By
From the * Department of Biochemistry and Molecular Biology, the Department of Physiology, the § Department of Geriatrics, and the
Department of Disease-related Gene Regulation Research
(Sandoz), Faculty of Medicine, The University of Tokyo, Tokyo 113, Japan; and the ¶ Pharmaceutical
Basic Research Laboratories (Aobadai), Japan Tobacco Inc., Kanagawa 227, Japan
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Abstract |
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Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse biological activities in addition to its well-known ability to stimulate platelet aggregation. Pharmacologic studies had suggested a role for PAF in pregnancy, neuronal cell migration, anaphylaxis, and endotoxic shock. Here we show that disruption of the PAF receptor gene in mice caused a marked reduction in systemic anaphylactic symptoms. Unexpectedly, however, the PAF receptor-deficient mice developed normally, were fertile, and remained sensitive to bacterial endotoxin. These mutant mice clearly show that PAF plays a dominant role in eliciting anaphylaxis, but that it is not essential for reproduction, brain development, or endotoxic shock.
Key words: platelet-activating factor; platelet-activating factor receptor; anaphylaxis; endotoxic shock; gene targeting ![]() |
Introduction |
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Platelet-activating factor (PAF1; 1-O-alkyl-2-acetyl- sn-glycero-3-phosphocholine) is a potent phospholipid mediator with various biological activities, including platelet activation, airway constriction, hypotension, and vascular permeation (1). PAF acts by binding to a G protein- coupled seven transmembrane receptor (5). Through G proteins, PAFR links to signal transduction mechanisms, including turnover of phosphatidylinositol, elevation in intracellular calcium concentration (6), and activation of many kinases, e.g., phosphatidylinositol-3 kinase (7), protein kinase C (8), and mitogen-activated protein kinase (9). We and others have cloned PAFR cDNAs of guinea pig (10), human (11), and rat (15) and a PAFR gene of mouse (16). A unique PAFR has been identified in each species by the molecular cloning technique, although possible existence of PAFR subtypes has been suggested by biochemical or pharmacologic methods (17, 18).
PAF is synthesized not only in various blood cells but also in many other tissues, e.g., early mammalian embryo (19), fetal lung (20), kidney (21), and brain (22) under physiological conditions. Thus, PAF, even at low concentrations, has been considered to constitutively regulate various physiological processes such as reproduction (19, 23), blood pressure (24), neural development (25), and hippocampal long-term potentiation (18). Despite extensive studies, the physiological importance of PAF in vivo remains largely unknown.
In addition to its role as a physiological mediator, PAF has been believed to be associated with the pathology of anaphylaxis and endotoxic shock. Systemic anaphylaxis is a rapid, often fatal type-I allergic reaction characterized by acute airway constriction, heart rate (HR) alteration, hypotension, and vascular leakage (26, 27); many anaphylactic symptoms can be mimicked by PAF injection in animals (4, 5). Indeed, PAF is released rapidly from antigen-stimulated mast cells (28) and basophils (29) in vitro. In studies in vivo, an increase in blood PAF level is also detected after antigen challenge to sensitized animals (30). PAFR antagonists, which inhibit the binding of PAF to the receptor, exert protective effects on anaphylaxis in animal models (30). However, the precise role of PAF among a lot of allergic mediators in the development of anaphylactic responses is unclear.
Endotoxic or septic shock is the result of activation of
the immune system by endotoxin/LPS, a component of
Gram-negative bacterial cell wall. In this state, macrophages
produce large amounts of cytokines such as TNF- and IL-6,
as well as other inflammatory mediators, including PAF,
that are released into the circulation (33, 34). Thus, these
events lead to hypotension, tissue injury, vascular leakage,
and frequently, death. As is the case in anaphylaxis, PAF
per se is capable of eliciting many symptoms associated
with endotoxic shock (4, 5). Further, it has been reported
that the blood PAF level increased during endotoxemia
(30, 35) and the PAF acetylhydrolase activity decreased in
plasma of patients with sepsis (36). The administration of
PAFR antagonists to animals and humans protects them from
the deleterious effects of endotoxin (30, 32, 37). Our
recent demonstration that transgenic mice overexpressing
PAFR are hypersensitive to endotoxin has suggested that
PAF plays an exaggerating role in the in vivo response to
endotoxin (41).
To date, our ability to address the pathophysiological role of PAF has largely depended on the use of structurally diverse PAFR antagonists. However, pharmacologic studies often encounter problems related to the specificity of drugs. Indeed, several PAFR antagonists are demonstrated to have an inhibitory effect on the activity of intracellular PAF acetylhydrolase, an inactivating enzyme of PAF (42, 43). Some other antagonists inhibit histamine effects through the interaction with its G protein-coupled receptor (40, 44).
To clearly identify both the nonredundant physiological role of PAF and the pathophysiological role of PAF without the use of possibly nonspecific PAFR antagonists, we generated animals in which the PAFR signaling was congenitally declined by targeted disruption of the PAFR gene in murine embryonic stem (ES) cells. We report here that the resulting PAFR-deficient mice were fertile and developed normally. Furthermore, although these mice showed the markedly attenuated anaphylactic symptoms, the responses to endotoxin remained intact. Thus, our studies demonstrate that PAF plays a dominant role in eliciting anaphylaxis, and is not essential for reproduction, brain development, or endotoxic shock.
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Materials and Methods |
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Reagents.
Materials and chemicals were obtained from the following sources.Targeted Disruption of Mouse PAFR Gene.
The murine PAFR genomic DNA was isolated by screening a
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Northern Hybridization.
Total RNA was prepared from 2% casein-induced neutrophils as previously described (16). The RNA (1 µg) was electrophoresed, transferred to a nylon membrane, and hybridized (16) with either the PAFR probe shown in Fig. 1 a or a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (Clontech, Palo Alto, CA).Radioligand Binding Assay.
Lung membranes were prepared and used for ligand binding assay as described (41). Peritoneal exudate macrophages were obtained using thioglycollate as described in reference 16. The cells were cultured in 6-well dishes at 106 cells/well. After incubation for 2 h, nonadherent cells were washed away with RPMI 1640 medium containing 10% heat-inactivated FCS (GIBCO BRL, Rockville, MD). Adherent macrophages were then cultured for 1 or 2 d. Binding of [3H]WEB 2086 (20 nM) or [3H]acetyl-PAF (10 nM) was competed with unlabeled WEB 2086 (20 µM) in Hepes-Tyrode's buffer (pH 7.4) containing 0.1% BSA (Hepes-BSA buffer) at 20°C for 90 min or 25°C for 60 min, respectively. The cells were washed three times with cold Hepes-BSA buffer and lysed with 5% Triton X-100. The radioactivity associated with the cells was measured by liquid scintillation counting.Measurement of Intracellular Calcium Concentration.
Neutrophils were loaded with 3 µM Fura-2AM (Dojindo, Kumamoto, Japan) at 37°C for 1 h and resuspended in calcium-free Hepes-Tyrode's buffer (pH 7.4) at 2 × 106 cells/ml. Intracellular calcium concentrations were measured using a fluorometer (CAF-110; JASCO, Tokyo, Japan) as described previously (45).Sensitization of Mice.
pafr+/+ and pafrMeasurement of Mean Arterial Pressure and HR.
Under urethane anesthesia (1.5 g/kg, intraperitoneal), mice were cannulated with PE-10 polyethylene tubing (Clay Adams, Parsippany, NJ) into the right femoral artery. The tubing was filled with heparinized saline and connected to a pressure transducer (TP-200T; Nihon Kohden, Tokyo, Japan). The blood pressure signals were amplified (AP-600G; Nihon Kohden) and stored into a tape recorder. HR was computed using a tachometer (AT-601G; Nihon Kohden) on the basis of an electrocardiogram. The data were fed into a computer (PC9801RX; NEC, Tokyo, Japan) to calculate the mean values. Body temperature was maintained at 37 ± 1°C with a heating pad by monitoring rectal temperature. The cannulated mice were injected with 200 µg/kg of PAF (10 ml/kg), 200 µg/kg of lyso-PAF (10 ml/kg), 15 mg/kg of endotoxin (1 ml/kg), or 150 µg of OVA (100 µl/head) via the right femoral vein.Bronchopulmonary Responses.
Total lung resistance (RL) was measured as previously described (41, 46, 47). Mechanically ventilated mice were challenged with 500 µg OVA via the jugular vein in a 100-µl bolus. The lungs were removed intact 5 min after the challenge of OVA. For histological analysis, the lungs were frozen in liquid nitrogen, fixed in Carnoy's solution, and stained with hematoxylin and eosin (46). In other experiments, the dissected lungs were weighed and placed in a gravity convection oven at 90°C for 72 h to determine dry weights for calculation of wet weight/dry weight ratios (46).Determination of Cytokine Levels.
Adherent peritoneal macrophages were prepared as described in reference 16 and were cultured on 48-well dishes at 2.5 × 105 cells/well in 250 µl of RPMI 1640 medium supplemented with 10% heat-inactivated FCS (Moregate, Melbourne, Australia). The cells were exposed to endotoxin (100 ng/ml) plus mouse rIFN- ![]() |
Results and Discussion |
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A gene-targeting construct was generated in which an open reading frame of the
G protein-coupled PAFR gene was inserted by a PGK-neo cassette (Fig. 1 a). The construct was electroporated into
E14-1 ES cells. Two targeted ES clones were identified after screening 670 ES clones resistant to both G418 and fluoroiodoadenosyluracine by Southern blot analysis of ScaI-digested genomic DNA using the 5' probe shown in Fig. 1 a
(data not shown). Further Southern blot analyses with the 3'
probe after BamHI digestion, and Neo probe and PAFR
probe after ScaI digestion, demonstrated the absence of rearrangements or extra insertional events in these two clones (data not shown). The targeted clones were injected into
C57BL/6 blastocysts, and the resulting male chimeras bred
to C57BL/6 females to produce mice heterozygous for the
disrupted PAFR allele. Crossings between F1 or F2 heterozygous mice under specific pathogen-free conditions
yielded 82 wild-type (pafr+/+), 140 heterozygous (pafr+/)
mutant, and 72 homozygous (pafr
/
) mutant mice, consistent with a normal 1:2:1 Mendelian distribution of offspring (
2 test). pafr
/
mouse lines were established from
both clones.
Inactivation of the PAFR gene was confirmed by genomic Southern analysis (Fig. 1 b) and by Northern analysis
of RNA isolated from neutrophils (Fig. 1 c). Neither peritoneal macrophages nor lung tissues from pafr/
mice displayed any detectable binding sites for the PAFR antagonist WEB 2086 (16, 48; data not shown), confirming the loss of
surface receptors. In pafr
/
mice, challenge with PAF no
longer induced an increase in intracellular calcium concentration in neutrophils (49) (Fig. 1 d) or a decrease in mean
arterial pressure (MAP; reference 50; Fig. 1 e). These results
provide compelling evidence that the PAFR plays an essential role in PAF-induced calcium mobilization in neutrophils and in systemic hypotension.
pafr/
mice were healthy in appearance and grew normally up to
1 yr of age. There were no changes in blood cell populations, differential leukocyte counts, or serum biochemical
parameters (data not shown). Light microscopic examination of major organs (brain, heart, lung, liver, kidney, pituitary, thyroid, adrenal, thymus, spleen, gastrointestinal tract,
bone, bone marrow, and skin) revealed no differences in
morphology between pafr+/+ and pafr
/
mice at 13-16 wk
old (data not shown). A requirement for PAF in neural cell
migration had previously been proposed based on the finding that mutation in the LIS1 gene encoding a subunit of
brain PAF acetylhydrolase causes Miller-Dieker lissencephaly (25). The apparent lack of brain malformation in pafr
/
mice suggests, however, that the LIS1 subunit may have
other function(s) relevant to brain development that are
unrelated to PAF. This is consistent with a recent report
that LIS1 reduces microtubule catastrophe events by interacting with tubulin (51).
PAF has also been proposed to play significant roles in
reproductive functions such as implantation and parturition
in various species (52). In fact, human preembryos produced by in vitro fertilization show an increased rate of implantation after exposure to PAF (53) and transgenic mice
that overproduce PAFR display reproductive disorders
(41). Hence, there existed a possibility that pafr/
mice
might be infertile. Contrary to this expectation, however, the homozygous pafr
/
mice were fertile and produced
healthy litters of normal size (6.2 ± 1.2 [pafr
/
× pafr
/
;
n = 5] versus 8.0 ± 0.7 [pafr+/
× pafr+/
; n = 7]; mean
litter size ± SEM, P
0.2, the unpaired t test [two-tailed]). These results suggest that PAF is not essential for
normal development and reproduction of mice, although
we cannot rule out the possible existence of additional
PAFR subtypes as previously suggested by biochemical and
pharmacologic experiments (17, 18). Furthermore, it is also
possible that there may be receptor-independent actions
of PAF.
MAP was measured through a catheter placed in the
femoral artery. There were no significant differences in the
baseline of both parameters between pafr+/+ and pafr/
mice (101 ± 5 versus 107 ± 4 mmHg; mean ± SEM, n = 5 each, P
0.4, the unpaired t test [two-tailed]). Although
intravenously administered PAF has a potent hypotensive
effect, as shown in Fig. 1 e, the lack of difference in MAP
between pafr+/+ and pafr
/
mice suggests that PAF may
not be a mediator involved in setting the basal tone of vascular smooth muscle. HR of pafr+/+ and pafr
/
mice were
also indistinguishable (586 ± 22 versus 560 ± 28 beats/ min; mean ± SEM, n = 5 each, P
0.5, the unpaired t
test [two-tailed]). However, on the contrary, we observed
in OVA-sensitized mice (see below) that HR of pafr
/
mice (648 ± 7 beats/min; mean ± SEM, n = 10) was significantly higher than that of pafr+/+ mice (570 ± 18 beats/
min; n = 12, P = 0.003, the Mann-Whitney U test),
whereas MAPs were comparable between pafr+/+ and pafr
/
mice. Sensitization for active anaphylaxis may be associated with many changes, e.g., in levels of antigen-specific Igs of different isotypes, numbers of various effector cells, and local concentrations of immunomodulatory cytokines. It is
possible that some of these changes cause the observed increase in the basal HR.
To directly evaluate the
pathophysiological role of PAF in systemic anaphylaxis,
pafr+/+ and pafr/
mice were sensitized with OVA in the
presence of adjuvants (26). This treatment caused mice of
both genotypes to develop comparable and significant levels of OVA-specific IgE and IgG1 (data not shown), which
mediate systemic anaphylaxis (27). The sensitized animals
were injected intravenously with OVA to elicit systemic anaphylaxis. Cardiovascular or bronchopulmonary parameters were measured to assess the severity of the shock. Soon
after challenge with OVA, all pafr+/+ mice developed severe and irreversible hypotension (Fig. 2 a). In this shock
state, HR response to OVA was biphasic. OVA caused a
rapid onset of tachycardia that reached a maximal increase
of ~20% after 10 min, followed by bradycardia (Fig. 2 b).
The tachycardia may be a compensatory response due to
baroreceptor reflex. Five of six pafr+/+ mice died within 25 min (Fig. 2), and the remaining mouse died after 48 min.
In contrast, the fall in MAP was much milder in pafr
/
mice compared with pafr+/+ mice (Fig. 2 a). Furthermore,
HR remained stable upon the administration of OVA (Fig.
2 b). Only one of seven pafr
/
mice died after 36 min. Lethality among pafr
/
mice monitored 50 min after the antigen challenge was significantly lower than that of pafr+/+
mice (P <0.005, as determined by Fisher's exact test).
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Next, RL was measured in tracheostomized, mechanically ventilated mice. All pafr+/+ mice showed a rapid increase in RL after the intravenous injection of OVA (Fig. 3 a).
In comparison to the response in pafr+/+ mice, antigen
challenge of pafr/
mice produced a significantly smaller
increase in RL (Fig. 3 a). Lung tissues in pafr+/+ mice given
OVA were characterized by airway constriction associated with folding of airway epithelium, alveolar wall thickening,
alveolar distortion, and edema (Fig. 3 b). Pulmonary edema
due to increased vascular permeability was further assessed
by measuring wet weight/dry weight ratios (Fig. 3 c). In
marked contrast to pafr+/+ mice, pafr
/
mice displayed no
response to the antigen challenge, either in the wet
weight/dry weight ratios (Fig. 3 c) or in lung histology (Fig. 3 b).
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These results clearly demonstrate a dominant role for
PAF in the anaphylactic responses. Because pafr/
mice
still show milder symptoms of anaphylaxis, chemical mediators other than PAF, such as eicosanoids (55), histamine,
or serotonin, probably also contribute to the anaphylactic
responses, albeit to a much lesser extent.
The results of this study elucidated a large contribution of PAF to allergen-induced airway obstruction. As for the effects of PAF on the bronchopulmonary system, we reported that PAFR overexpressing mice show a remarkable airway hypersensitivity to methacholine (41). Thus, PAF is likely to play an important role in the pathophysiology of the bronchopulmonary system. However, the murine airways are somehow unresponsive to the intravenously administered PAF (41, 50, 56), whereas PAF given in the same manner elicited severe hypotension in mice (Fig. 1 e; reference 50). This is in contrast to our present data from immunologically stimulated mice which shows that PAF is deeply involved in both hypotension and bronchoconstriction. The murine airways, therefore, do not appear to respond to the blood-released PAF, which exerts deleterious effects on the cardiovascular system. Only PAF produced in the lung may act on the airways in a paracrine fashion.
Endotoxic Shock.To assess the importance of PAF and
PAFR in the development of endotoxic shock, PAFR-deficient mice were examined for their sensitivity to endotoxin using three kinds of assays. First, pafr+/+ and pafr/
mice were treated with endotoxin given intravenously at a
dose range of 7.5-30 mg/kg. As shown in Table 1, there
was no significant difference in lethality in this model between pafr+/+ and pafr
/
mice. At each dose, mice of both
genotypes exhibited apparently equivalent symptoms of endotoxic shock: lethargy, piloerection, and diarrhea. We
next measured MAP of anesthetized mice in endotoxemia for 100 min as shown in Fig. 2 a. pafr
/
mice showed hypotensive responses to endotoxin that were indistinguishable from those of pafr+/+ mice (Fig. 4). The drop in MAP
during endotoxic shock was slow but progressive, in contrast to the rapid drop observed during anaphylactic shock
(Fig. 2 a). Finally, the ability of pafr
/
macrophages to respond to endotoxin was evaluated. Thioglycollate-stimulated peritoneal macrophages were exposed to endotoxin
to induce expression of TNF-
and IL-6. Levels of these
cytokines in the culture medium were measured by ELISA.
In pafr+/+ and pafr
/
macrophages, endotoxin induced
equivalent levels of TNF-
(10,250 and 10,544 pg/ml, respectively) and IL-6 (4,352 and 4,314 pg/ml, respectively).
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During endotoxic shock, a strong hypotensive factor, nitric oxide (NO), is synthesized. A PAFR antagonist is reported to inhibit the endotoxin-stimulated NO production
in cultured murine macrophages (57), suggesting that endotoxin-induced hypotension is mediated by PAF and NO
in this order. However, our results demonstrate that endotoxic shock can be elicited without the action of PAF. Contrary to our expectation based on the previous pharmacologic reports, PAFR-deficient mice failed to acquire
resistance to endotoxin. The intact susceptibility to endotoxin is consistent with our present data that macrophages
from PAFR-deficient mice produced TNF- and IL-6, the
inflammatory cytokines induced by endotoxin, to the same
extent as those from wild-type mice. Since we have previously reported an increased susceptibility to endotoxin in PAFR overexpressing mice (41), it is very intriguing that
PAFR-deficient mice show normal sensitivity to endotoxin. It may be that PAF functions in vivo not as an essential, but as an exaggerating factor for endotoxic shock.
There are noteworthy pharmacologic reports exhibiting
that ID50 values of various PAF antagonists for death due to
anaphylaxis are nearly equal to those for PAF-induced death (32), whereas the doses of PAFR antagonists for inhibiting endotoxin-induced death are usually >10 times
higher than those for inhibiting PAF-induced death (39).
These data may suggest that PAFR antagonists attenuate
the symptoms of anaphylaxis and endotoxic shock in different ways. The latter might be PAFR independent.
Although PAF has pleiotropic biological activities in vitro, the effects of deleting the PAFR
gene were restricted to anaphylactic responses, i.e., PAFR-deficient mice were fertile and grew normally and were as
sensitive to endotoxin as the wild-type mice. Thus, we
conclude that although PAF is a dominant anaphylactic mediator, it may not be required for reproduction, neural development, or endotoxic shock. PAFR antagonists should, therefore, be useful in preventing anaphylactic responses in humans
without causing serious mechanism-based side effects. Recent
mouse models demonstrated that neither IgE (26) nor its
high-affinity receptor, FcRI (27), is required for active
systemic anaphylaxis, further supporting the validity of PAF
as a target mediator for preventing and treating the disorder.
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Footnotes |
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Address correspondence to Takao Shimizu, Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail: tshimizu{at}m.u-tokyo.ac.jp
Received for publication 20 January 1998 and in revised form 10 March 1998.
We thank I. Suganuma, M. Kumai (Japan Tobacco), Y. Hara, Y. Matsumoto, H. Sakamoto, Y. Suzuki, C. Suzuki, and M. Ito (The University of Tokyo) for technical assistance, D. Saffen (The University of Tokyo), H. Bito (Kyoto University), and M. Ohara for critical reading of the manuscript, A. Takaoka (Taisho Pharmaceutical) for instruction in determining the serum Ig levels, and N. Oigawa for secretarial assistance. We are also grateful to T. Izumi (The University of Tokyo) for valuable suggestions and M. Noma, H. Sasai, Y. Nishi, and T. Matsumoto (Japan Tobacco) for their support.
This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture and the Ministry of Health and Welfare of Japan, by grants from Human Science Foundation, Senri Life Science Foundation and the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan, and by Toyota Physical and Chemical Research Institute (to S. Ishii).
1Abbreviations used in this paper ANOVA, analysis of variance; ES, embryonic stem; HR, heart rate; LTB4, leukotriene B4; MAP, mean arterial pressure; NO, nitric oxide; PAF, platelet-activating factor; RL, total lung resistance.
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