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Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Samuel C. Johnson Medical Research Building, Scottsdale, AZ 85259
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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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A growing body of literature now exists suggesting that the release of
eosinophil secondary (sometimes referred to as specific) granule
proteins (ESGPs) is critical to eosinophil-mediated activities and may
be responsible for many of the observed pathologies associated with
allergic respiratory disease. Potential deleterious effects of ESGPs
include not only activities leading to airway damage and lung
dysfunction (e.g., AHR) (10), but also direct effects on
several other cell types. On a molar basis, the most abundant human
ESGPs are eosinophil major basic proteins (MBPs). MBPs are small
cationic proteins (
12 kDa; pI 10–12) that colocalize with the
characteristic electron-dense crystalline core of eosinophil secondary
granules (11). In the mammalian orders examined to date,
including Primata (humans) and Rodentia (mice), MBPs are encoded by two
genes that share considerable sequence identity (12, 13, 14).
The relative function(s) of each MBP in a given species is not known.
For example, the reported differences in biological activities between
the human MBPs (hMBP-1 and -2) are nominal, and assessments of the
proteins stored in the granule show that nearly all the MBP in
eosinophils is hMBP-1 (10:1) (13). Moreover, many
clinical studies have shown that the deposition of hMBP-1 is a
diagnostic marker of the inflammation occurring in the lungs of
asthmatics, and accompanies airway epithelial damage, lung remodeling,
and perturbations of lung function (15).
The correlative association between AHR and MBP-1 (and therefore eosinophils) has been documented in several mammalian species, including humans (15, 16, 17, 18). Several studies have defined a potential causative mechanism resulting in AHR by linking eosinophil effector function, mediated by MBP-1 release, and muscarinic receptor dysfunction (17, 18, 19, 20). However, the link between eosinophils and AHR using mouse models of pulmonary inflammation is more tenuous and controversial. For example, many studies disassociate AHR from the recruitment (and presumably activation) of eosinophils to the lung. These studies can be divided into three groups: 1) studies that demonstrate that airway eosinophilia can occur without AHR (21); 2) mouse models that show that AHR occurs in the absence of a significant airways eosinophilia (22, 23, 24, 25, 26); and 3) studies that disassociate the kinetics of eosinophil recruitment to the lung and the development of AHR (27). Moreover, several studies in the mouse link AHR directly with T cell activities independently of eosinophil effector function (25, 27, 28, 29, 30, 31, 32). Therefore, while a preponderance of evidence suggests that eosinophil effector functions are responsible for Ag-induced AHR, data from in vivo studies using mouse models have, in some cases, been in conflict with this hypothesis.
To define the specific role(s) of mMBP-1 in the development of Ag-induced AHR, we created a gene knockout mouse line deficient for this protein. These mice display baseline perturbations of airway reactivity (relative to wild-type littermates) in response to methacholine, but show no homeostatic phenotype when airway reactivity is assessed by serotonin provocation. Furthermore, regardless of the nonspecific stimulus used to assay reactivity, the loss of mMBP-1 did not obviate Ag-induced AHR in an OVA sensitization/challenge model. These data suggest that although mMBP-1 expression, even at the low levels observed in naive mice, is sufficient to alter uniquely muscarinic receptor function(s), the AHR occurring in murine models of asthma is not causatively linked to mMBP-1 release from eosinophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A mMBP-1 gene targeting vector was constructed using genomic DNA
fragments flanking a 1.55-kb region that includes exons 2, 3, and 4 of
this gene (12). The targeting vector replaces these exons
with a neomycin resistance cassette (PGK-neo) and also
includes a herpes simplex virus thymidine kinase gene (HSV-TK) as a
negative selection marker (Fig. 1
A). GK-129 embryonic stem
(ES) cells (4 x 107) (33) were
electroporated with 25 µg of linearized DNA and subjected to positive
selection for PGK-neo with G418 (400 µg/ml) and negative
selection for the presence of the HSV-TK marker with gancyclovir (2
µM). Clones containing heterozygous disruptions of the gene were
identified by Southern blot analysis with a probe (Probe 1) flanking
the 5' homology region (i.e., not contained within the targeting
construct). The presence of a single integration event was verified by
hybridization with a neo-derived DNA fragment (Probe 2).
Gene-targeted cells were injected into C57BL/6J blastocysts to create
chimeric animals that were subsequently bred to 129/SvJ mice, allowing
the transmission of this allele on a 129 inbred background
(129/Ola/Hsdx129/SvJ). All mice were maintained in microisolator cages
housed in a specific pathogen-free animal facility. The sentinel cages
within this animal colony were negative for viral Abs and the presence
of known mouse pathogens. Protocols and studies involving animals were
conducted in accordance with National Institutes of Health and Mayo
institutional guidelines.
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A:
primer P1, 5'-ATGCCACTGTGAGACAGGGTAAGG-3'; primer P2,
5'-CAGATGAAGAGCAGACGCTC-3'; and primer P3, 5'-GAACCAGCTGGGGCTCGAG-3'.
Primers P1 and P2 anneal uniquely to the wild-type locus and produce an
874-bp PCR amplicon. Primers P1 and P3 anneal to the targeted locus and
produce a 683-bp PCR amplicon. Southern blots were prepared with
GeneScreen Plus membranes (NEN, Boston, MA) and hybridized with the
32P-labeled DNA probes identified in Fig. 1. Determination of mMBP-1 gene expression
RT-PCR to identify potential mMBP-1 transcripts from specific
tissues were performed using template cDNA derived from the reverse
transcription of 1 µg of total RNA (bone marrow, liver, lung, and
spleen). PCR were performed using a primer set from exon 2 (P4,
5'-TCTACTTCTGGCTCTTCTAGTCGGG-3') and exon 6 (P5,
5'-GACACAGTGAGATAGACGCCAGTG-3') to generate a 631-bp amplicon. The
reaction program consisted of an initial denaturation step of 94°C
for 5 min, followed by 30 cycles of 94°C (30 s), 60°C (1 min),
72°C (2 min), and a final 72°C (5-min) extension period. Parallel
PCR using primers derived from the 
-actin gene showed that all
samples of RNA were reverse transcribed.
Electron microscopy of peripheral eosinophils
Eosinophils for microscopy were generated from peritoneal cavity exudates of mice sensitized/challenged with Mesocestoides corti protein extract (12). Exudate cells (20–40% eosinophils) were collected by lavage and concentrated by low speed centrifugation (8 min/1000 rpm, 4°C). The leukocytes in this pellet were resuspended directly in Trump’s fixative (4% formaldehyde, 0.5% glutaraldehyde, and 84 mM NaH2PO4, pH 7.2) and stored at room temperature before application to electron microscopy grids.
OVA-induced allergic airway inflammation
Assessments of lung function and histopathology were performed on OVA-sensitized/saline-treated vs OVA-challenged 8- to 16-wk-old mice. All mice were immunized by two i.p. injections (100 µl) of OVA (20 µg; crude grade IV, Sigma) complexed with 2.25 mg of Imject Alum (Al(OH)3/Mg(OH)2; Pierce, Rockford, IL) on days 0 and 14. Sensitized mice were then challenged with an OVA aerosol (1% OVA prepared in saline) for 20 min on days 24, 25, and 26 using an ultrasonic nebulizer (De Vilbiss, Somerset, PA); control animals received a saline-only aerosol. The mice were assessed for pulmonary cellular infiltrates, histopathologies, and lung function on day 28.
Assessments of allergic airway inflammation: pulmonary cellular infiltrates and histological changes in the lung
The methods and analyses assessing cellular infiltrates
associated with bronchoalveolar lavage (BAL) and collagen digests of
whole lungs were described previously (34).
Histopathologic changes in the lungs, including mucus cell content of
the airway epithelium, were assessed from tissues excised and fixed in
10% formalin (lungs were inflated with a fixed volume (0.5 ml) of
fixative). The lung samples were washed free of formalin with 1x PBS
and subsequently dehydrated through an ethanol series before
equilibration in xylene and embedding in paraffin. Sections (4 µm)
were mounted on slides and stained with either periodic acid-Schiff
(counterstained with hematoxylin/methylgreen) or alcian blue, pH 2.5
(counterstained with nuclear fast red). Parasagittal sections were
analyzed by brightfield microscopy. The mucus content in the airway
epithelium of mice from different groups was based on the evaluation of
40 airways (both proximal and distal)/mouse (n = 5
animals/group). Relative comparisons of mucus content were made between
cohorts of animals using an imaging program (Image ProPlus) quantifying
the data as an airway mucus index: [(average alcian blue staining
intensity of the airway epithelium) x (area of airway epithelium
staining with alcian blue)]/[(total area of the conducting airway
epithelium) x (total number of airways assessed)].
Immunohistochemical detection of eosinophils in paraffin-embedded tissue
Sections of lung tissue (4 µm) were assessed for the infiltration of eosinophils using mAbs specific for individual secondary granule proteins. mAbs were raised against native mMBP-1 and the collective group of murine eosinophil-associated RNases (mEARs) using protein purified from peripheral blood eosinophils derived from an IL-5 transgenic line of mice (35). Individual Abs were selected on the basis of reactivity to native protein in an ELISA format as well as their utility as a detection reagent using formalin-fixed paraffin sections. The specificity of the mMBP-1 (rat mAb 14.7.4) and mEARs (rat mAb 32.1.3) protein A-Sepharose-purified Abs was determined by competition assays with purified native protein. Immunohistochemical staining was performed with diaminobenzidene-peroxidase detection reagents according to the manufacturer’s instructions (Vector, Burlingame, CA). Briefly, lung sections were deparaffinized and washed in PBS (1x PBS), and endogenous peroxidase activity was quenched in a methanol buffer (80% methanol/0.6% hydrogen peroxide) in preparation for Ab staining. Methanol-peroxide-treated sections were washed with 1x PBS, digested (10 min, 25°C) with pepsin (Zymed, San Francisco, CA), and blocked by incubation at room temperature in 1.5% normal goat serum for 30 min. The blocked sections were treated with the rat mAbs at final concentrations of 0.4 µg/ml (diluting the Abs in 1.5% normal goat serum/PBS for 1 h at room temperature). The slides were subsequently washed free of primary Ab with several changes of 1x PBS, and a secondary reagent (polyclonal rabbit anti-rat IgG Abs conjugated to HRP (Dako, Carpinteria, CA)) was bound at 13 µg/ml (room temperature, 1 h) before histochemical development using diaminobenzidene-peroxidase. The sections were counterstained with methyl green in preparation for photomicroscopy.
Determination of airway reactivity in response to provocation with nonspecific stimuli
AHR was assessed by inducing airflow obstruction with either a methacholine or serotonin aerosol using a noninvasive protocol (34, 36). This methodology uses unrestrained conscious mice that are placed into the main chamber of a plethysmograph (Buxco Electronics, Troy, NY). Pressure differences between this chamber and a reference chamber were used to extrapolate minute volume, tidal volume, breathing frequency, and enhanced pause (PENH). PENH is a dimensionless parameter that is a function of total pulmonary airflow in mice (i.e., the sum of the airflows in the upper and lower respiratory tracts) during the respiratory cycle of the animal. This parameter closely correlates to lung resistance as measured by traditional invasive techniques using ventilated animals (36). Dose-response data were plotted as the percent baseline PENH vs the log10 of the methacholine or serotonin solution (milligrams per milliliter) used to generate the aerosol. There were no statistically significant differences (Tukey-Kramer highest significant difference test) in observed baseline PENH values (±SEM) between each cohort of animals used in these studies: wild-type (+/+) saline, 0.70 ± 0.07; wild-type (+/+) OVA, 0.63 ± 0.08; mMBP-1-/- saline, 0.51 ± 0.02; and mMBP-1-/- OVA, 0.52 ± 0.04.
Statistical analyses
Pairs of groups were compared using Student’s t tests. The p value for significance was set at 0.05, and values for all measurements are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported the identification and cloning of mMBP-1,
the murine orthologue of hMBP-1 (12). Genomic clones
representing this locus were used to develop a targeting construct
designed to replace exons 2, 3, and 4 of the mMBP-1 gene with a
neomycin resistance cassette (Fig. 1A). These exons include
the signal sequence associated with protein secretion, the
propolypeptide precursor that is proteolytically cleaved, and the
N-terminal 54 aa of the mature mMBP-1 protein stored in the secondary
granule. The targeting construct was electroporated into GK129 ES
cells, and clones heterozygous for the induced mMBP-1 mutation were
identified by Southern blot as shown in Fig. 1B. Targeted ES
cell clones were injected into C57BL/6J blastocysts, and
highly chimeric male mice were bred with 129/SvJ females to generate
germline mice. Homozygous mMBP-1 knockout mice on a 129 background were
generated by interbreeding of heterozygous siblings (Fig. 1C). We have not observed any anomalies associated with the
breeding of mMBP-1-/- mice. Homozygous animals
were produced from these sibling intercrosses at the expected Mendelian
frequency, and matings of homozygous mice produced litters of a size
and frequency equivalent to wild-type or heterozygous animals.
Gene activity was assessed using RT-PCR in the lung, liver, spleen, and
bone marrow. These data show that homozygous mutant mice did not
accumulate mMBP-1 transcripts in any of the tissues examined (Fig. 1D). Expression of the mMBP-1 gene was restricted to
wild-type and mMBP-1+/- mice, and as reported
previously (35), steady state transcripts were limited to
known sites of eosinophilopoiesis (i.e., bone marrow and spleen).
Loss of mMBP-1 has ultrastructural effects on eosinophil secondary granules
The number of secondary granules in mature eosinophils (derived
from the peritoneal cavity of mice sensitized/challenged with a
parasite (Mesocestoides corti) protein extract
(12)) appeared to be unaffected by deletion of the mMBP-1
gene. The average number of granules per cross-sectional area, as
determined by electron microscopy, was the same in wild-type (29.9
± 3.6) and mMBP-1-/- (32.1 ± 3.8)
eosinophils. In addition, the size of the granules in each group of
mice (i.e., cross-sectional area) was indistinguishable. However,
Wright-Giemsa-stained mMBP-1-/- eosinophils
lacked much of the intense color associated with the secondary granules
of these cells, suggesting that although the granules were present,
they no longer had enough cationic character to bind eosin efficiently
(data not shown). The identification of secondary granules in
eosinophils is based primarily on electron microscopy showing the
presence of an electron-dense crystalline core uniquely associated with
this granule. In human eosinophils, electron immunogold staining using
an anti-hMBP-1 Ab has shown that hMBP-1 localizes to the
crystalline core (11). An examination of electron
micrographs of eosinophils recovered from the peritoneal cavity of
M. corti-sensitized/challenged mice revealed that
mMBP-1-deficient animals lack the electron-dense core associated with
secondary granules (Fig. 2). This
observation shows that the core structure in mouse eosinophil
granules is dependent on the presence of mMBP-1, but does not exclude
the possibility that the core may also be dependent on other molecules
contained within the secondary granule, including the other MBP gene
family member (i.e., mMBP-2). It is also noteworthy that the secondary
granules of heterozygous animals contain electron-dense cores that were
identical with the granule cores from wild-type mice. Although we have
no quantitative data with regard to mMBP-1 production in heterozygous
mice, this observation suggests that core formation is not sensitive to
the 50% decrease in mMBP-1 synthesis that one might expect in
mMBP-1+/- mice.
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The importance of mMBP-1 to eosinophil effector function was
assessed using an established OVA model of allergic pulmonary
inflammation, which included i.p. sensitization of the mice with OVA
and an adjuvant, followed by several OVA aerosol challenges of the lung
(see Materials and Methods). Fig. 3A compares the number and
composition of leukocytes found in the BAL fluid of wild-type and
mMBP-1-/- mice in response to an aerosol
challenge with saline or OVA. Whereas aerosol challenge with saline
alone had no effect on the homoeostatic levels of cells in the lumen of
either wild-type or mMBP-1-/- mice (i.e.,
eosinophils were not recruited to the lumen and comprise <1% of the
cells in the airway), the pulmonary inflammation associated with OVA
challenge in both wild-type and mMBP-1-/- mice
was accompanied by the selective recruitment of eosinophils to the
airway lumen. The recruitment of eosinophils to the airways of
OVA-challenged mMBP-1-/- mice was
indistinguishable from that in wild-type littermates. Eosinophil
trafficking to the interstitial regions of the lung in
mMBP-1-/- mice was also unaffected relative to
that in wild-type littermates, as differential counts of leukocytes
recovered from collagen-digested whole lungs showed equal numbers of
eosinophils recruited to the lung in response to OVA challenge (Fig. 3B).
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). These data also demonstrated that
mMBP-1-containing eosinophils are a homeostatic resident population
within the parenchyma of wild-type mice at baseline (i.e., OVA
sensitized/saline challenged; Fig. 4A), that increases
dramatically in response to an OVA aerosol provocation (Fig. 4B). In contrast, this mAb failed to detect the presence of
mMBP-1-containing eosinophils in the lungs of knockout mice, either at
baseline (Fig. 4C) or after an OVA aerosol provocation
(i.e., OVA-sensitized/OVA-challenged; Fig. 4D). However, the
spatial distribution of eosinophils within the pulmonary interstitium
of mMBP-1-/- mice was nonetheless unaffected as
a consequence of the absence of mMBP-1. Recruited eosinophils were
identified in knockout mice by immunohistochemistry using a rat mAb
specific for another eosinophil secondary granule protein group, the
mEARs (rmAb 32.1.3). These data demonstrated that eosinophils were
recruited to perivascular and peribronchial regions of the lungs of
both OVA-treated wild-type and mMBP-1-/- mice
with no difference in the relative distribution of these cells between
either group of mice (Fig. 4, E and F).
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OVA-treated wild-type and mMBP-1-/- mice
each displayed the same airway changes, including
bronchiolar-associated leukocyte aggregates, airway epithelial cell
hypertrophy, and goblet cell hyperplasia (Fig. 5, A and C, vs Fig. 5, B and D, respectively). Furthermore, the
periodic acid-Schiff-stained sections of Fig. 5 also show that airway
epithelial mucus content of both wild-type and
mMBP-1-/- mice increased dramatically in
response to OVA sensitization/challenge. A quantitative assessment of
airway epithelial mucus demonstrates that the extent of OVA-induced
mucus production in wild-type and mMBP-1-/-
mice is identical (Fig. 5E). In summary,
mMBP-1-/- mice displayed no differences in
OVA-induced pulmonary histopathology relative to wild-type animals,
suggesting that many, if not all, histologic changes associated with
allergic inflammation in mouse models are not contingent upon the
release of mMBP-1 from eosinophils recruited to the lung.
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The relationship between the development of AHR and mMBP-1
expression was initially examined by assessing baseline methacholine
responsiveness in naive animals. Surprisingly, the methacholine
dose-response curves (means of single-animal measurements) showed that
the threshold dose for methacholine-induced airflow changes in
wild-type mice was lower than the dose required to induce equivalent
changes in knockout mice (Fig. 6A). Moreover, the absolute
magnitude of these methacholine-induced changes was significantly
higher in wild-type mice relative to mMBP-1-/-
littermates. This baseline phenotype, however, was observable only with
methacholine; no airflow changes were observed between wild-type and
mMBP-1-/- mice in response to another
nonspecific stimulus (serotonin provocation; Fig. 6B). The
unique ability of methacholine to induce airflow perturbations in naive
mMBP-1-/- mice suggests that mMBP-1 is a
homeostatic ligand of one or more pulmonary muscarinic receptors.
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compiles data
comparing either methacholine (A) or serotonin
(B) dose-dependent reactivity changes in
OVA-sensitized/challenged wild-type and
mMBP-1-/- mice (n =
8–10/group) relative to control groups. In response to methacholine
provocation (Fig. 7A), three striking differences regarding
the absolute values of the responses obtained from each genotype are
apparent. 1) Saline-treated mMBP-1-/- mice were
hyporeactive relative to saline-treated wild-type mice (effective dose
200% (ED200) = 36.2 ± 6.9 vs
12.5 ± 1.1 mg/ml, respectively; p < 0.03),
confirming that in the absence of mMBP-1 the observed baseline
reactivity in response to methacholine is attenuated relative to that
in wild-type animals. 2) The threshold dose for methacholine-induced
airflow changes in OVA-sensitized/challenged
mMBP-1-/- mice was also two to three times the
amount of methacholine necessary to produce the same changes in
OVA-treated wild-type mice (ED200 = 11.0 ±
1.6 vs 4.3 ± 0.2 mg/ml, respectively; p <
0.03)). 3) Airway reactivity in response to the methacholine dose
inducing the maximal observed PENH difference between wild-type and
mMBP-1-/- mice (i.e., 25 mg/ml) is markedly
less (50% decrease) in both saline- and OVA-treated
mMBP-1-/- mice relative to the responses
observed in wild-type mice (p < 0.02). The
implication of these differences is that the relative shift in the
methacholine dose-response curves is the same in both genotypes. OVA
provocation (relative to a genotype-matched saline control group) of
either wild-type (ED200 = 4.3 ± 0.2 vs
12.5 ± 1.1 mg/ml, respectively; p < 0.01) or
mMBP-1-/- (ED200 =
11.0 ± 1.6 vs 36.2 ± 6.9 mg/ml, respectively;
p < 0.01) mice resulted in an 60% decrease in the
threshold dose for methacholine-induced airflow changes. Thus,
allergen-induced increases in methacholine-mediated airflow changes
(i.e., AHR) occur in the absence of mMBP-1. This lack of an effect on
allergen-induced airflow changes in mMBP-1 knockout mice was confirmed
using serotonin as an alternative nonspecific stimulus (Fig. 7B). OVA-sensitized/challenged wild-type and
mMBP-1-/- mice each displayed identical
increases in serotonin-induced airflow changes relative to naive
control groups of mice, which displayed no differences in their
baseline responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This apparent difference observed between MBP-1 effector function in patients with asthma and the murine models may reflect the chronic inflammatory character of this disease state in humans. Human MBP-1 deposition probably occurs over long periods of time (i.e., months/years) and cumulatively correlates with both AHR and histopathologic airway epithelial changes, whereas extensive (and chronic) eosinophil degranulation does not occur in the 96-h challenge interval of most OVA protocols. However, our unpublished data assessing murine vs human eosinophil respiratory burst and release of EPO in vitro demonstrate that mouse eosinophil degranulation is greater than an order of magnitude less under conditions that elicit this response from human cells. These data imply that the incongruities between human patients and mouse models of asthma are probably not an issue of the OVA model, but reflect species differences in eosinophil effector function. We would propose that degranulation occurs more readily in human eosinophils and is required, because of unique effector functions that were/are evolutionarily advantageous to primates and not rodents. The resolution of this quandary will probably have significant implications regarding other roles of eosinophils in mouse models of human disease, as some, but not all, effector functions will have been conserved during mammalian evolution.
The data presented here do not preclude the participation of other ESGPs in the development of Ag-induced lung dysfunction. However, the demonstration that OVA-induced AHR in the mouse occurs independently of mMBP-1 suggests a parsimonious explanation of previous studies that showed a disassociation of eosinophil effector function and AHR; Ag-induced AHR in mouse models of asthma may not be causatively linked to the release of ESGPs. Studies in which AHR occurred in the absence of a significant airways eosinophilia (22, 23, 24, 25) or that disassociated AHR from eosinophil recruitment to the lung (27) were apparently assessing an mMBP-1-independent phenomena. This lack of an observed effect mediated by the release of the most abundant ESGP suggests that AHR is either dependent on other less abundant ESGPs, eosinophil effector functions other than degranulation, or is a pathophysiologic response mediated by the activity of another cell type(s). As noted in several studies, activated CD4+ T cells recruited to the lung in response to allergen challenge were likely candidates to mediate eosinophil-independent effects (25, 30, 31). Furthermore, genetic studies linking AHR and T cells (29, 32) or studies linking AHR and T cell-derived cytokines that have little or no direct effect on eosinophil proliferation and/or function (e.g., pulmonary expression of IL-13) (27, 28), further support a relationship between AHR and T cells. The implicit conclusion that arises is that a direct causative link between eosinophil degranulation (i.e., the release of ESGPs such as mMBP-1) and the development of AHR remains an elusive, and potentially unattainable, goal in the mouse.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, MCP Hahnemann University, 2900 Queen Lane, Philadelphia, PA 19129.
3 Address correspondence and reprints requests to Dr. James J. Lee or Dr. Nancy A. Lee, Department of Biochemistry and Molecular Biology, SCJMRB-RESEARCH, Mayo Clinic Scottsdale, 13400 East Shea Boulevard, Scottsdale, AZ 85259.
4 Abbreviations used in this paper: AHR, airway hyper-responsiveness; mMBP-1, murine major basic protein-1; hMBP-1, major basic protein-1; human ESGP, eosinophil secondary granule protein; PGK-neo, neomycin resistance cassette; HSV-TK, herpes simplex virus thymidine kinase gene; +/+, wild-type; +/-, heterozygous; -/-, homozygous; EAR, eosinophil-associated RNase; BAL, bronchoalveolar lavage; PENH, enhanced pause; ED200, effective dose 200%; ES, embryonic stem.
Received for publication May 22, 2000. Accepted for publication August 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J. J. Lee, D. Dimina, M. P. Macias, S. I. Ochkur, M. P. McGarry, K. R. O'Neill, C. Protheroe, R. Pero, T. Nguyen, S. A. Cormier, et al. Defining a Link with Asthma in Mice Congenitally Deficient in Eosinophils Science, September 17, 2004; 305(5691): 1773 - 1776. [Abstract] [Full Text] [PDF]
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H. W. J. Young, J. G. Molina, D. Dimina, H. Zhong, M. Jacobson, L.-N. L. Chan, T.-S. Chan, J. J. Lee, and M. R. Blackburn A3 Adenosine Receptor Signaling Contributes to Airway Inflammation and Mucus Production in Adenosine Deaminase-Deficient Mice J. Immunol., July 15, 2004; 173(2): 1380 - 1389. [Abstract] [Full Text] [PDF]
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 K. Clark, L. Simson, N. Newcombe, A. M. L. Koskinen, J. Mattes, N. A. Lee, J. J. Lee, L. A. Dent, K. I. Matthaei, and P. S. Foster Eosinophil degranulation in the allergic lung of mice primarily occurs in the airway lumen J. Leukoc. Biol., June 1, 2004; 75(6): 1001 - 1009. [Abstract] [Full Text] [PDF]
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E. Forbes, T. Murase, M. Yang, K. I. Matthaei, J. J. Lee, N. A. Lee, P. S. Foster, and S. P. Hogan Immunopathogenesis of Experimental Ulcerative Colitis Is Mediated by Eosinophil Peroxidase J. Immunol., May 1, 2004; 172(9): 5664 - 5675. [Abstract] [Full Text] [PDF]
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macrophage; Lym, lymphocyte; Eos, eosinophil; Neu, neutrophil; Mono,
monocyte. *, p < 0.05,
wild-type/mMBP-1-/- saline control groups vs OVA-treated
mice.
