Polymorphonuclear Leukocytes for Enhanced Release of Superoxide by Lipopolysaccharide: Possible Mechanism of These Actions - Infection and ...
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INFECTION AND IMMUNITY, Mar. 1994, p. 922-927 Vol. 62, No. 3
0019-9567/94/$04.00+0
Copyright ©) 1994, American Society for Microbiology
Pentoxifylline and CD14 Antibody Additively Inhibit Priming of
Polymorphonuclear Leukocytes for Enhanced Release of
Superoxide by Lipopolysaccharide: Possible
Mechanism of These Actions
KOZO YASUI,I* ATSUSHI KOMIYAMA,' THADDEUS F. P. MOLSKI,2 AND RAMADAN I. SHA'AFI2
Department of Pediatrics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan,' and
Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 060302
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Received 18 June 1993/Returned for modification 28 July 1993/Accepted 10 December 1993
Lipopolysaccharide (LPS) primes human polymorphonuclear leukocytes (PMN) for enhanced O2- produc-
tion in response to stimulation by N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Serum factor is
essential for priming at lower concentrations of LPS. Complexes of LPS and LPS-binding protein are
recognized by CD14 on PMN. We investigated the effects of a monoclonal antibody against CD14 (MY4) and
of pentoxifylline (POF), a membrane fluidizer, alone and in combination, on LPS-LPS-binding protein
enhancement of PMN superoxide production. LPS plus serum potentiated the fMet-Leu-Phe-induced
activation of phospholipase D evidenced by increased phosphatidic acid formation. Phosphatidic acid
formation and O2- production were inhibited by MY4 and POF. Our results suggest that the actions of these
agents occur at an early step in the excitation-response sequence. In the absence of a second stimulus, LPS plus
serum caused an increase in the amount of Gia2 associated with the membrane via CD14. POF, however, had
no effect on Gja2 in the membrane. POF alone significantly changed the affinity (KD) of the fMet-Leu-Phe
receptor of PMN (from 25.2 ± 4.5 nM to 15.2 ± 2.4 nM [P < 0.01; n = 4]) at 37°C. The differences between
the sites of action of MY4 and POF may lead to cooperation by these agents for inhibition of priming by LPS
plus serum for enhanced 02 production. Clinical use of the antibody and POF may diminish tissue damage
caused by PMN in clinical endotoxic shock.
The lipopolysaccharide (LPS) of gram-negative bacteria inhibits the priming action of LPS and serum complexes on
exerts profound effects on the host immune systems, including human neutrophils (35, 37).
activation of B lymphocytes (14) and macrophages (18, 22, 25). Pentoxifylline (POF) is a methylxanthine that was intro-
Exposure of neutrophils to endotoxin (LPS) primes the cells duced in 1984 for the treatment of peripheral vaso-occlusive
for enhanced release of microbicidal metabolites, including disease. POF is a membrane fluidizer which decreases the
superoxide anion (02-) and hydrogen peroxide (8, 13). The blood viscosity and can also modulate neutrophil functions (2,
increase in oxidative metabolism might permit increased resis- 4, 16, 17, 30, 31). Its precise mechanism of action is not fully
tance to bacterial infection but also could predispose the host understood. However, a change in neutrophil membrane flu-
to increased oxidative tissue damage (8, 13), which plays an idity may be associated with this phenomenon (21).
important role in endotoxic shock (24). It is possible that anti-CD14 MAb and POF cooperate in
It has been found that serum factors are required for the regulating the oxygen metabolite production by neutrophils
priming effect of LPS on neutrophils (1). The serum factors primed by LPS and serum. In order to clarify this possibility,
preserve LPS from inactivation by an enzyme and shift the LPS the effects of anti-CD14 MAb and POF alone and in combi-
dose-response curve in neutrophil priming to the left (1). In nation upon superoxide anion production in LPS-primed che-
vivo, LPS concentrations of 0.01 to 1 ng/ml are sufficient to moattractant-stimulated neutrophils were investigated. In ad-
induce symptoms of sepsis (20), while high LPS concentrations dition, phospholipase D (PLD) activity, Gix2 translocation,
(100 ng/ml to 1 ,ug/ml) are required to enhance the oxidative and chemoattractant receptors were examined in treated neu-
burst directly in vitro (8, 13). trophils.
LPS binds to proteins such as lipopolysaccharide-binding
protein (LBP) in serum (32, 34). Complexes of LPS and LBP MATERIALS AND METHODS
accelerate the priming of superoxide anion production in
neutrophils (34). CD14, a differentiation antigen of leukocytes, Reagents. [9,10-(n)3H]myristic acid (53 Ci/mmol) was pur-
recognizes LPS-LBP complexes on the surface of monocytes, chased from Amersham (Arlington Heights, Ill.). 1 5I-labeled
and blockade of CD14 with monoclonal antibodies (MAbs) protein A (NEX-146) and 3H-labeled N-formyl-methionyl-
prevents synthesis of tumor necrosis factor alpha by human leucyl-phenylalanine (3H-fMet-Leu-Phe) were from New
whole blood incubated with LPS (36). CD14 is also expressed England Nuclear (Boston, Mass.). LPS-free bovine serum
on neutrophils (35), and blockade of CD14 with a MAb albumin (BSA), cytochrome c (type IV), fMet-Leu-Phe,
1-palmitoyl-2-oleoyl phosphatidic acid (PA), 1,2-dioleoyl-sn-
glycerol, dimethyl sulfoxide, N-ethylmaleimide, diisopropyl-
fluorophosphate, EGTA [ethylene glycol-bis(,B-aminoethyl-
*
Corresponding author. Mailing address: Department of Pediatrics, ether)-N,N,N',N'-tetraacetic acid], HEPES (N-2-hydroxyethyl-
Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, piperazine-N'-2-ethanesulfonic acid), leupeptin, phenylmeth-
Japan. Phone: 81-263-35-4600. Fax: 81-263-36-6158. ylsulfonyl fluoride, superoxide dismutase, and POF were from
922VOL. 62, 1994 INHIBITION OF PRIMING BY LIPOPOLYSACCHARIDE 923
Sigma Chemical (St. Louis, Mo.). Phosphatidyl ethanol (PEt) with iodine vapor, and radioactivity was determined by liquid
was from Funakoshi (Tokyo, Japan). Silica gel 60 thin-layer scintillation spectrometry.
chromatography plates were from Merck & Co. (Darmstadt, 3H-fMet-Leu-Phe binding to PMN. The fMet-Leu-Phe bind-
Federal Republic of Germany). LPS (protein content, less than ing to cells was assayed by a modification of the method of
1%), purified from Escherichia coli 055:B5, was from Difco Snyderman and Fudman (28) as previously described (38).
Laboratories (Detroit, Mich.). Mouse anti-CD14 MAb MY4 Binding of 3H-fMet-Leu-Phe was performed with 1.5 x 10"
(immunoglobulin G2b [IgG2b]) was from Coulter (Hialeah, cells in 0.15 ml of HBSS buffer in the absence (total binding)
Fla.). fMet-Leu-Phe was prepared as a concentrated stock or presence (nonspecific binding) of a 1,000-fold-greater
solution in dimethyl sulfoxide and stored at - 20°C. The cells amount of unlabeled fMet-Leu-Phe for 30 min. The specific
were exposed to no more than 0.1% dimethyl sulfoxide, which binding was defined as the total binding minus the nonspecific
had no effect on superoxide production. Antibody was dis- binding. Each experiment was performed in duplicate or
solved in sterilized water and stocked at 4°C. LPS (5 mg/ml) triplicate. The standard error of the binding measurements was
was dissolved in sterile, pyrogen-free water and then diluted to less than 5% in all cases.
appropriate concentrations with Hanks balanced salt solution Preparation of plasma membranes and immunoblotting.
(HBSS). An attenuated solution of POF was negative in the PMN were subjected to nitrogen cavitation and ultracentrifu-
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Limlululs assay, including HBSS, serum, and dextran. gation (12). Cells were disrupted in buffered sucrose solution
Cell preparation. Heparinized (10 U/ml) whole blood was (1 mM EGTA, 1 mM diisopropyl-fluorophosphate, 0.1 mM
obtained from healthy adult donors, and polymorphonuclear leupeptin, 0.1 mM phenylmethylsulfonyl fluoride) at 350 lb/in2
leukocytes (PMN) were isolated by using a Histopaque (LPS for 20 min at 4°C (1 lb/in2 = 6.9 kPa). Cell lysates were
free; Sigma) gradient as described by Aida and Pabst (1). The centrifuged at 40,000 x g for 10 min to pellet the plasma
remaining erythrocytes were lysed by hypotonic shock. The membranes and nuclei. Pellets were then resuspended in buffer
cells were resuspended in sterilized HBSS without Ca2+ and A (75 mM Tris-HCl [pH 7.4], 25 mM MgCl2), underlaid with
Mg2 . The last supernatants of cell suspensions were checked 10 ml of 50% sucrose (50 mM Tris-HCI [pH 7.4], 10 mM
for the presence of endotoxin by the Limulus amebocyte lysate MgCl,), and centrifuged at 800 x g for 20 min at 4°C. The
test. Serum was prepared from platelet-poor plasma by the supernatants containing the plasma membrane were harvested,
addition of a 2% fluid volume of 1 M calcium chloride followed 20 ml of buffer A was added, and centrifugation was carried
by incubation for 3 h at 37°C to allow clot formation. Serum out at 40,000 x g for 10 min. The pellets were resuspended in
was heated at 56°C for 30 min to inactivate the complement sucrose solution. After quantitation of proteins, aliquots (100
system. RI) containing 50 p.g of membrane proteins were prepared for
Superoxide anion production. Superoxide anion (O, ) pro- immunoblotting as described previously (1 1). The protein (100
duction was determined at 37°C by the method of Cohen and RI) was boiled with 50 [lI of stop solution (9% [wt/vol] sodium
Chovaniec (6). The release of 0°, was measured as the dodecyl sulfate [SDS], 6% [vol/vol] 2-mercaptoethanol, 10%
change in A550 from the baseline, using a Beckman model [vol/vol] glycerol, and a trace amount of bromophenol blue dye
DU-50 spectrophotometer. The reaction proceeded for 2 min in 0.196 M Tris HCI [pH 6.7]). The mixture was electrophore-
after addition of the stimulant, fMet-Leu-Phe (1 [LM), or sed through an SDS-5 to 15% polyacrylamide gradient gel, and
buffer (control). The reaction was stopped with 0.5 mM the proteins were transferred to a nitrocellulose sheet. GQ2, the
N-ethylmaleimide. The generation of O0- was calculated by ot subunit of the corresponding G protein, was determined with
subtracting the absorbance in the presence of superoxide anti-Gio-2 serum (AS7; a gift from Allen M. Spiegel, National
dismutase (1 mM) from that of a duplicate sample without Institutes of Health, Bethesda, Md.), and the antibodies bound
superoxide dismutase and then dividing the value by 21.1 x to the nitrocellulose sheet were detected with '25I-labeled
103/cm/mol/liter for the molar extinction coefficient. Results protein A (final concentration, I VLCi/ml in 5% [wt/vol] BSA).
are expressed as nanomoles per 107 cells per 2 min. Blots were developed on Kodak X-Omat films overnight at
Radiolabeling of PMN and measurements of lipids. PLD - 70°C. Molecular masses were determined by comparison
activity was assessed by labeling PMN (5 x 10'/ml) for 60 min with Bio-Rad standards.
with [3H]myristic acid (1 pLCi/ml) in a 37°C, 5% CO2, humid- Statistics. Results are presented as means ± standard
ified incubator (5). After removal of unincorporated isotope, deviations. Each assay was performed in duplicate or triplicate.
cells were rinsed twice in Ca2+- and Mg2+-free HBSS contain- Statistical significance was determined by Student's t test. A P
ing 1.0 mg of LPS-free BSA per ml and then incubated in the value of924 YASUI ET AL. INFECT. IMMUN.
A 50
SUPEROXIDE
PRODUCTION
(nmole) 40
in
107 PMN, 2min
30
20
10 2
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01
c F
0 5 10 0 oi 1i1.0 10 100
B MY 4 PENTOXIFYLLINE
(pg/mi) (pg/mf)
FIG. 2. Effect of incubation with LPS plus serum for 30 min at 37°C
FMLP on 02- production by PMN stimulated with fMet-Leu-Phe (1 p.M).
The modulation of 02- production by PMN primed with LPS (10
ng/ml) and 1% serum for 30 min at 37°C and stimulated with
fMet-Leu-Phe for 2 min at 37°C by MY4 (left) or POF (right) is shown.
PMN were pretreated at 0°C for 15 min with the indicated concentra-
2 min tion of the antibody prior to incubation with buffer (open symbols) or
LPS plus serum (closed symbols). POF was present for 30 min during
FIG. 1. Time courses of 02- production in PMN primed with LPS the incubation. Data are means ± standard deviations from four
(10 ng/ml) and 1% serum (A) and control PMN (B) stimulated with separate experiments.
fMet-Leu-Phe (1 ,uM).
treated PMN stimulated with fMet-Leu-Phe. These results are
concentration, LPS did not prime PMN in the absence of summarized in Fig. 2. The cells were preincubated at 0°C for 15
serum (data not shown). LPS (10 ng/ml)-primed PMN in the min with MY4 prior to incubation with or without LPS plus 1%
presence of 1% serum released 4.5 ± 0.4 times (range, 3 to 8 serum at 37°C for 30 min. The MAb MY4 (2.5 ,ug/ml) did not
times; n = 10) more 02- than did similarly prepared control affect superoxide production in unprimed PMN stimulated
cells. Small amounts of serum were effective for priming, with 1 ,uM fMet-Leu-Phe (10.8 ± 0.6 nmol/2 min/107 PMN
half-maximum priming was observed with 0.2% serum, and the without MY4 and 10.2 ± 0.4 nmol/2 min/107 PMN with MY4
priming reached a plateau with 1.0% serum (data not shown). [n = 4]). MY4 (2.5 ,ug/ml) did reduce superoxide production in
Maximum priming was observed between 30 and 45 min after PMN primed with LPS (10 ng/ml) plus 1% serum and stimu-
the addition of LPS (data not shown). No priming was lated with fMet-Leu-Phe (45.3 ± 3.5 versus 21.2 ± 2.8 nmol/2
observed when PMN were incubated without LPS in the min/107 PMN [n = 4; P < 0.01]). Serum factor was necessary
presence of 1% serum, compared with untreated cells (Table for priming PMN at LPS concentrations ofVOL. 62? 1994 INHIBITION OF PRIMING BY LIPOPOLYSACCHARIDE 925
2.0
tE
-
-......
40
g.a)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~........... 0
0
0
a)
*' 1.5-
/~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....
~
~~~ ~ ~ ~
i6~~~~~~~~~~~~~fr....
~ ~ ~ ~ ~ ~~~.... m
~
~~ 0
~~ ~ ~ l)..
~~.
anti-.
LE 1-
d~~~~~~~~~~~..
0-
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15_m
o0
buffer~~~~~~~~~~~~........
experi-~~...............
I
different.I...........
0-L
0 5 10
0n Minutes
poten-~~~~~~~..............
plus~~~~~~~~~~~~..........
In....
observed~~~....
FIG. 5. Time courses of PA production in PMN stimulated with 1
he..
~~~~..
~ ~ ~ ~ ~
,uM fMet-Leu-Phe. The cells were incubated at 37°C for 30 min with
should~~~~~~............. LPS (10 ng/ml) plus 1% serum (open circle) or buffer (closed circle).
In other cases, either PMN were pretreated at 0°C for 15 min with
MY4 (2.5 p.g/ml) (open square) or POF (10 ,ug/ml) was present during
the incubation with LPS plus serum (closed square). The cross
indicates treatment with MY4 and POF followed by LPS plus serum.
Data are from one experiment and are representative of results in
three separate experiments. The mean basal level of 'H-PA in PMN
was 1,500 ± 220 dpm/2.5 x 105 cells.
ethanol resulted in an increase in the formation of PEt without
formation of PA and diacyl-glycerol (data not shown). From
these results, we concluded that LPS and serum potentiated
PLD activation in human PMN. MY4 or POF inhibited
potentiation of PA formation by LPS plus serum. The combi-
nation of MY4 and POF additively inhibited potentiation of
PA formation by LPS plus serum.
fMet-Leu-Phe binding. From the specific binding of 3.3 to 50
nM 3H-fMet-Leu-Phe to PMN, we calculated the KD and the
number of binding sites per cell. For control PMN at 4°C, the
Priming Inhibitor KD of PMN (n = 4) was 18.5 ± 1.2 nM and there were 12,700
+ 1,200 receptors per cell. Incubation with POF (10 pLg/ml) or
None None
MY4 (2.5 ,ug/ml) for 30 min did not affect fMet-Leu-Phe
None MY4 h binding; for incubation with POF the K,, was 17.9 ± 1.5 nM
None POF
"ZIP: and there were 12,500 ± 1,500 receptors per cell, and for
ZI::p incubating with MY4 the KD was 18.6 ± 0.9 nM and there
None MY 4 +POF were 12,000 + 2,000 receptors per cell. At 37°C, the K,, of
LPS None control PMN (n = 4) was 25.2 ± 4.5 nM and there were 52,000
+ 4,000 receptors per cell. POF changed the KD to 15.2 ± 2.4
LPS MY4 nM (n = 4; P < 0.01) and the number of receptors per cell to
LPS POF
H~~~~ 55,000 + 3,000, but MY4 did not affect fMet-Leu-Phe binding
LPS MY 4 +POF to cells.
Effect of LPS on the amount of Giao2 in the membrane. We
compared the amounts of the guanine nucleotide regulatory
0 10 20 30 40 50 protein Giot which transduces the action of the fMet-Leu-Phe
fMet-Leu-Phe-stimulated 02 production receptor, under various conditions. We previously showed that
(nmol/l X107 cells, 2min) the interaction of the LPS-serum complex with CD14 causes
FIG. 4. Effect of anti-CD14 antibody (MY4) and POF on °2-
Gict2 translocation (37). We measured the amount of Giox2
protein in the membrane by immunoblotting (Western blots)
production by PMN after stimulation with ,uM fMet-Leu-Phe for 2 using antiserum that specifically reacts with the Giot2 subunit
min. The cells were pretreated at 0°C for 15 min with MY4 (2.5 ,ug/ml)
prior to incubation with buffer (None) or LPS (10 ng/ml) plus 1% (Fig. 6). The cells were incubated with LPS (10 ng/ml) plus 1%
serum (LPS) at 37°C for 30 min. POF (10 ,ug/ml) was present for 30 serum and/or POF (10 ,ug/ml) or MY4 (10 ,ug/ml) for 30 min.
min during the incubation at 37°C in some instances. Results are Autoradiographs of the resulting blots were quantified by laser
means ± standard deviations from four different experiments. scanning densitometry. The Gicx, level in the plasma mem-926 YASUI ET AL. INFEcr. IMMUN.
KDa We previously reported that LPS in combination with serum,
in the absence of a second stimulus, caused an increase in the
amount of Gia2 (a subunit of G protein) in the membrane
43- 4nommaw 4000mool
during interaction with CD14 (37). The guanine nucleotide
411momw 'Isloolow
- --
Gi a2 regulatory protein (G protein) plays an important role in
29- regulating 02 production (26, 33); G protein transduces the
action of fMet-Leu-Phe, and an anti-CD14 MAb inhibits
translocation of G protein. This finding suggests that LPS-
18.4- serum complexes prime PMN through CD14 by translocating
cell components.
Control POFMY4 MY4 LPS POF POF also inhibits 02 production and the activation of PLD
+ + + in PMN primed by LPS plus serum, yet preincubation with
LPS POF LPS POF inhibits 02 production even in control PMN (2, 17, 30,
FIG. 6. Autoradiograph showing immunoblotting of GaOt2 protein 31) but has no effect on the amount of Gict2 in the PMN
from PMN membranes treated with POF (10 jig/ml) or LPS (10 ng/ml) membrane. Hence, there may be another mechanism of action.
Downloaded from http://iai.asm.org/ on January 20, 2021 by guest
plus 1% serum (LPS). An anti-CD14 MAb (MY4) was preincubated at POF decreases superoxide production by PMN stimulated with
0°C for 15 min prior to the incubation at 37°C for 30 min. POF was
present during the incubation. Membrane protein was analyzed by fMet-Leu-Phe but not that by PMN stimulated with phorbol
SDS-polyacrylamide gel electrophoresis followed by immunoblotting myristate acetate (30). This suggests that POF acts through a
with anti-Gi2 serum (AS/7). A representative experiment is shown. pathway bypassed by phorbol myristate acetate, such as a
receptor on the membrane. POF does not affect the binding of
fMet-Leu-Phe to PMN at 4°C but changes the affinity of the
brane clearly increased in the presence of LPS and serum. binding at 37°C. Inasmuch as membrane fluidity is largely
Neither LPS nor serum alone increased the level of Gia2 (data involved in the modulation of the chemoattractant receptor
not shown). When cells were incubated with the anti-CD14 (9), POF, a membrane fluidizer, may make the PMN mem-
MAb for 15 min at 0°C before the addition of LPS and serum brane more fluid and modify a cell function(s) by modulation
and then incubated for 30 min at 37°C, the antibody against of receptors. The numbers of receptors and the binding affinity
CD14 reduced the increase in the amount of Giot2 associated for the chemotactic peptide fMet-Leu-Phe were not found to
with the membrane treated with LPS and serum, but POF had be increased in LPS-primed cells (13). Bessler and coworkers
no effect on the amount of Gia2. This finding suggests that (2) and Hill and coworkers (16) have indicated that POF
interaction of LPS-serum with CD14 causes the translocation increases intracellular levels of cyclic AMP (cAMP) in PMN.
of an intracellular component (such as G protein) to the Accumulation of cAMP has an inhibitory effect on superoxide
plasma membrane. production (2). However, LPS plus serum had no effect on
intracellular levels of cAMP (data not shown).
From the results presented here, several conclusions can be
DISCUSSION drawn regarding the regulation of priming by LPS plus serum
LPS in combination with human serum primes human PMN of PMN 02 production stimulated with fMet-Leu-Phe. First,
for enhanced°2- production in response to fMet-Leu-Phe LPS plus serum primes PMN at an early step in excitation-
stimulation. In the absence of serum, high concentrations of response coupling. Second, blocking CD14 with a MAb and
LPS and longer incubation times are required to potentiate the exposure to POF additively inhibit priming of PMN for
02 production by fMet-Leu-Phe (1). A serum factor shifts enhanced release of superoxide by LPS plus serum. Third, the
the LPS dose-response curve to lower concentrations, and 30 antibody and POF may have different mechanisms for regulat-
min of incubation is enough to achieve full priming by LPS and ing PMN functions primed by LPS plus serum.
serum. Control PMN, not exposed to LPS, showed no priming The data presented here may be of clinical significance for
when the cells were stimulated with fMet-Leu-Phe in the patients with endotoxic shock. The maximum reported dose of
presence of serum. This result indicates that our preparative POF administered to humans to date has been about 3 mg/kg
techniques had not led to spuriously primed PMN and that given intravenously, resulting in a peak blood POF concentra-
priming by LPS was able to occur after isolation. CD14, a tion of 2 to 5 ,ug/ml (27). A 15-mg/kg dose in mice results in a
55-kDa glycoprotein on the surface of neutrophils, binds with peak blood POF concentration of about 15 to 20 ,ug/ml, which
LPS-LPB complexes, and PMN contain an intracellular pool of is tolerable in neonatal mice (19). Furthermore, POF inhibits
CD14 that is rapidly upregulated upon stimulation with some release of tumor necrosis factor following LPS stimulation of
agonists (35). We showed here and in a previous study (37) monocytes (29). Tissue damage, the result of the uncontrolla-
that blocking CD14 with a MAb prevented PMN priming for ble generation of oxygen metabolites and cytokines by phago-
enhanced 02- production by LPS and the serum factor. cytes, may play an important role in endotoxic shock. We
Accordingly, it is reasonable to hypothesize that the priming believe that blockade of CD14 on the cells and clinical use of
action of LPS plus serum is mediated through CD14. POF may diminish production of these oxygen metabolites and
LPS plus serum potentiated fMet-Leu-Phe-induced activa- improve outcomes in this condition.
tion of PLD, an enzyme closely connected to the activation of
an oxidative burst at an early sterp in stimulation by fMet-Leu- ACKNOWLEDGMENTS
Phe (10, 15, 23). Preparation of [ H]myristic acid-labeled PMN
takes 120 min, which is enough time for temporal adaptation in This work was supported in part by NIH grants GM-37694, Al-
24935, and AI-09648.
fMet-Leu-Phe receptors (7). It is hard to compare PLD
activation simply in such cells; however, we found significantly REFERENCES
increased PA formation in PMN treated with LPS plus serum. 1. Aida, Y., and M. J. Pabst. 1990. Priming of neutrophils by
Antibody against CD14 reduced the activation of PLD primed lipopolysaccharide for enhanced release of superoxide: require-
by LPS plus serum. This indicates that the action of CD14 also ment for plasma but not for tumor necrosis factor-a. J. Immunol.
occurs at an early step in the excitation-response process. 145:3017-3025.VOL. 62, 1994 INHIBITION OF PRIMING BY LIPOPOLYSACCHARIDE 927
2. Bessler, H., R. Gilgal, M. Djaldetti, and I. Zahavi. 1986. Effect of Wilmore. 1988. Detection of circulating tumor necrosis factor after
pentoxifylline on the phagocytic activity, cAMP levels, and super- endotoxin administration. N. EngI. J. Med. 318:1481-1486.
oxide anion production by monocytes and polymorphonuclear 21. Newton, J. A., E. R. Ashwood, K. D. Yang, N. H. Augustine, and
cells. J. Leukocyte Biol. 40:747-754. H. R. Hill. 1989. Effect of pentoxifylline on developmental changes
3. Bligh, E. A., and W. J. Dyer. 1959. A rapid method of total lipid in neutrophil cell surface mobility and membrane fluidity. J. Cell.
extraction and purification. Can. J. Biochem. Physiol. 37:911-918. Physiol. 140:427-431.
4. Buescher, E. S., S. M. Mcllheran, S. M. Banks, and S. Vadhan-Raj. 22. Pabst, M. J., and R. B. Johnston, Jr. 1980. Increased production of
1990. Alteration of the functional effects of granulocyte-macroph- superoxide anion by macrophages exposed in vitro to muramyl
age colony-stimulating factor on polymorphonuclear leukocytes by dipeptide or lipopolysaccharide. J. Exp. Med. 151:101-114.
membrane-fluidizing agents. Infect. Immun. 58:3002-3008. 23. Rossi, F., M. Grzeskowiak, V. Della Bianka, F. Calzetti, and G.
5. Cabot, M. C., C. J. Welsh, H.-T. Cao, and H. Chabbott. 1988. The Gandini. 1990. Phosphatidic acid and not diacylglycerol generated
phosphatidylcholine pathway of diacylglycerol formation stimu- by phospholipase D is functionally linked to the activation of the
lated by phorbol diesters occurs via phospholipase D activation. NADPH oxidase by FMLP in human neutrophils. Biochem.
FEBS Lett. 233:153-157. Biophys. Res. Commun. 168:320-327.
6. Cohen, H. J., and M. E. Chovaniec. 1978. Superoxide generation 24. Rothstein, J. L., and H. Schreiber. 1988. Synergy between tumor
by digitonin-stimulated guinea pig granulocytes. J. Clin. Invest. necrosis factor and bacterial products causes hemorrhagic necrosis
and lethal shock in normal mice. Proc. Natl. Acad. Sci. USA
Downloaded from http://iai.asm.org/ on January 20, 2021 by guest
61:1081-1087.
7. English, D., H. E. Broxmeyer, T. G. Gabig, L. P. Akard, D. E. 85:607-611.
Williams, and R. Hoffman. 1988. Temporal adaptation of neutro- 25. Schumann, R. R., S. R. Leong, G. W. Flaggs, P. W. Gray, S. D.
phil oxidative responsiveness to n-formyl-methionyl-leucyl-phenyl- Wright, J. C. Mathison, P. S. Tobias, and R. J. Ulevitch. 1990.
alanine: acceleration by granulocyte-macrophage colony stimulat- Structure and function of lipopolysaccharide binding protein.
ing factor. J. Immunol. 141:2400-2406. Science 249:1429-1431.
8. Forehand, J. R., M. J. Pabst, W. A. Phillips, and R. B. Johnston, 26. Sha'afi, R. I., and T. F. P. Molski. 1988. Activation of the
Jr. 1989. Lipopolysaccharide priming of human neutrophils for an neutrophil. Prog. Allergy 42:1-64.
enhanced respiratory burst: role of intracellular free calcium. J. 27. Smith, R. V., E. S. Waller, J. T. Doluisio, M. T. Bauza, S. K. Puri,
Clin. Invest. 83:74-83. I. Ho, and H. B. Lassman. 1986. Pharmacokinetics of orally
9. Gallin, J. I., and B. E. Seligmann. 1984. Mobilization and adap- administered pentoxifylline in humans. J. Pharm. Sci. 75:47-52.
tation of human neutrophil chemoattractant fmet-leu-phe recep- 28. Snyderman, R., and E. J. Fudman. 1980. Demonstration of a
tors. Fed. Proc. 43:2732-2736. chemotactic factor receptor on macrophages. J. Immunol. 124:
10. Gelas, P., V. V. Tscharner, M. Record, M. Baggiolini, and H. 2754-2757.
Chap. 1992. Human neutrophil phospholipase D activation by 29. Strieter, R. M., D. G. Remick, P. A. Ward, R. N. Spengler, J. P.
N-formylmethionyl-leucyl-phenylalanine reveals a two-step pro- Lynch III, J. Larrick, and S. L. Kunkel. 1988. Cellular and
cess for the control of phosphatidylcholine breakdown and oxida- molecular regulation of tumor necrosis factor-alpha production by
tive burst. Biochem. J. 287:67-72. pentoxifylline. Biochem. Biophys. Res. Commun. 155:1230-1236.
11. Gomez-Cambronero, J., C.-K. Huang, V. A. Bonak, E. Wang, J. E. 30. Sullivan, G. W., H. T. Carper, W. J. Novick, Jr., and G. L. Mandell.
Casnellie, T. Shiraishi, and R. I. Sha'afi. 1989. Tyrosine phospho- 1988. Inhibition of the inflammatory action of interleukin-1 and
rylation in human neutrophil. Biochem. Biophys. Res. Commun. tumor necrosis factor (alpha) on neutrophil function by pentoxi-
162:1478-1485. fylline. Infect. Immun. 56:1722-1729.
12. Gomez-Cambronero, J., M. Yamazaki, F. Metwally, T. F. P. 31. Thiel, M., H. Bardenheuer, G. Poch, C. Madel, and K. Peter. 1991.
Molski, V. A. Bonak, C.-K. Huang, E. L. Becker, and R. I. Sha'afi. Pentoxifylline does not act via adenosine receptors in the inhibi-
1989. Granulocyte-macrophage colony-stimulating factor and hu- tion of the superoxide anion production of human polymorpho-
man neutrophils: role of guanine nucleotide regulatory proteins. nuclear leukocytes. Biochem. Biophys. Res. Commun. 180:53-58.
Proc. Natl. Acad. Sci. USA 86:3569-3573. 32. Tobias, P. S., K. Soldau, and R. J. Ulevitch. 1986. Isolation of a
13. Guthrie, L. A., L. C. McPhail, P. M. Henson, and R. B. Johnston, lipopolysaccharide-binding acute phase reactant from rabbit se-
Jr. 1984. The priming of neutrophils for enhanced release of rum. J. Exp. Med. 164:777-793.
oxygen metabolites by bacterial lipopolvsaccharide: evidence for 33. Volpi, M., P. H. Naccache, T. F. P. Molski, J. Shefcyk, C.-K.
increased activity of the superoxide-producing enzyme. J. Exp. Huang, M. L. Marsh, J. Munoz, E. L. Becker, and R. I. Sha'afi.
Med. 160:1656-1671. 1985. Pertussis toxin inhibits fMet-Leu-Phe but phorbol ester-
14. Hammerling, U., A. F. Chin, and J. Abbott. 1976. Ontogeny of stimulated changes in rabbit neutrophils. Role of G proteins in
murine B lymphocytes: sequence of B-cell differentiation from excitation response coupling. Proc. Natl. Acad. Sci. USA 82:2708-
surface-immunoglobulin-negative precursors to plasma cells. Proc. 2712.
Natl. Acad. Sci. USA 73:2008-2011. 34. Vosbeck, K., P. S. Tobias, H. Mueller, R. A. Allen, K.-E. Arfors,
15. Hardy, S. J., B. S. Robinson, A. Poulos, D. P. Harvey, A. Ferrante, R. J. Ulevitch, and L. A. Sklar. 1990. Priming of polymorphonu-
and A. W. Murray. 1991. The neutrophil respiratory burst; re- clear granulocytes by lipopolysaccharides and its complexes with
sponses to fatty acids, N-formylmethionylleucylphenylalanine and lipopolysaccharide binding protein and high density lipoprotein. J.
phorbol ester suggest divergent signalling mechanisms. Eur. J. Leukocyte Biol. 47:97-104.
Biochem. 198:801-806. 35. Wright, S. D., R. A. Ramos, A. Hermanowski-Vosatka, P. Rock-
16. Hill, H. R., N. H. Augustine, J. A. Newton, A. 0. Shigeoka, E. well, and P. A. Detmers. 1991. Activation of the adhesive capacity
Morris, and F. Sacchi. 1987. Correction of a developmental defect of CR3 on neutrophils by endotoxin: dependence on lipopolysac-
in neutrophil activation and movement. Am. J. Pathol. 128:307-314. charide binding protein and CD14. J. Exp. Med. 173:1281-1286.
17. Krause, P. J., E. G. Maderazo, J. Contrino, L. Eisenfeld, V. C. 36. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, and J. C.
Herson, N. Greca, P. Bannon, and D. L. Kreutzer. 1991. Modu- Mathinson. 1990. CD14, a receptor for complexes of lipopolysac-
lation of neonatal neutrophil function by pentoxifylline. Pediatr. charide (LPS) and LPS binding protein. Science 249:1431-1433.
Res. 29:123-127. 37. Yasui, K., E. L. Becker, and R. I. Sha'afi. 1992. Lipopolysaccharide
18. Kurt-Jones, E. A., D. I. Beller, S. B. Mizel, and E. R. Unanue. 1985. and serum cause the translocation of G-protein to the membrane
Identification of a membrane-associated interleukin I in macro- and prime neutrophils via CD14. Biochem. Biophys. Res. Com-
phages. Proc. Natl. Acad. Sci. USA 82:1204-1209. mun. 183:1280-1286.
19. Maderazo, E. G., S. Breaux, C. L. Woronick, and P. J. Krause. 38. Yasui, K., M. Masuda, T. Matsuoka, M. Yamazaki, A. Komiyama,
1990. Efficacy, toxicity and pharmacokinetics of pentoxyfylline and T. Akabane, M. Hasui, Y. Kobayashi, and K. Murata. 1988.
its analogs in experimental Staphylococcus aureus infections. An- Abnormal membrane fluidity as a cause of impaired functional
timicrob. Agents Chemother. 34:1100-1106. dynamics of chemoattractant receptors on neonatal polymorpho-
20. Michie, H. R., K. R. Manogue, D. R. Spriggs, A. Revhaug, S. I. nuclear leukocytes: lack of modulation of the receptors by a
Dwyer, C. A. Dinarello, A. Cerami, S. M. Wolff, and D. W. membrane fluidizer. Pediatr. Res. 24:442-446.You can also read
