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Departments of
*
Medicine and
Pathology, University of Washington, Seattle, WA 98195; and
ICOS Corporation, Bothell, WA 98021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In normal human volunteer subjects, inhalation of aerosolized PAF produces many of these same effects including dose-dependent bronchoconstriction and nonspecific airway hyperresponsiveness (13). Studies with asthmatic subjects have found that plasma PAF levels increase after allergen challenge (14) and are significantly elevated during asthmatic attacks compared with basal levels measured in the absence of symptoms (15). Higher concentrations of PAF are found in sputum (16) and bronchoalveolar lavage (BAL) fluid from asthmatic patients than from subjects without asthma (17). Patients with asthma also have increased PAF receptor mRNA levels in their lungs (18).
The potent biologic activities of PAF require that the mediator’s synthesis and subsequent degradation be tightly regulated. In plasma, the conversion of PAF to biologically inactive lyso-PAF is catalyzed by a 43-kDa protein known as PAF-acetylhydrolase (PAF-AH) (19). The plasma form of human PAF-AH has been cloned and expressed as a recombinant protein (rPAF-AH) (19, 20). PAF-AH is a group VII phospholipase A2 that hydrolyzes the sn-2 bond of PAF, limiting its half-life to a few minutes (19, 21). Biochemical studies have demonstrated that rPAF-AH has substrate specificity and enzymatic activity comparable to that of native plasma PAF-AH (20). rPAF-AH suppresses PAF-induced inflammation in vivo. After i.v. administration, rPAF-AH inhibits PAF-induced pleurisy and paw edema in rats (20). A genetically linked plasma PAF-AH deficiency has been found in 4% of the Japanese population (22). This deficiency is attributable to a missense mutation in exon 9 of the plasma PAF-AH gene (V279F) in which phenylalanine replaces valine 279 (23). The V279F mutation causes a complete loss of PAF-AH enzyme activity (23). In the Japanese population, the prevalence of the V279F mutation is higher in asthmatic than in control subjects; patients homozygous for this mutation are more likely to develop the most severe form of asthma (24).
Despite the findings linking PAF to the etiology of asthma, clinical trials of potent PAF receptor antagonists in the treatment of asthma have been disappointing. A novel alternate approach to limiting PAF’s effects in asthma is elevating endogenous plasma PAF-AH levels by the administration of rPAF-AH. To test the therapeutic potential of rPAF-AH in asthma, we examined the effect of exogenously administered rPAF-AH in a murine model of allergen-induced asthma (25, 26). In this model, airway inflammation was induced in OVA-sensitized mice by intranasal (i.n.) OVA challenge administered on 3 consecutive days. Twenty-four hours after the last i.n. OVA challenge, airway inflammation and hyperreactivity to methacholine were evaluated. We found that rPAF-AH treatment inhibits mucus hypersecretion, eosinophil influx into the lungs, and airway hyperreactivity to methacholine after allergen challenge.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (6–8 wk of age; D&K, Seattle, WA), were used in all experiments. All animal study protocols were approved by the University of Washington Animal Care Committee.
Allergen induction of eosinophil infiltration
To induce eosinophil infiltration into murine airway tissue, mice were sensitized and later challenged with OVA (Pierce, Rockford, IL) as the allergen. Mice were immunized with OVA (100 µg) complexed with aluminum potassium sulfate in a 0.2-ml volume, administered by i.p. injection on days 0 and 14 as previously described (25). On days 14, 25, 26, and 27, mice were anesthetized with 0.2–0.3 ml of ketamine (6.5 mg/ml) and xylazine (0.44 mg/ml) diluted in normal saline. The OVA, rPAF-AH (100 µg)/OVA, and rPAF-AH (200 µg)/OVA groups all received 100 µg OVA in 0.05 ml normal saline by the i.n. route on day 14 and 50 µg of OVA in 0.05 ml normal saline by the i.n. route on days 25, 26, and 27. The control group received normal saline with aluminum potassium sulfate by the i.p. route on days 0 and 14 and 0.05 ml normal saline by the i.n. route on days 14, 25, 26, and 27.
Administration of rPAF-AH
The rPAF-AH (100 µg)/OVA group received a rPAF-AH dosage of 100 µg/day, whereas the rPAF-AH (200 µg)/OVA group received a dosage of 200 µg/day. The initial rPAF-AH dose was administered by i.p. injection 30 min before challenge with OVA on day 25. Previous pharmacokinetic studies indicated that the plasma half-life of rPAF-AH in the mouse was in the range of 6–8 h. Therefore, to maintain plasma rPAF-AH levels greater than 5 µg/ml during the course of the allergen challenge, mice received additional rPAF-AH dosages on days 26 and 27 by the i.p. route 30 min before OVA treatment. rPAF-AH was supplied by ICOS (Bothell, WA).
Pulmonary function testing
On day 28, 
24 h after the last i.n. administration of either
normal saline or OVA, pulmonary mechanics in response to an i.v.
infusion of methacholine were evaluated in the mice by a
plethysmographic method (25) modified from previously
described methods (27, 28). After pentobarbital anesthesia
(70–90 mg/kg by the i.p. route), the jugular vein was cannulated, the
trachea was intubated and connected to a Harvard ventilator, and the
thorax was opened by a thoracotomy as previously described
(25). The animal was placed in a supine position in one
compartment of a two-chamber whole-body plethysmograph; the dead space
of the system was 0.025 ml. The following minute ventilation maintained
normal arterial blood gases: tidal volume, 0.2 ml/20 gm; frequency, 120
breaths/min; and positive-end expiratory pressure, 2.5–3.0 cm
H2O (29). Copper mesh in the
plethysmograph served as a heat sink for rapid gas compression during
each tidal breath. Because the plethysmograph is a closed system, a
change in box pressure (Pbox) of the first
chamber (measured by a sensitive transducer (±0.7 cm
H20)) represented the change in box volume
(
vol = Pbox);
Pbox is equivalent to lung pleural pressure. The
system was calibrated by delivering a known volume of 0.2 ml of air at
a frequency of 120/min such that a calibration factor was equal to
0.2 ml/Pbox. Lung volume was equal to
Pbox with each breath multiplied by the
calibration factor. Ambient pressure swings and temperature increases
in the first chamber were offset as previously described
(25). Pressure movements at the opening of the tracheal
tube (Paw) were measured by another transducer
referenced to Pbox to determine transpulmonary
pressure (Ptp = Paw -
Pbox). After initial inflation to a
Paw of 30–35 cm H2O, the
lungs were inflated at least once 1–2 min before each measurement to
prevent partial collapse. An analogue-to-digital data acquisition
system (Strawberry Tree, Sunnyvale, CA) was employed to sample
Ptp and Pbox at 5 ms
intervals with a smoothing function employed to dampen background noise
as previously described (25). Flow was calculated by the
change in volume from Pbox, point-to-point/5
ms.
Data from seven consecutive breaths collected three times during the
first 10 min were used to calculate basal pulmonary function.
Methacholine was then infused by hand delivery into the jugular vein
over 10 s at a concentration of 120 µg/kg after 10 min of
ventilation. Dynamic compliance (Cdyn) was
determined for both the control period and during the peak response to
methacholine challenge. Tracheal tube resistance (0.63 cm
H2O x ml-1 x s) was
subtracted from all airway resistance measurements.
Cdyn was calculated as the change in tidal volume
(Vt) divided by the difference between Ptp at
end-inspiration and end-expiration when flow is zero
(Cdyn = Vt/Ptp). At
the completion of pulmonary function testing, each mouse was
exsanguinated by cardiac puncture; plasma samples were collected from
all mice in each experimental group and stored at -70°C until assay
of rPAF-AH.
Bronchoalveolar lavage
After tying off the left lung of the mainstem bronchus, the right lung was lavaged three times with 0.4 ml of normal saline. The three BAL fluid samples collected from each animal were pooled, and the number of cells in a 0.05-ml aliquot was determined using a hemocytometer. The remaining sample was centrifuged at 4°C for 10 min at 200 x g, and the supernatant was stored at -70°C until assay of rPAF-AH and IL-5 levels. The cell pellet was resuspended in normal saline containing 10% BSA, and BAL fluid cell smears were made on glass slides. To stain eosinophils, dried slides were stained with Discombe’s diluting fluid (0.05% aqueous eosin and 5% (v/v) acetone in distilled water) for 5–8 min, rinsed with water for 0.5 min, and counterstained with 0.07% methylene blue for 2 min.
Lung histology
The trachea and left lung (upper and lower lobes) were collected
and fixed in Carnoy’s solution at 20°C for 15 h. After embedding
in paraffin, the tissues were cut into 5-µm sections. Eosinophils
were stained in the lung tissue with Discombe’s solution as described
above. The number of eosinophils per unit airway area (2200
µm2) was determined by morphometric analysis as
previously described (25, 30). Airway mucus was identified
by the following staining methods: hematoxylin and eosin, methylene
blue, mucicarmine, toluidine blue, alcian blue, and alcian
blue/periodic acid-Schiff (PAS) reaction as previously described
(25). Mucin was stained with mucicarmine solution. Mucin
and sialic acid-rich nonsulfated mucosubstances were stained
metachromatically with toluidine blue (pH 4.5) and acidic mucin, and
sulfated mucosubstances were stained with alcian blue (pH 2.5) and PAS
reaction (25).
Occlusion of the airway diameter by mucus was assessed on a
semiquantitative scale ranging from 0 to 5+ (25). For each
mouse, 10 airway sections randomly distributed throughout the left lung
were assessed for mucus occlusion by morphometric analysis by
individuals blinded to the protocol design. Each airway section was
assigned a score for airway diameter occlusion by mucus based on the
following criteria: 0, no mucus; 1, 10% occlusion; 2, 30%
occlusion; 3, 50% occlusion; 4, 80% occlusion; 5, 90–100%
occlusion (25). Mucus occlusion was assessed
morphometrically in 8–10 airways randomly distributed throughout the
lungs of each mouse.
Immunocytochemistry (ICC)
Esterase-positive macrophages in the lung sections were detected
by the 
-naphthyl acetate histochemical method (31). The
phenotype of the lung esterase-positive macrophages was assessed by
ICC. The number of CD11b- and CD11c-positive macrophages per unit
airway area (2200 µm2 in perivascular,
periairway, and alveolar regions of the lungs) was determined by
morphometric analysis as previously described (25, 30).
The lung sections were incubated for 30–60 min at room temperature
with the primary Ab, either rat IgG mAb against mouse CD11b/CD18
(Mac-1) (100 µg/0.5 ml, clone M1/70 (9)) purchased from
Boehringer Mannheim, (Indianapolis, IN) or hamster IgG mAb against
mouse CD11c (>1 µg/ml, secreted from hybridoma cell line N418CHB224;
American Type Culture Collection, Manassas, VA; and kindly provided by
Dr. Andrew G. Farr, University of Washington, Seattle, WA), which
reacts with CD11c from mouse dendritic cells). After incubation with
the primary Ab, the sections were incubated for 20 min with the
respective second Ab, either biotinylated goat anti-rat or
biotinylated goat anti-hamster IgG (10 µg/ml in 5% nonfat dry
milk with 1% goat serum; Jackson ImmunoResearch, West Grove, PA).
After washes, the sections were then incubated for 15–45 min at room
temperature with HRP solution of the ABC Elite Kit (Vector
Laboratories, Burlingame, CA) and were counterstained with 2% aqueous
methyl green, dehydrated in a series of ethanol concentrations up to
100%, cleared in xylene, and mounted with Permount (Fisher Scientific,
Pittsburgh, PA).
Assay of IL-5 in BAL fluid
Levels of IL-5 in the BAL fluid were determined by ELISA using EM IL-5 kit (Endogen, Woburn, MA). The IL-5 kit has a sensitivity of <5 pg/ml. Dilutions of the BAL fluid were made starting at 1:2. IL-5 standards, ranging from 20 to 320 pg/ml, were also assayed. A standard polynomial equation (y = a + bc + cx2) was used to create a standard curve for the IL-5 standards. Concentrations of IL-5 in the BAL fluid were calculated from the line equation and the sample dilution factor.
Assay of rPAF-AH levels in plasma and BAL fluid
Levels of rPAF-AH in the plasma and BAL fluid were determined by a standard enzyme immunoassay (EIA) method described below. The EIA is specific for human PAF-AH and does not detect endogenous murine PAF-AH, which is present in the plasma and possibly in the BAL fluid samples. For the EIA, Immulon 4 "C" plates (Dynex Technologies, Chantilly, VA) were coated with 125 µl of the rPAF-AH-specific mAb 90G11D diluted to 3.0 µg/ml; after removing the coating solution, wells were blocked. Murine plasma and BAL fluid samples were plated in triplicate at 100 µl/well into 90G11D-coated plates. After incubation at 37°C for 30 min, plates were washed, 100 µl volume of a second rPAF-AH-specific mAb, biotin-labeled 90F2D, was added to each well, and plates were incubated for 30 min at 37°C. After extensive washing, 100 µl of Streptavidin-HRP (Pierce) was added to each well, and absorbance at 450/630 nm was read on an EL 312e Bio-kinetic microplate reader (Bio-Tek, Winooski, VT) within 30 min of adding the stop reagent. Data analysis was performed by calculating the mean A450 for each of the standards, test samples, and assay control samples. A standard polynomial equation (y = a + bc + cx2) was used to create a standard curve for the rPAF-AH standards. Concentrations of rPAF-AH in the sample specimens were calculated using the line equation, the sample mean A450, and the sample dilution factor.
Statistical analysis
The data are presented as the mean ± SE of the mean. A Student’s two-tailed t test was used to compare data for BAL fluid eosinophil counts and airway mucus between the different experimental groups. For the evaluation of pulmonary mechanics, a Fisher protected least significant difference test was used to compare values for Cdyn between experimental groups. A one-way ANOVA (Dunn’s method) was used to compare IL-5 levels in the BAL fluid between the experimental groups. Differences were considered statistically significant for p values <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On day 28, plasma and BAL fluid samples were collected 24 h
after administration of the final rPAF-AH dosage and were assayed for
rPAF-AH levels. In an effort to maintain plasma rPAF-AH levels above 5
µg/ml (10-fold over the endogenous PAF-AH level in plasma), for
the duration of the allergen challenge, a dosage of 100 µg/day was
selected for intervention in this murine asthma model. We also
determined whether increasing the rPAF-AH dosage to 10 mg/kg/day (200
µg/mouse/day) had additional effects in this model. In the rPAF-AH
(100 µg)/OVA group, which received a rPAF-AH dosage of 100 µg/day
on days 25, 26, and 27, the mean plasma concentration was 5.7 ±
0.7 µg/ml on day 28. Increasing the rPAF-AH dosage to 200 µg/day in
the rPAF-AH (200 µg)/OVA group resulted in mean plasma rPAF-AH levels
of 7.4 ± 2.8 µg/ml on day 28. BAL fluid samples from the
rPAF-AH (100 µg)/OVA and rPAF-AH (200 µg)/OVA groups did not have
detectable levels of rPAF-AH. As expected, rPAF-AH was not detected in
any plasma (or BAL fluid) samples collected from the saline and OVA
groups.
Effect of rPAF-AH on eosinophil and macrophage recruitment into the lungs
Twenty-four hours after the final i.n. OVA challenge, BAL was
performed on the right lung of all animals from each experimental
group. The mean number of eosinophils in BAL fluid collected from the
saline group was 0.01 ± 0.003 x 105
cells (Fig. 1
C). After OVA
treatment, the number of eosinophils in the BAL fluid from the OVA
group increased 285-fold to 2.85 ± 0.68 x
105 (saline group vs OVA group; p
= 0.0005). Pretreatment with either 100 µg/day or 200 µg/day of
rPAF-AH reduced eosinophil infiltration into the BAL fluid by 74%
(p = 0.0095; rPAF-AH (100 µg)/OVA vs OVA) and
by 80% (p = 0.05; rPAF-AH (200 µg)/OVA vs
OVA), respectively (Fig. 1C). rPAF-AH (200 µg/day) also
significantly reduced eosinophil infiltration into the lung parenchyma
and alveoli of OVA-sensitized/challenged mice (Fig. 2, C vs B; Fig. 3; Fig. 4,
B vs A; and Fig. 5,
B vs A).
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A). rPAF-AH treatment induced a
significant increase in macrophages in the lungs of the OVA-treated
mice (Fig. 4, B vs A; Fig. 5, B vs
A; and Fig. 6). The number of
esterase-positive macrophages was increased in the perivascular,
periairway, and alveolar areas of the lungs of the rPAF-AH-treated
mice (Fig. 6). ICC of the lung tissue was performed to phenotype the
lung macrophages. A significant increase in both CD11b- and
CD11c-positive macrophages in each of these lung regions was seen in
OVA-sensitized/challenged mice treated with rPAF-AH (200 µg/day)
compared with OVA treatment alone (Fig. 6).
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A marked increase in mucus-secreting airway cells and airway mucus
as detected histochemically was observed in OVA-treated mice compared
with saline controls (Fig. 2, B vs A; Fig. 3; and
Fig. 7A). rPAF-AH (200 µg/day) significantly reduced the
percentage of airway cells staining positive for mucus glycoproteins in
the lungs of the OVA-treated mice (Fig. 3 and Fig. 7, B vs A). Daily
i.p. administration of rPAF-AH during allergen challenge also reduced
occlusion of the airways resulting from mucus secretion (Fig. 3 and
Fig. 7, B vs A). In the OVA group, a mean score
of 3.38 ± 0.30 was seen representing 60–80% occlusion of the
airways by mucus (Fig. 3). With a rPAF-AH dosage of 200 µg/day, the
airway occlusion score was significantly reduced to 2.36 ± 0.19
(Fig. 3).
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In the saline group, the mean BAL fluid IL-5 levels were 0.5
± 0.2 pg/ml (Fig. 8). In the OVA group,
a 52-fold increase in BAL fluid IL-5 levels were seen with a mean
concentration of 22.2 ± 3.9 pg/ml. In the rPAF-AH (100 µg)/OVA
group, the rPAF-AH dosage of 100 µg/day had no effect on IL-5 levels
in the BAL fluid with a mean of 26.1 ± 5.8 pg/ml. However, in the
rPAF-AH (200 µg)/OVA group, mean BAL fluid levels of IL-5 decreased
to 9.7 ± 3.1 pg/ml, a 56.3% reduction compared with the
OVA-treated group; however, this effect was not statistically
significant compared with the OVA-treated group (rPAF-AH (200 µg)/OVA
group vs OVA group; p = 0.1).
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Airway reactivity was evaluated on day 28, which was 24 h
after the third i.n. challenge. In the OVA group, airway
hyperreactivity was seen after challenge with methacholine with a
significant decrease in Cdyn compared with the
saline group (saline group vs OVA group; p = 0.001)
(Fig. 9). rPAF-AH at 100 µg/day
(rPAF-AH (100 µg)/OVA group) given before OVA on days 25, 26, and 27
did not significantly reduce bronchial hyperresponsiveness to
methacholine in the OVA-sensitized and challenged mice. In contrast,
rPAF-AH at 200 µg/day significantly decreased the
methacholine-induced lung response (rPAF-AH (200 µg)/OVA group vs OVA
group; p = 0.0438).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasma levels of rPAF-AH greater than 5 µg/ml were achieved 24 h after the final rPAF-AH administration at both 100 µg/day and 200 µg/day dosages. The 100 µg/day dosage resulted in mean plasma rPAF-AH levels of 5.7 ± 0.7, whereas plasma rPAF-AH levels of 7.4 ± 2.8 µg/ml were achieved with the 200 µg/day dosage. These plasma rPAF-AH levels are sufficient to block PAF-mediated inflammation in other animal models (20). rPAF-AH was not detected in BAL fluid collected from mice treated with rPAF-AH, indicating that extravasation of the enzyme into the alveolar space did not occur in the model. This suggests that degradation of PAF by rPAF-AH, which reduces eosinophil infiltration and mucus secretion in the airways, occurs within the blood vessels. Although rPAF-AH treatment at a dosage of 100 µg/day did not reduce airway hyperresponsiveness or affect IL-5 levels in BAL fluid, eosinophil infiltration into the lungs was inhibited. When the dosage was increased to 200 µg/day, reduction in BAL fluid IL-5 levels and airway hyperreactivity to methacholine was observed.
PAF is a potent chemotactic and chemokinetic factor for eosinophils
(32). PAF promotes eosinophil recruitment to sites of
allergic inflammation through activation of the eosinophil adhesion
molecules, the ß1 integrin very late Ag-4
(4ß1; CD49d/CD29), and
the ß2 integrin CD11/CD18, which interact with
their respective ligands on endothelial cells, VCAM-1, and ICAM-1
(33). The increased adherence of eosinophils to cultured
HUVEC has been shown in vitro when the eosinophils are activated by PAF
(34). Eosinophil activation by PAF also induces the
selective transendothelial migration of eosinophils across unstimulated
HUVEC (35). In an in vitro model of human eosinophil
transmigration through basement membrane components, both an
eosinophil-activating cytokine (e.g., IL-5) and a specific
chemoattractant (e.g., PAF) are required for the eosinophil
transmigration (36). Thus, inactivation of PAF by rPAF-AH
may interfere with a variety of PAF-induced mechanisms of eosinophil
recruitment including reduction in IL-5, which primes eosinophils for
migration into allergic inflammatory sites.
Unexpectedly, increased numbers of CD11b- and CD11c-positive macrophages were recovered in the lung parenchyma and alveoli of the rPAF-AH-treated allergic mice. The CD11c immunostaining of these cells suggests that they are either dendritic cells or activated macrophages (37, 38). The airways of patients with asthma have an increased number of dendritic cells (39), and dendritic cells are found in lung parenchymal germinal centers after allergen challenge (40). The mechanism(s) and effects of the observed influx into the lungs of CD11b- and CD11c-positive mononuclear cells in our murine asthma model are unknown. Lung macrophages may play a protective role in limiting pulmonary inflammatory responses to allergens and other stimuli (41, 42, 43, 44, 45, 46). Human alveolar macrophages after IgE-dependent activation suppress lymphocyte proliferation (42). Human monocyte-derived macrophages can ingest senescent leukocytes (46), and hydrogen peroxide-induced lung injury is reduced by alveolar macrophage antioxidants (44). In patients with the adult respiratory distress syndrome (ARDS), the number of alveolar macrophages in BAL fluid increases (both as a percentage of total cells and in absolute numbers) in survivors of ARDS (43).
Another potent anti-inflammatory effect exhibited by rPAF-AH treatment in this murine asthma model was blockade of airway mucus release. In rodent, feline, and human lung explants, in vitro administration of PAF induces mucus glycoprotein release (47, 48, 49). Similarly, in vivo administration of PAF by either inhaled or i.v. routes stimulates airway mucus release in ferrets (5, 6). A portion of PAF receptor-induced mucus glycoprotein secretion is mediated indirectly by release of leukotrienes, 5-lipoxygenase pathway products of arachidonic acid metabolism (48).
PAF induces bronchoconstriction directly by action on airway smooth muscle PAF receptors and indirectly by induction of other bronchoconstrictor molecules such as thromboxane A2 and cysteinyl leukotrienes (1, 50). In mice, PAF-induced airway hyperreactivity to acetylcholine is controlled by a single gene and is not dependent on PAF-induced hyperpermeability (51). Transgenic mice overexpressing the PAF receptor have increased airway responsiveness to methacholine compared with their littermate controls (52). Prior studies in animal models (50, 53) and in some studies of normal human volunteers have shown that PAF inhalation can increase bronchial hyperreactivity for prolonged periods (13, 54). However, other studies have failed to demonstrate increased airway hyperreactivity after PAF inhalation in normal (55, 56) or asthmatic subjects (57, 58). Although the PAF receptor antagonists WEB2086 (59) and U.K.-74505 (60) do not reduce airway responsiveness after allergen challenge, the more potent PAF antagonist Y-24180 significantly reduces bronchial hyperresponsiveness in patients with asthma, as determined by improvement in the provocative concentration (PC) of methacholine, producing a 20% fall in the 1-s forced expiratory volume (FEV1) from control (PC20-FEV1) values (61).
In our asthma model, airway reactivity was evaluated on day 28, which was 24 h after the third i.n. challenge of BALB/c mice with OVA. Mice in the OVA group demonstrated airway hyperreactivity when challenged with methacholine (i.e., Cdyn was significantly reduced compared with the control group). A dissociation between the effects of rPAF-AH on airway eosinophilia and hyperresponsiveness was observed. Although eosinophil influx into the lungs was inhibited comparably by the two doses of rPAF-AH employed, airway hyperreactivity was only affected by the highest dose (200 µg/day). These data showing a discordance between airway eosinophilia and hyperreactivity are consistent with our prior work demonstrating that 5-lipoxygenase and 5-lipoxygenase-activating protein inhibitors of leukotriene synthesis inhibit allergen-induced lung eosinophil recruitment without affecting bronchial hyperresponsiveness to methacholine (25). Similarly, inhibition of lung eosinophilia by anti-IL-5 mAb treatment fails to affect airway hyperreactivity in some (62) but not all murine asthma models (63). Using a CD49d mAb in our mouse model of asthma, we have found that local, intrapulmonary blockade of CD49d by i.n. administration of the Ab blocks all evidence of lung inflammation and airway hyperreactivity to methacholine (64). In contrast, systemic administration of CD49d mAb to block CD49d on circulating leukocytes prevents only airway eosinophilia and not bronchial hyperresponsiveness (64). Overall, these studies demonstrate that allergen-induced airway hyperreactivity may develop in the absence of eosinophilia.
Thus, PAF is likely an important mediator of both the airway hyperreactivity and late-phase inflammation that occur in this allergen-induced model of asthma. These data suggest that rPAF-AH treatment may reduce allergen-induced airway disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William R. Henderson, Jr., Department of Medicine, Box 356523, University of Washington, 1959 Northeast Pacific Street, Seattle, WA 98195-6523. E-mail address:
3 Current address: Department of Pathology, 304th Hospital of People’s Liberation Army, Beijing, People’s Republic of China.
4 Abbreviations used in this paper: PAF, platelet-activating factor; BAL, bronchoalveolar lavage; PAF-AH, PAF-acetylhydrolase; i.n., intranasal; Pbox, box pressure; Ptp, transpulmonary pressure; Cdyn, dynamic compliance; PAS, periodic acid-Schiff; ICC, immunocytochemistry; EIA, enzyme imunoassay.
Received for publication October 22, 1999. Accepted for publication January 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma. J. Clin. Invest. 100:3083.[Medline]
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 H. S. Seo, J. H. Kim, and M. H. Nahm Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid Clin. Vaccine Immunol., April 1, 2006; 13(4): 452 - 458. [Abstract] [Full Text] [PDF]
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 S. Katz, R. Harris, J. T.-Y. Lau, and A. Chau The use of gene expression analysis and proteomic databases in the development of a screening system to determine the value of natural medicinal products. Evid. Based Complement. Altern. Med., March 1, 2006; 3(1): 65 - 70. [Abstract] [Full Text] [PDF]
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 F Y Liew, M Patel, and D Xu Toll-like receptor 2 signalling and inflammation Ann Rheum Dis, November 1, 2005; 64(suppl_4): iv104 - iv105. [Full Text] [PDF]
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 S. M. Vinson, A. Rickard, J. S. Ryerse, and J. McHowat Neutrophil Adherence to Bladder Microvascular Endothelial Cells following Platelet-Activating Factor Acetylhydrolase Inhibition J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1241 - 1247. [Abstract] [Full Text] [PDF]
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 M. Patel, D. Xu, P. Kewin, B. Choo-Kang, C. McSharry, N. C. Thomson, and F. Y. Liew TLR2 Agonist Ameliorates Established Allergic Airway Inflammation by Promoting Th1 Response and Not via Regulatory T Cells J. Immunol., June 15, 2005; 174(12): 7558 - 7563. [Abstract] [Full Text] [PDF]
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S. Ishii, T. Nagase, H. Shindou, H. Takizawa, Y. Ouchi, and T. Shimizu Platelet-Activating Factor Receptor Develops Airway Hyperresponsiveness Independently of Airway Inflammation in a Murine Asthma Model J. Immunol., June 1, 2004; 172(11): 7095 - 7102. [Abstract] [Full Text] [PDF]
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A. McKay, B. P. Leung, I. B. McInnes, N. C. Thomson, and F. Y. Liew A Novel Anti-Inflammatory Role of Simvastatin in a Murine Model of Allergic Asthma J. Immunol., March 1, 2004; 172(5): 2903 - 2908. [Abstract] [Full Text] [PDF]
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 M. A. Elovitz, Z. Wang, E. K. Chien, D. F. Rychlik, and M. Phillippe A New Model for Inflammation-Induced Preterm Birth: The Role of Platelet-Activating Factor and Toll-Like Receptor-4 Am. J. Pathol., November 1, 2003; 163(5): 2103 - 2111. [Abstract] [Full Text] [PDF]
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G. C. Koo, K. Shah, G. J. F. Ding, J. Xiao, R. Wnek, G. Doherty, X. C. Tong, R. B. Pepinsky, K.-C. Lin, W. K. Hagmann, et al. A Small Molecule Very Late Antigen-4 Antagonist Can Inhibit Ovalbumin-induced Lung Inflammation Am. J. Respir. Crit. Care Med., May 15, 2003; 167(10): 1400 - 1409. [Abstract] [Full Text] [PDF]
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A. Iwata, K. Nishio, R. K. Winn, E. Y. Chi, W. R. Henderson Jr., and J. M. Harlan A Broad-Spectrum Caspase Inhibitor Attenuates Allergic Airway Inflammation in Murine Asthma Model J. Immunol., March 15, 2003; 170(6): 3386 - 3391. [Abstract] [Full Text] [PDF]
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I. Kolleck, P. Sinha, and B. Rustow Vitamin E as an Antioxidant of the Lung: Mechanisms of Vitamin E Delivery to Alveolar Type II Cells Am. J. Respir. Crit. Care Med., December 15, 2002; 166(12): S62 - 66. [Abstract] [Full Text] [PDF]
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 L. Whittaker, N. Niu, U.-A. Temann, A. Stoddard, R. A. Flavell, A. Ray, R. J. Homer, and L. Cohn Interleukin-13 Mediates a Fundamental Pathway for Airway Epithelial Mucus Induced by CD4 T Cells and Interleukin-9 Am. J. Respir. Cell Mol. Biol., November 1, 2002; 27(5): 593 - 602. [Abstract] [Full Text] [PDF]
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T. NAGASE, S. ISHII, H. SHINDOU, Y. OUCHI, and T. SHIMIZU Airway Hyperresponsiveness in Transgenic Mice Overexpressing Platelet Activating Factor Receptor Is Mediated by an Atropine-Sensitive Pathway Am. J. Respir. Crit. Care Med., January 15, 2002; 165(2): 200 - 205. [Abstract] [Full Text] [PDF]
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N. R. HENIG, M. L. AITKEN, M. C. LIU, A. S. YU, and W. R. HENDERSON Jr. Effect of Recombinant Human Platelet-activating Factor-Acetylhydrolase on Allergen-induced Asthmatic Responses Am. J. Respir. Crit. Care Med., August 1, 2000; 162(2): 523 - 527. [Abstract] [Full Text] [PDF]
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