-36702, AM-27214, HL-28235, AI-18042,
and CA-28166. Dr. Terkeltaub received fellowship support from the
Canadian Arthritis Society and the Medical Research Council of
Canada. Dr. Curtiss is an Established Investigator of the American
Heart Association.
Robert Terkeltaub, MD: Assistant Professor of Medicine;
Dianne Smeltzer: Research Technician; Linda K. Curtiss, PhD:
Associate Member; Mark H. Ginsberg, MD: Associate Member.
Submitted for publication March 8, 1985; accepted in revised form August 1, 1985.
Arthritis and Rheumatism, Vol. 29, No. 3 (March 1986)
number of inflammatory cell types to certain inflammatory surfaces, and this effect appears to be due to
inhibition of physical association of crystals with cell
Acute gouty inflammation is largely dependent
on interactions between monosodium urate crystals
and cells (1-3). The self-limited nature of acute gout
and the variable intensity of inflammation associated
with intraarticular urate crystals (2,445) might therefore be due to the appearance in gouty joint fluid of
substances which interfere with crystal-cell interaction. Plasma and serum markedly inhibit certain
neutrophil and platelet responses to urate crystals in
vitro (7,8), but lipoprotein depletion abrogates the
inhibitory effect of plasma on urate-induced neutrophil
oxidative and cytolytic responses (9). Low density
lipoprotein (LDL) is the major inhibitory plasma
lipoprotein (9). LDL is excluded from normal joint
fluid on the basis of its large size (10,ll). However,
LDL gains access to inflamed joints as a consequence
of enhanced synovial permeability (10-15); thus, variations in intrasynovial LDL may be capable of modulating the intensity of acute gouty inflammation in
LDL could inhibit neutrophil stimulation by
urates in a number of ways, including inhibition of
physical interaction of urates with neutrophils or inhibition of subsequent events required for phagocytosis,
cell stimulation, or lysis. In this study we have addressed the mechanism, stimulus specificity, and cellular specificity of LDL’s inhibitory activity. We have
found that LDL is capable of inhibiting the responses
of other cell types to urates, that LDL inhibits cellular
responses to particulates other than urate crystals, and
that LDL directly inhibits physical association of urate
crystals with cells.
Urate crystals were prepared under sterile conditions
and characterized as previously described (9). Crystals were
used in unheated form and ranged from 3 - 3 0 ~ in length.
Silica crystals (lop, unheated) were donated by the Pennsylvania Glass Sand Corporation, Pittsburgh, PA. 51Crwas
purchased from Amersham, Arlington Heights, IL, and
labeled 5-hydroxytryptamine creatinine sulfate (3H- and 14Cserotonin) was obtained from New England Nuclear, Boston, MA. Purified human a-thrombin was generously donated by Dr. J. Fenton (NY State Department of Health).
Imipraniine, globulin-free bovine serum albumin (BSA), and
cytochalasin B were obtained from Sigma, St. Louis, MO.
IgG (Human Cohn fraction 11) was purchased from ER
Squibb and Sons, New York, NY.
Fifteen milligrams per milliliter of Cohn fraction I1
was heat-aggregated at 63°C for 30 minutes and stored at
-70°C until use. Acid-soluble calf skin collagen was obtained from Calbiochem-Behring, San Diego, CA and
solubilized in modified Tyrode’s buffer containing 0.5%
(volume/volume) acetic acid. All other chemicals were reagent grade .
]Buffers. Modified Tyrode’s solution contained NaCl
(8 gdliiter), KC1 (0.195 g d i t e r ) , NaHCO3 (1.02 gmfliter),
MgzC16H20 (0.213 gmfliter), BSA (1 gdliter), and glucose
(1 g d i t e r ) . Hanks’ balanced salt solution (HBSS) was
obtained from Flow Laboratories, McLean, VA.
Plasma lipoprotein depletion and isolation. Lipoproteindepleted plasma (LPDP) was prepared by ultracentrifugation
(9). Plasma containing 20 mM EDTA was adjusted to a density
of 1.25 g d m l with solid KBr and centrifuged at 45,000 revolutions per minute for 20 hours at 4°C in a fixed-angle 50Ti rotor
(Beckman Instruments, Spinco Div., Palo Alto, CA). The
lipoprotein-containing supernatant was removed and the
infranatamt taken as LPDP. Sham-depleted plasma was obtained by mixing the supernatant and infranatant. Both LPDP
and sham LPDP were exhaustively dialyzed against calciumfree 10 mtMphosphate buffered saline, pH 7.4, and their protein
concentrations measured and equalized by addition of buEer.
Lipoprotein depletion was verified by >90% reduction of
cholesterol content of LPDP versus sham LPDP.
Lipoproteins were isolated by sequential ultracentrifugation of pooled plasma from fasting normal subjects,
using solid KBr for density adjustment, as previously described (16). The lipoprotein fractions assessed in this study
were: LIIL, d = 1.019-1.063 g d m l ; high density lipoprotein
(HDL), id = 1.063-1.25 g d m l . The fractions were dialyzed
against (11.45M NaCl containing 0.3 mM EDTA and 0.0005%
a-tocopherol, filter-sterilized, and stored sterile. All
lipoprotein concentrations were expressed on the basis of
protein a s measured by a modification of the Lowry assay,
using a BSA standard (17). The lipoproteins used displayed
a consistent apoprotein composition as judged by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (16).
Concurrent measurement of phagocytosis and
neutrophil luminol-dependent chemiluminescence (CL).
Neutrophils were isolated from human blood by the method
of Boyum (18), utilizing Ficoll-Hypaque density gradients
and dextran sedimentation with modifications as previously
described (19). These preparations contained >95% neutrophils. CL assays were performed by using previously described modifications (20) of the method of Wilson et al (21)
and monitored in a Beckman LS-8OOO liquid scintillation
counter (Beckman Instruments, Fullerton, CA) operated
in the single photon counting mode. The assay conditions
were: lo6 cells per assay in a final volume of 0.5 ml HBSS
containing 0.02M HEPES, 0.25% (weightholume) BSA, and
2 pM luminol (Aldrich, Milwaukee, WI). Urate crystals (25
mg/ml) were preincubated with equal volumes of LPDP,
sham LPDP, and buffer and washed as previously described
At time 0, the preincubated crystals were added to
the neutrophil suspensions, and CL was monitored at 22°C at
0.25-minute intervals for 15 minutes. Maximum uratestimulated CL has previously been observed at this time
point (9). CL values were determined as counts per minute
per lo6 cells, after correction for background CL obtained in
the absence of stimulation. At 15 minutes, the crystal-cell
suspensions were fixed with an equal volume of cold 2%
paraformaldehyde in 0.1M sodium phosphate, pH 7.4, and
maintained at melting ice temperature for 1 hour. Samples
were examined under polarized light microscopy. A minimum of 200 cells was counted for each sample.
The percentage of cells with apparent intracytoplasmic monosodium urate crystals was determined by previously described methods (23,24). Samples containing 5
pg/ml of cytochalasin B as an inhibitor of phagocytosis were
included in each experiment as controls for background
superimposition of crystals and cells. Inhibition was determined by the formula:
% inhibition = 1 - - x 100
where Ri represents the result in the presence of added
inhibitor and & represents the result in the absence of
Neutrophil degranulation assay. Isolated neutrophils
in HBSS-HEPES-BSA (6 x lo6 celldml) and urate crystals
(2.5 mglml) in a total volume of 0.5 ml were incubated in 5-ml
polypropylene tubes at 37°C for 1 hour with continuous
shaking. Incubations were terminated by addition of 2 ml of
iced HBSS and centrifugation at 300g for 10 minutes at 4°C.
Supernatants were decanted for assay of a-mannosidase as
previously described (7). Certain tubes were terminated
immediately as 0-time blanks. Cell suspensions lysed by
addition of Triton X-100in HBSS (final concentration 0.1%,
v/v) and sonication were used to determine total enzyme
activity. Results were expressed as percentage of total
enzyme activity, by the formula:
Enzyme release - 0-time blanks
x 100.
Total enzyme
Platelet preparation and secretiodlysis studies. All
platelet preparation was performed in plasticware. Plateletrich plasma (PRP) was prepared, as described previously
Table 1. Concurrent measurement of urate crystal phagocytosis and neutrophil chemiluminescence
(CL), using washed crystals (1 mg/ml) previously exposed to buffer, calcium-free lipoprotein-depleted
plasma (LPDP), or sham LPDP*
Crystals exposed to
Buffer (samples containing
5 pglml cytochalasin B)
Fraction of
cells with internalized
crystals (%,
mean f SE)
f 1.2
16.3 t 2.6
4.1 f 0.2
Inhibition of
(%, mean
f SE)
97.5 '' 0.3
* 0.8
Maximum CL
(cpm x
mean t SE)
Inhibition of
CL (%, mean
? SE)
2.04 2 0.52
0.55 f 0.16
15.6 ? 5.5
78.0 t 3.5
* See Materials and Methods for details.
(25), from acid citrate dextrose-anticoagulated whole blood
from healthy volunteers who had not recently taken aspirin.
Platelets were prelabeled with 3H-serotonin (2 pCi/ml of
PRP) or "Cr (10 pCi/ml of PRP) and ''C-serotonin (0.5
pCilml of PRP), as previously described (26). The cells were
them centrifuged at 2,400 rpm for 20 minutes at 10°C in an
International PR6 centrifuge (IEC, Boston, MA) and the
supernatant discarded. The platelet pellet was resuspended
in 2 ml of modified Tyrode's solution, pH 6.5, and gel-filtered
over Sepharose 2B, as previously described (26). The platelet-containing fractions (<7 ml) were pooled and diluted to
lo!' cells/ml with modified Tyrode's buffer. Imipramine (2
pM) was added to the platelet suspension to prevent
reuptake of serotonin in release assays (27).
Stimuli and inhibitors (0.2 ml) were added simultaneously to platelets (0.1 ml, at a final concentration of 3.3 x
10" cellshl) for 30 minutes at 37°C with constant agitation.
After the incubation, 0.3 ml of 4% paraformaldehyde in 0.1M
sodium phosphate buffer, pH 7.4, was added to halt secretion (26). The samples were immediately centrifuged in a
prechilled Sorvall RT 6000 centrifuge at 2,800 rpm (1,000g)
for 20 minutes at 4°C. The platelet-free supernatant (0.15 ml)
was mixed with 3 ml of liquid scintillation cocktail (Scinti-A;
Packard, Downers Grove, IL) and counted in a Searle Delta
300 liquid scintillation spectrometer. Percent release with
correction for background was calculated as previously
described (26).
Assay of association of platelets with urate crystals.
Gel-filtered, 'lcr-labeled platelets were diluted to a concentration of 4 x 108/ml. Platelets (0.125 ml) were incubated
with agitation at 4°C for 15 minutes with buffer, 2 mg/ml
urate crystals, and added inhibitors in modified Tyrode's
buffer which also contained 6 mM EDTA, to inhibit platelet
aggregation. At 15 minutes, samples were centrifuged at 30g
for 5 minutes or 1,OOOg for 15 minutes at 4°C. Supernatants
were carefully decanted, and the radioactivity of the pellets
and supernates counted in a Searle 1195 gamma scintillation
spectrometer. Percent urate-induced "Cr-platelet sedimentation was calculated by subtraction of the percent of platelets sedimenting in buffer alone from the percent sedimenting
in the presence of 2 mg/ml crystals. Inhibition of sedimentation was calculated as was described above for effects on
LDL inhibition of phagocytosis of urate crystals
by neutrophils. LDL is the major inhibitor in plasma of
neutrophil oxidative and cytolytic responses to urate
crystals (9). The lipoprotein could act by inhibiting
crystal-cell contact, initial transduction events, ingestion of crystals, or events subsequent to crystal
ingestion. We concurrently quantitated the neutrophil
respiratory burst (using luminol-dependentchemiluminescence) and apparent phagocytosis of crystals under
polarized light microscopy. Precoating of crystals with
calcium-free plasma (sham LPDP) equally diminished
phagocytosis and neutrophil luminol-dependent CL
responses, relative to responses obtained with naked
crystals (Table 1). Plasma inhibitory activity was
largely removed by lipoprotein depletion. Isolated
LDL was a potent inhibitor of crystal phagocytosis by
neutrophils (Figure l), and it reconstituted the inhibitory activity of lipoprotein-depleted plasma for urateinduced CL (9).
In the presence and absence of 5 pg/ml
cytochalasin B as an inhibitor of phagocytosis, neutrophi1 degranulation (a-mannosidase release) stimulated
by 2.5 mg/ml urate crystals was inhibited by addition
of 80 pdml LDL, by 59.8% (mean +- SE 13.2 2 0.4%
release versus 5.3 & 0.3% release) with cytochalasin B
and by 54.3% (30.8 ? 0.5% release versus 14.1 5 1%
release) without cytochalasin B. Degranulation stimulated by serum opsonized zymosan (9) (10' particles/ml) was not inhibited by the same concentration
of LDL, either in the presence of cytochalasin B (1 1.1
? 0.4% release versus 11.2 f 0.1% release) or in the
absence of cytochalasin B (25.2 & 2.5% release versus
26 ? 1.4% release). Therefore, LDL not only blunted
crystal ingestion by the neutrophils, but it also specifically inhibited stimulation of the cells in the absence
100 r
6ot Y
Figure 1. Effect of low density lipoprotein (LDL) on urate crystal
phagocytosis by neutrophils. Urate crystals were incubated with
neutrophils, as described in Materials and Methods, in the presence
or absence of 5-500 pg/ml LDL. Fixed neutrophils were counted for
the presence of apparent intracellular crystals, and inhibition by
LDL was quantitated relative to phagocytosis in buffer alone (8% of
neutroplhils contained intracellular crystals). Data are expressed as
mean % inhibition and range of duplicate determinations.
was the major plasma lipoprotein inhibitor. To further
analyze the specificity of the LDL effect, the inhibitory activities of urate-induced 'H-serotonin release of
LDL, LPDP, and BSA were compared. LDL was at
least 1,000-fold more potent than LPDP or BSA, in
inhibiting crystal-induced platelet serotonin release
(Figure 3), under conditions in which LDL at a concentration of only 10 pg/ml gave maximal inhibition of
this response.
Because urate-induced release of serotonin occurs via both secretion and lysis (30), we analyzed the
effects of LDL on these 2 processes. We studied cells
that were labeled with 51Cr and ''C-serotonin and
exposed to urate crystals (8 mg/ml) in the presence of
cytochalasin B (3 pg/ml). Under these conditions,
urate-induced 'lCr loss was consistently <3% of the
total, as previously reported (30), whereas 14Cserotonin secretion ranged from 1848% in 3 separate
t eo -
of phagocytosis, implying that LDL could influence
the earliest responses of cells to the crystals.
LDL and plasma inhibition of urate-induced
platelet secretion. We studied the effects of LDL on
platelet responses to urate crystals, in order to determine if' these effects were limited to neutrophils. When
urate crystals were incubated with platelets, a dosedependent release of serotonin occurred (28). A
submaximal dose of 4 mg/ml urate crystals was chosen
for release assays. Addition of plasma (sham LPDP) to
the crystal-cell mixture resulted in a dose-dependent
abrogation of platelet 3H-serotonin release (Figure 2).
LPDP exerted much less inhibition, and this inhibition
was not clearly dose-related.
'To determine the class of lipoproteins suppressing the platelet response, LPDP reconstituted to physiologic concentrations (29) of HDL (1.5 mg/ml) and
LDL (0.9 mg/ml), respectively, was added to the
crystal-cell suspension. Readdition of LDL alone reconstituted the bulk of the inhibitory activity of whole
plasma, whereas HDL failed to reconstitute inhibitory
activity (Table 2). Thus, as with neutrophils (9), LDL
70 60 -
30 -
Dilution of Plasma
Figure 2. Effect of plasma lipoprotein depletion on plasma inhibition of urate crystal-induced platelet serotonin release. Urate crystals were added to gel-filtered platelets and incubated as described in
Materials and Methods. Percent of total 3H-serotonin released per
cell suspension was quantitated. Urate crystals stimulated 65.4%
release of serotonin in the buffer alone. Percent inhibition (and SE)
of crystal-induced serotonin release by simultaneously added
lipoprotein-depleted plasma (LPDP) and sham-depleted plasma is
Table 2. Platelet inhibitory activity of lipoprotein-depleted plasma
(LPDP) after reconstitution with high density lipoprotein (HDL; 1.5
mg/ml) or low density lipoprotein (LDL; 0.9 mg/ml)*
release (%,
mean SE)
Addition to
Inhibition of
response (%,
mean 2 SE)
65.4 * i s
1.9 * 1.1
Sham-depleted plasma
90 al
A Silica crystals
80 -
0 Urate crystals
Heat-aggregated IgG
97.2 f 1.7
26.8 0.9
17.5 f 4.4
91.2 f 2.2
47.8 f 0.5
53.9 2 2.51
5.3 f 1.4
100 -
0 Thrombin
en 60Lc
* 'The inhibition by LPDP, sham-depleted plasma, and selectively
reconstituted LPDP (all at 1:2 final dilution) of platelet 'H-serotonin
release induced by 4 mg/ml urate crystals was measured in triplicate,
as described in Materials and Methods.
80 -
0 10
Concentration of Inhibitor Ipgimll
Figure 3. Semilogarithmic plot of the relative potencies of inhibition of urate-induced platelet serotonin release by low density
lipoprotein (LDL), lipoprotein-depleted plasma (LPDP), and bovine
serum albumin (BSA). Inhibitors were coincubated with urate
crystals and platelets, in triplicate, as described in Materials and
Methods. Urate crystals stimulated 66% release of serotonin in
buffer alone. Values shown are mean inhibition and SE.
experiments. LDL (25450 pg/ml) progressively abrogated urate-induced ''C-serotonin secretion under
these conditions (data not shown). LDL also abrogated the small degree of 51Cr loss which occurred
(data not shown). Thus, LDL is a major plasma
inhibitor of both platelet secretory and lytic responses
to1 monosodium urate crystals; its effects are not
limited to neutrophils.
Mechanism and stimulus specificity of LDL effects on platelets. To evaluate the stimulus specificity
of LDL inhibition, its effects on platelet responses to a
number of other soluble and particulate stimuli were
assessed. LDL (22.5-450 pg/ml) progressively abrogated monosodium urate and silica crystal-induced
serotonin release, but secretory responses to thrombin, acid-soluble collagen, and heat-aggregated IgG
were not inhibited (Figure 4). Similarly, with neutro-
Concentration of LDL (pglmll
Figure 4. Low density lipoprotein (LDL) inhibition of platelet
response to various stimuli. Platelet suspensions labeled with 'Hserotonin were incubated in triplicate with 4 mg/ml silica crystals
(19.9% serotonin release), 4 mg/ml urate crystals (80% serotonin
release), 1 unit/ml thrombin (85.4% serotonin release), 0.5 mg/ml
acid-soluble collagen (86.8% serotonin release), and 5 mg/ml heataggregated IgG (100% serotonin release). The mean inhibitory
activity for 'H-serotonin release with added LDL is shown.
phils, isolated LDL reduced CL responses to silica,
calcium pyrophosphate and hydroxyapatite crystals,
and lp polystyrene (latex) beads (data not shown), but
not to a number of other soluble and particulate
agonists (9). LbL's inhibitory effects on cellular responses therefore appeared to be specific for certain
Because LDL binds to urate crystals (9) and
stimulates a wide range of cellular responses to them,
it may act to inhibit initial physical association of
crystals and cells. To test this, we used low-speed
centrifugation to separate free platelets from those
bound to urate crystals. Utilizing I4C-labeled urate
crystals at 2 mg/ml, we established that 98.6 t 0.1% of
the crystals (mean t SE, n = 3) were sedimented in
Tyrode's-dextrose-BSA buffer by centrifugation at 30g
for 5 minutes. The proportion of 14C-urate crystals
sedimented under these conditions was not significantly altered by the presence of LDL, HDL, or LPDP
(at concentrations up to 1 mg/ml). Under the same
2 orders of magnitude less active in inhibition of
crystal-induced platelet sedimentation.
2 3 4 5 6 7 8 9 10
Urate Crystal Concentration (mglml)
0 1
Low density lipoprotein is a potent inhibitor of
the neutrophil cytolytic and oxidative responses to
monosodium urate crystals (9). These cellular responses are believed to be closely linked to phagocytosis of these crystals, which is followed by dissolution of the phagolysosomal membrane and lysis of
the neutrophil from within (8,23,3 1-33). We have
shown that LDL not only diminishes apparent
phagocytosis of urate crystals by neutrophils (Table 1
and Figure l), but it also suppresses secretory responses of nonphagocytic platelets (Figure 2) and
cytochalasin B-treated neutrophils. Thus, LDL may
predominantly act in the earliest stages of crystal-cell
interaction, rather than in late, transduction events.
Furthermore, LDL's effects are neither urate crys-
Figure 5. Dose-dependent platelet sedimentation by urate crystals.
"Cr-labeled platelets were incubated in duplicate with 0-10 mg/ml
urate crystals in the presence of 3 mM EDTA as described in
Materials and Methods. 51Cr in pellets and carefully decanted
supernatants was counted in a gamma scintillation counter. Data are
expressed as mean % of platelets pelleted and range of duplicate
conditilons, 35.2 ? 2% (mean
SE, n = 12) of
"Cr-labeled platelets were sedimented in the absence
of crystals, and sedimentation was not inhibited by
additioin of LDL, HDL, or LPDP.
'When urate crystals (1-10 mg/ml) were added to
the platelet suspension, there was a dose-dependent
increase in the sedimentation of "Cr-labeled platelets,
up to a maximum of 97% (Figure 5 ) . These experiments were conducted at 4°C in the presence of 6 mM
EDTA, to inhibit platelet responsiveness (including
aggregation and lysis). Under these conditions, we
confirmed that no detectable urate-induced platelet
lysis or secretion occurred, as assessed by comparing
"Cr loss and 3H-serotonin release in the supernatant
in parallel samples sedimented at 1,OOOg. Single platelets adherent to urate crystals were seen in formaldehyde-fixed pellets examined under polarized light
We studied inhibition of platelet sedimentation
in the presence of 2 mg/ml urate crystals, since this
gave a submaximal response (Figure 5). LDL at
10-1,000 pg/ml was a potent inhibitor of this phenomenon (Figure 6). HDL and LPDP were approximately
Concentration of Inhibitors (pglml)
Figure 6. Inhibition of physical crystal-platelet association by low
density lipoprotein (LDL). "Cr-labeled platelets were incubated in
triplicate with buffer or 2 mglml urate crystals, with added LDL,
lipoprotein-depleted plasma (LPDP), or high density protein (HDL)
as described in Materials and Methods. Background sedimentation
was quantitated and subtracted from urate-induced sedimentation.
Values shown are % inhibition (and SE) of urate-induced platelet
sedimentation by added inhibitors (95.8% of platelets sedimented in
the presence of crystals and buffer alone).
tal--specific nor cell-specific since platelet responses to
silica crystals (Figure 4) and neutrophil responses to
silica, calcium pyrophosphate and hydroxyapatite
crystals, and latex beads (not shown) are also inhibited.
The phagocytic process is known to be rapid
and to involve initial particle-cell contact, followed by
recognition and an increase in the adhesiveness of the
plasma membrane to the particle and the formation of
pseudopodia at the point of contact (reviewed in refs.
34 and 35). The pseudopodia, binding to recognized
sites distributed about the entire particle surface
(36,37), surround it and fuse. Cytoskeletal assembly
and contraction, under the influence of cytosolic ionized calcium, mediates this process (34).
LDL is a large molecule, with an apparent
Stokes’ radius of 100-120A (38). Particle-bound LDL
thus might inhibit phagocytosis via steric hindrance of
contact, attachment, and engulfment. Monosodium
urate crystals bind to a number of identified platelet
membrane glycoproteins, and this interaction not only
mediates platelet stimulation by urate crystals (26), but
could also conceivably mediate physical plateletcrystal association. Thus, LDL’s effects may arise via
interference with particle-binding to membrane
glycoproteins. Alternatively, constituents of particlebound LDL could alter plasma membrane or intracellular metabolic processes required for firm adherence or ingestion.
We chose to study LDL’s effects on platelet
responses since these cells, like neutrophils, secrete
granular contents and manifest changes in shape,
including formation of pseudopodia, in response to
stimulation by urate crystals (28,30). Furthermore,
platelets could be easily separated from urate crystals
in suspension by low-speed centrifugation, enabling
quantitation of early physical crystal-cell association.
LDL was a potent inhibitor of urate crystal
association with platelets (Figure 6). Since these studies were performed under nonsecretory, nonlytic, and
nonaggregating conditions, the results suggest that
LDL largely acts via interference with initial crystal--cell contact and/or adherence. It should be noted
that LDL’s inhibitory activity for crystal-cell association at a concentration of 10 pg/ml (Figure 6) was
somewhat less than its inhibitory activity for crystalinduced serotonin release (Figures 3 and 4). This may
be solely because the crystal-platelet association was
quantitated under conditions (4”C, 6 mM EDTA)
where platelet responsiveness to urates was blunted.
Alternatively, inhibitory activities of LDL other than
inhibition of physical association (e.g., effects on
membrane lysis and membrane triggering associated
with binding of crystals to membrane glycoproteins
[261) could be contributing also at low LDL concentrations.
In summary, low density lipoprotein inhibits a
broad range of responses of a number of cell types to
certain phlogistic particles, and this inhibition appears
to be largely due to inhibition of physical association
of the particle with the cell. Studies are in progress to
elucidate the responsible constituent(s) of LDL, including the role of the neutral and polar lipids and the
apoprotein(s) of LDL. It is conceivable that LDL, long
recognized as a deleterious factor in the pathogenesis
of atherosclerosis, may prove to have some beneficial
effects in certain forms of particulate-induced inflammation.
We are grateful to Jody Martin and Anna Latham for
their excellent technical assistance, and to Nancy McCarthy
for expert secretarial help.
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physical, crystals, interactions, lipoprotein, inhibits, low, inflammatory, phlogiston, density, cells
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