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Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, AZ 85259;
Department of Cell Biology, Cleveland Clinic Foundation, Lerner Research Institute, Cleveland, OH 44195; and
Allergic Diseases Research Laboratory, Departments of Immunology and Medicine, Mayo Clinic, Rochester, MN 55905
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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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The prominent effector functions mediated by eosinophils appear to result from the release of abundant cationic proteins from morphologically distinct cytoplasmic granules through a process known as degranulation (18). The most abundant of these granule proteins (by mass) is a cationic heme-containing protein identified as eosinophil peroxidase (EPO).3 EPO appears to be specific for the eosinophil cell type and represents nearly 25% of the total protein mass of the secondary granule (19). The release of EPO, together with the generation of the reduced oxygen species (i.e., superoxide and hydrogen peroxide) resulting from the respiratory burst of activated eosinophils (20), provides a potent defense mechanism (e.g., peroxidase-mediated generation of hypohalous acids (21)) capable of killing invading pathogens such as bacteria (22) and multicellular helminth parasites (23, 24). Moreover, EPO activities have been shown to be potentially immunoregulative, triggering mast cell degranulation (25), activating platelets (26), and increasing the respiratory burst/phagocytic capabilities of macrophages (27). A series of provocative studies examining the biochemistry of EPO activity also suggested that the release of this granule protein during allergic inflammatory responses results in oxidative damage of resident lung proteins by nitration or bromination of tyrosine residues (28, 29). The generation of 3-bromotyrosine is particularly important because this was shown to be an EPO-specific product that increases >10-fold in bronchoalveolar lavage (BAL) fluid recovered from asthmatic, but not healthy control, subjects after segmental allergen challenge (30). Moreover, nearly 100-fold increases in 3-bromotyrosine content were observed in BAL fluid recovered from subjects with severe asthma who had been admitted to an intensive care unit compared with nonasthmatic patients (31).
Cases of EPO deficiency (Presentey’s anomaly) have been documented in a number of patients and are characterized by the absence or attenuation of cytochemical staining for eosinophil-derived peroxidase and a significant loss in the volume of the eosinophil secondary granule occupied by the electron-translucent matrix (32). However, notable clinical symptoms were absent in these cases, as the identification of EPO-deficient patients occurred randomly during studies unrelated to asthma or allergic disease (33). A similar EPO deficiency was also described in a unique substrain of New Zealand White (NZW) mice (34). This deficiency in NZW mice led to comparable effects (i.e., relative to human EPO-deficient patients) on the electron microscopic ultrastructure of eosinophil secondary granules. Unfortunately, the random genetic aberration occurring in NZW mice is not well characterized and may include perturbations of other loci in addition to EPO.
We have created a null allele at the EPO locus in the mouse to define specific effector functions mediated by this eosinophil granule protein during allergic pulmonary disease. Eosinophils from homozygous null EPO mice (EPO-/-) were shown to be deficient of peroxidase activity and displayed the same decrease in the electron-translucent matrix of the secondary granules observed in both NZW mice and EPO-deficient patients. OVA sensitization/aerosol challenge of wild-type and EPO-/- mice demonstrated that the EPO deficiency had no effects on the development of airway pathologies including OVA-induced airway hyperresponsiveness (AHR) following methacholine provocation. Furthermore, the extent of EPO-dependent modification of BAL proteins from OVA-challenged mice was <10% of the values reported from humans following either allergen challenge or asthma-related respiratory failure. The lack of visual evidence of eosinophil degranulation in mouse OVA models, together with no observable differences (i.e., relative to wild-type littermates) in the OVA-mediated pathologies occurring in EPO-/- mice or mice deficient for major basic protein (MBP)-1 (35), suggest that the release of eosinophil secondary granule proteins (ESGPs) is not a prominent effector function in mouse models of respiratory inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An EPO gene-targeting vector was constructed using
129/SvJ-derived genomic DNA flanking a 3.9-kb fragment containing exons
6, 7, and 8 of this gene (36). The targeting vector
replaces these exons with a neomycin resistance cassette (PGK-neo) and
also includes the diphtheria toxin A gene as a negative selection
marker (Fig. 1
A). The
targeting construct (25 µg) was linearized and introduced into 4
x 107 GK-129 embryonic stem (ES) cells
(37) via electroporation. Transfectants were selected on
the basis of growth in the presence of 400 µg/ml G418. Surviving
GK-129 ES cell clones were analyzed by Southern blot of
SpeI-digested DNA to identify heterozygous disruptions of
the EPO gene using a probe flanking the 5' homology region
of the targeting vector (probe 1). A single integration event was
verified by Southern blot of BamHI/BclI-digested
DNA using a probe derived from the neomycin resistance gene (probe 2;
data not shown). Targeted GK-129 ES cells from clones 6 and 57 were
injected into C57BL/6J blastocysts to generate chimeric animals that
were bred to 129/SvJ mice, allowing the transmission of this allele on
a 129-inbred background (129/Ola/Hsd x 129/SvJ). Mice were
maintained in microisolator cages housed in a specific pathogen-free
facility. The sentinel cages within this animal colony surveyed
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 Foundation institutional
guidelines.
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A. The wild-type allele was identified as a 750-bp PCR
amplicon using sequence information derived from exon 8 (primer 1
(P1), 5'-TGAAACCCCCAAACTGACGG-3') and exon 9
(primer 2 (P2), 5'-ACAGAGCTAAGCGGGACGTG-3'). The
targeted EPO locus was identified as a 1000-bp PCR amplicon
using P2 and a primer derived from the
neomycin-resistance gene (primer 3 (P3),
5'-CATCGAGCGAGCACGTACTC-3'). PCRs were assembled in 100-µl
volumes using the GeneAmp XL-PCR kit (PerkinElmer Applied Biosystems,
Foster City, CA) and a cycling strategy of 94°C for 5 min followed by
30 cycles of 94°C (1 min), 57°C (2.5 min), 72°C (2 min), and a
final extension of 72°C for 7 min. RNA isolation and RT-PCR analysis
Total RNA was prepared from bone marrow, liver, lung, and spleen
of +/+, EPO+/-, or
EPO-/- animals by homogenization in a
guanidinium isothiocyanate buffer followed by acid phenol extraction
and ethanol precipitation (38). RNA samples were treated
with RNase-free DNase I (RQ1; Promega, Madison, WI) for 30 min at
37°C, followed by phenol/chloroform extraction and ethanol
precipitation before reverse transcription reactions. Total RNA (1
µg) was reverse transcribed with SuperScript II reverse transcriptase
(Life Technologies, Rockville, MD) in the presence of anchored
oligo(dT) primers (dT16VN where
V = A, C, or G and N = A, C, G, or T)
according to the manufacturer’s instructions. PCRs were performed
using cDNA derived from one-tenth of the reverse transcriptase
reactions (i.e., cDNA derived from 100 ng total RNA) in 50-µl
reactions with 1 µM of EPO-specific primers and 5 U
Taq polymerase (Boehringer Mannheim, Indianapolis, IN).
EPO transcripts were identified in the samples as a 263-bp
amplicon. The location and orientation of the RT-PCR primers
(P1 and P4,
5'-TGAAAACATTGGCTACACGG-3') are shown in Fig. 1A. A
-actin primer set (Stratagene, La Jolla, CA) was used as a positive
control for the RT-PCR. All PCRs were performed in duplicate using 100
ng total RNA in the absence of reverse transcription as a negative
control (data not shown). The PCRs were conducted using a thermocycle
profile of 94°C for 5 min followed by 30 cycles of 94°C (30 s),
58°C (2 min), 72°C (3 min), and a final extension of 72°C for
10 min.
Induction of peritoneal cavity eosinophilia
Large numbers of eosinophils (>2 x 106, 20–30% eosinophils) were induced in the peritoneal cavity of mice via a sensitization/challenge protocol using a whole-protein extract from the helminth Mesocestoides corti, MCA) (39). Briefly, +/+, EPO+/-, or EPO-/- animals were initially sensitized with a 400-µl s.c. injection of 8 x 109 killed pertussis organisms (Michigan Department of Public Health, Lansing, MI) and 250 µg MCA suspended in sterile saline. Twenty-one days later mice were challenged with a 300-µl i.p. injection of 200 µg MCA in sterile saline. Peritoneal cavity cells were harvested 3 days later by lavage with 1x PBS, 5% FCS, and 20 U/ml heparin. Recovered leukocytes were washed several times by centrifugation/resuspension in 1x PBS before use in subsequent experiments. Differential counts of cytospin preparations of these cells reveal that neutrophils composed <1% of all samples examined.
Cytochemical detection of EPO
Cytospin preparations of peritoneal cavity cells harvested from MCA-sensitized/challenged mice were prepared using a Cytospin3 (Shandon Scientific, Cheshire, U.K.). The slides were subsequently fixed for 30 s at 4°C in a formalin-acetone buffer (0.75 mM Na2HPO4, 7.5 mM KH2PO4 (pH 7.5), 45% (v/v) acetone, and 10% (w/v) formaldehyde), washed in tap water for several minutes, and stained (10 min at room temperature) for EPO activity using a diaminobenzidine (DAB)-H2O2 phosphate buffer (6 mM Na2HPO4, 286 mM KH2PO4 (pH 7.4), 2 mM (3',3')-DAB tetrahydrochloride (Sigma, St. Louis, MO), 0.01% H2O2 (Sigma), and 8 mM NaCN (Aldrich, Milwaukee, WI)) as described earlier (36). The concentration of CN- included in this reaction buffer was selected to differentially inhibit the activity of myeloperoxidase found in other leukocyte types that may be present. The staining reactions were terminated by washing under tap water, and the slides were counterstained with Harris hematoxylin (Sigma) before dehydration through an ascending series of ethanol washes, incubation in xylene, and mounting with Permount (Fisher Scientific, Pittsburgh, PA).
Endpoint colorimetric assessment of EPO activity
The amount of EPO activity contained within cells or recovered fluids was determined by an endpoint colorimetric assay, as described previously (40). To determine the amount of EPO activity within cells, peritoneal cavity cells and/or leukocytes recovered from BAL fluid were washed in Kreb’s buffer (118 mM NaCl, 25 mM NaHCO3, 11 mM glucose, 1 mM NaH2PO4·H2O, 5 mM KCl, 0.5 mM MgCl2·6H2O, and 2.5 mM CaCl2·6H2O (pH 7.4)) and counted on a hemocytometer. The number of eosinophils in a given leukocyte population was determined by differential cell counts of cytospin preparations. Dilutions ranging from 102 to 104 eosinophils were seeded in 50-µl volumes in a 96-well microtiter plate, and EPO activity was measured in each lysate through the addition of 75 µl o-phenylenediamine-H2O2 buffer (50 mM Tris-HCl (pH 8), 0.1% Triton X-100, 4 mM H2O2, and 10 mM of the EPO (vs myeloperoxidase)-specific substrate o-phenylenediamine). The reactions were allowed to proceed at room temperature for 30 min before the addition of 50 µl stop buffer (2N H2SO4). Absorbance of each sample was measured at 490 nm using a Vmax Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA). EPO activity present in BAL fluid was assessed by incubating undiluted cell-free BAL fluid with o-phenylenediamine-H2O2 buffer at 37°C for 30 min (41). Colorimetric readings due to EPO activity were calculated as the difference in absorbance measurement of each reaction with a duplicate reaction conducted in the presence (30 mM) of the peroxidase inhibitor 3-amino-1,2,4-triazole. The fraction of EPO released from airway eosinophils was estimated by measuring the amount of EPO activity in cell-free BAL fluid as a function of total EPO activity in the BAL (i.e., the sum of EPO in the BAL fluid and EPO associated with airway eosinophils). EPO activity was calculated from a standard curve established using 1 x 102-5 x 105 eosinophils purified as a homogeneous population (99% pure) from an IL-5-transgenic mouse line (42). The A490 reading of each sample (i.e., the BAL fluid and BAL cell pellet derived from each mouse) was used to obtain the equivalent amount of EPO contained in a known number of eosinophils as determined from this standing curve.
Electron microscopy of leukocytes
Peritoneal cavity exudates of mice sensitized/challenged with M. corti protein extract were collected by lavage and concentrated by low speed centrifugation (1000 rpm, 4°C). The leukocytes of this low speed pellet were resuspended directly in Trump’s fixative (4% formaldehyde, 0.5% gluteraldehyde, and 84 mM NaH2PO4 (pH 7.2)) and stored at room temperature before application on electron microscopy grids.
Induction of allergic airway inflammation
Wild-type and EPO-/- mice littermates, age 8–12 wk, were sensitized by two i.p. injections of either saline or 20 µg OVA (grade IV, Sigma)-2.25 mg Imject Alum (Al(OH)3-Mg(OH)2; Pierce, Rockford, IL) in 100 µl saline on days 0 and 14. Mice were challenged on days 24, 25, and 26 by 20-min inhalations of an aerosol generated by nebulization of a 1% OVA solution prepared in saline (control mice received 20-min aerosol challenges of saline). Mice were assessed for pulmonary cellular infiltrates, histopathologies, and lung function on day 28.
Assessments of allergic airway inflammation: pulmonary cellular infiltrates and Ag-induced histopathologies
The number and specificity of cell types recruited to the
airspaces were determined by BAL as previously described
(43). Histopathologic changes to the airways and
parenchyma, including mucus cell content of the epithelium, were
assessed from lungs excised and fixed in 10% buffered formalin
overnight at 4°C (lungs were inflated with a fixed volume (0.5 ml) of
fixative). The lung samples were washed free of formalin with 1x PBS
and dehydrated through an ascending ethanol series before equilibration
in xylene and embedding in paraffin. Parasagittal sections (4 µm)
were stained with periodic acid-Schiff reagent (PAS) and counterstained
with hematoxylin-methyl green to assess mucus production and goblet
cell metaplasia. Three sections per lung were examined, and the mucus
content of 
40 airways (proximal and distal) per section were
measured from groups of six animals. An imaging program (Image ProPlus;
Media Cybernetics, Silver Spring, MD) was used to quantify the area and
intensity of PAS staining per airway. The data were quantified as a
mucus index: [(average PAS staining intensity of the airway
epithelium) x (area of airway epithelium staining with
PAS)/(total area of the conducting airway epithelium) x (total
number of airways assessed)].
Immunocytochemical detection of eosinophils
Sections of lung tissue (4 µm) were assessed for the infiltration of eosinophils using a rat mAb (14.7.4) specific for murine MBP-1 (35). Immunocytochemical staining was performed with DAB-peroxidase detection reagents (Vector Laboratories, Burlingame, CA) as described previously (35).
Determination of bromotyrosine levels in the lung
The 3-bromotyrosine content of proteins recovered from the BAL and whole-lung homogenates of mice was determined (relative to the precursor amino acid, i.e., tyrosine) by stable isotope-dilution gas chromatography/mass spectrometry (44). Sample preparation and specific methodologies used were as described earlier (29, 30). The 3-bromotyrosine levels in human subjects were derived from data of earlier clinical studies assessing oxidative damage of pulmonary proteins occurring in asthma patients (30, 31).
Assessment of AHR in response to methacholine challenge
AHR was assessed by inducing airflow obstruction with a methacholine aerosol using a noninvasive protocol (43, 45). 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 was shown to closely correlate with airway resistance as measured by traditional invasive techniques using ventilated animals (43). Dose-response data were plotted as the percent baseline PENH vs the log10 of the methacholine solution (mg/ml) used to generate the aerosol. There were no statistically significant differences (Tukey-Kramer honestly significant difference test) observed among the baseline PENH values (± SE) of the different animal groups used in these studies: wild-type (+/+) saline, 0.51 ± 0.03; wild-type (+/+) OVA, 0.49 ± 0.03; EPO-/- saline, 0.52 ± 0.04; and EPO-/- OVA, 0.55 ± 0.05.
Statistical analyses
Unless otherwise indicated, 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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Mouse eosinophils have been shown to express, and subsequently
store in secondary granules, a peroxidase that is uniquely associated
with this cell type (36). This ESGP is encoded by a single
gene that represents the mouse orthologue of human EPO (36, 46). A targeting construct was generated from 129/SvJ
-genomic clones representing the EPO locus and designed to replace
exons 6, 7, and 8 of this gene with a neomycin-resistance cassette
(Fig. 1A). These exons contain the conserved histidine and
arginine residues essential to catalytic activity and are present in
all members of this gene superfamily (36, 47, 48).
Moreover, these exons also contain six half-cystine residues involved
in disulfide linkages and 11 residues whose location within 4.5 
of the heme prosthetic group suggest that they are essential to
stabilize this motif (49). This targeting construct was
transfected into GK-129 ES cells, and heterozygous disruptions of the
EPO gene were identified by Southern blot as shown in Fig. 1B. Targeted ES cell clones were injected into C57BL/6J
blastocysts, and the resulting chimeric males were bred to 129/SvJ
females to transmit this mutant allele through the germline. Homozygous
EPO-knockout mice were generated by sibling intercross (Fig. 1C) and, thus, remained on a pure 129 genetic background.
The targeted EPO gene was transmitted to offspring at the
expected Mendelian frequency, and matings of
EPO-/- mice produced litters of a size
and frequency equivalent to wild-type or heterozygous animals. All
studies described in this report were performed with
EPO-/- and wild-type siblings derived
from matings of EPO+/- mice.
RT-PCR was used to assess gene expression in
EPO-/- mice and to confirm that these
animals did not express EPO transcripts. Fig. 1D
shows that expression of EPO was restricted to wild-type and
EPO+/- mice and occurred only in bone
marrow and spleen, tissues previously shown to be sites of
eosinophilopoiesis and EPO gene expression
(36).
The loss of EPO results in ultrastructural changes of the eosinophil secondary granule
In contrast to the loss of MBP-1 (35), which results
in the elimination of the electron dense core of the secondary granule,
EPO-/- peritoneal cavity exudate cells
recovered following sensitization/challenge of mice with a
whole-protein extract from the helminth M. Corti-contained
granules with both the electron dense core structure and electron
translucent matrix surrounding this core (Fig. 2). Moreover, the average number of
granules per cross-sectional area was found to be the same in wild-type
and EPO-/- eosinophils (23.4 ± 5.2
vs 27.5 ± 2.3, respectively; n = 4 mice, 12
eosinophils per mouse). However, the loss of EPO (a constituent of the
matrix) reduces the fraction of the granule occupied by the matrix such
that granules recovered from EPO-/-
eosinophils are 57% the size of wild-type granules, indicating that
the matrix volume results, in part, by the physical presence of
EPO.
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The loss of EPO activity in knockout mice was demonstrated by
cytochemical staining of peritoneal cavity exudate cells recovered
following sensitization/challenge of mice with a whole-protein extract
from the helminth M. corti. Fig. 3A shows that eosinophils from
EPO-/- mice were devoid of peroxidase
activity (i.e., CN--resistant peroxidase
activity (DAB staining) was present only in eosinophils from wild-type
and EPO+/- mice). The loss of peroxidase
activity in cells from EPO-/- mice was
further demonstrated using an endpoint colorimetric assay detecting EPO
in lysates of the peritoneal cavity exudates (Fig. 3B). The
peroxidase activity of EPO+/- eosinophils
was roughly 50% of that found in wild-type eosinophils, suggesting
that EPO levels in eosinophils are proportional to the number of
wild-type EPO loci present. Eosinophils recovered from
homozygous knockout mice derived from ES cell clone 6 or 57 were shown
to be EPO deficient using both the peroxidase cytochemical detection
methodology and the endpoint colorimetric assay. Thus, mice derived
from either ES clone were considered equivalent, and subsequent studies
were performed on animals derived from clone 57.
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shows that
although small numbers of eosinophils are homeostatic resident cells in
the lungs of both wild-type and EPO-/-
mice, OVA challenge resulted in an influx of eosinophils into the
peribronchial/perivascular regions of both genotypes. The localization
and number of eosinophils (150–200 eosinophils per millimeter
squared) in the lungs after OVA challenge were
indistinguishable between wild-type and
EPO-/- mice. As suggested by earlier
studies demonstrating the colocalization of EPO activity and
peroxidase-mediated oxidation products in eosinophils recruited to the
lung (31, 50), 3-bromotyrosine levels (as a fraction of
unmodified tyrosine residues present) increased in whole-lung
homogenates of OVA-treated wild-type mice relative to saline-challenged
controls (Fig. 5). In contrast, the
3-bromotyrosine/tyrosine levels in whole-lung homogenates were
undetectable in saline-treated control or OVA-treated
EPO-/- mice, the latter despite an
Ag-induced pulmonary eosinophilia equivalent to wild-type mice
(Fig. 5).
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In response to OVA provocation, equivalent numbers of eosinophils
were recruited to the BAL fluid of wild-type and
EPO-/- mice in a time-dependent fashion
following the last OVA aerosol challenge (data not shown). Maximal
recruitment of eosinophils occurred in both genotypes 48 h after
the final aerosol challenge (Table I).
Moreover, a detailed differential assessment of cells recruited to the
BAL 48 h after challenge revealed that the loss of EPO had no
effects on the recruitment of any leukocyte types relative to
OVA-treated wild-type controls (Fig. 6);
aerosol challenge with saline alone had no effect on the homeostatic
levels of cells in the airspaces of mice of either genotype.
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Wild-type and EPO-/- mice displayed
identical airway changes in response to OVA sensitization/challenge.
For example, PAS staining of lung sections demonstrated that the airway
epithelia of both wild-type and EPO-/-
mice exhibit increased mucus content in response to OVA challenge (Fig. 7). Moreover, this increase was
indistinguishable between genotypes and occurred in the bronchi as well
as the more distal airways (i.e., bronchioles).
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The potential role(s) of EPO in the development of Ag-induced AHR
to methacholine provocation was assessed by whole-body plethysmography
of wild-type and EPO-/- mice. The
resulting dose response curves (means of single-animal measurements;
n = 12–16 mice per group) are presented in Fig. 8. These data compare the responses of
wild-type and EPO-/- mice that have been
challenged with saline or OVA. The dose responses of both
saline-challenged groups were identical, indicating no significant role
of EPO at baseline. Furthermore, OVA-sensitized/challenged wild-type
and EPO-/- mice each displayed identical
increases in methacholine-induced airflow changes relative to the
saline-challenged control groups (i.e., AHR), indicating responses to
allergen-challenge in both genotypes. In addition, no significant
differences were observed in the dose-response curves of
OVA-sensitized/aerosol-challenged wild-type and
EPO-/- mice. Thus, the Ag-induced AHR in this
OVA model of asthma occurs independently of EPO.
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Assessments of EPO in cell-free BAL relative to EPO activity
associated with eosinophils recruited to the airways demonstrated that
the recruitment of airway eosinophils is not accompanied by a
significant release of EPO activity into the BAL (Table I). EPO
activity in the BAL increased as a function of airway eosinophil number
(i.e., time after challenge; data not shown). At the point of maximal
eosinophil airway recruitment (i.e., 48 h after challenge), only
nominal amounts of EPO were found in the cell-free BAL relative to the
activity contained within recruited airway eosinophils (i.e., 9% of
total activity). No measurable EPO was detected in BAL fluid from
EPO-/- mice. Furthermore, this limited
release of EPO by eosinophils recruited to the airway lumen of
OVA-treated mice is reflected by nominal 3-bromotyrosine/tyrosine
levels in proteins from the BAL (Fig. 9).
This result is a significant departure from observations of asthma
patients because the BAL fluid of both severe asthmatics in an
intensive care unit, as well as mild asthmatics 48 h after
segmental Ag challenge, each displayed >10-fold higher
3-bromotyrosine/tyrosine levels relative to the mouse and/or healthy
control subjects (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Data from studies using the mouse as a model system of allergic
pulmonary disease increasingly demonstrate that eosinophil
degranulation may not occur in Ag-challenge protocols
(62). For example, eosinophils recruited to the lung
remain intact, with no electron microscopic evidence of degranulation
or cfegs in the airway lumen, and the literature abounds with studies
demonstrating a lack of extracellular matrix-associated ESGP deposition
in the lung using immunohistochemical/immunofluorescence staining with
granule protein (MBP)-specific Ab reagents (Fig. 4 and Refs. 35, 63). Furthermore, the paucity of studies in the literature
(64) describing eosinophil-mediated tissue damage in the
mouse (e.g., cilliostasis and epithelial sloughing) also suggests that
although mouse eosinophils contain ESGPs that share considerable
degrees of sequence identity with their human counterparts (36, 39, 65, 66, 67), these proteins are apparently not released (and
therefore do not execute effector functions) in OVA models of
respiratory inflammation. This lack of degranulation in the mouse is
supported by our observations of knockout mice deficient for genes
encoding the abundant ESGPs, MBP-1, and now also EPO. The loss of MBP-1
in mice had no effect on the development of pulmonary pathologies in an
OVA-challenge model. These observations extended beyond pulmonary
histopathologies and airway changes, as MBP-1-deficient mice also
displayed OVA-induced AHR after provocation with nonspecific stimuli
(35). The data reported here show that the OVA-induced
pulmonary pathologies in EPO-/- mice are
similarly not attenuated relative to wild-type littermates as a
consequence of this ESGP deficiency.
Assessments of BAL fluids from OVA-treated wild-type mice indicate that
only nominal amounts of free EPO activity are present (i.e., 9% of
the EPO present in the eosinophils recruited to the airspaces). In
addition, only nominal levels of the EPO-specific oxidation product
3-bromotyrosine are observed in BAL-derived proteins. These data imply
either that eosinophils recruited to the lung are each only releasing a
small fraction of the available EPO (i.e., piecemeal degranulation) or
that a subset of the recruited eosinophils are undergoing an overt
release of EPO (i.e., cytolysis). Photo and electron microscopic
evidence showing the integrity of eosinophils recruited to the lung
(see, for example, Ref. 62) suggests that piecemeal
degranulation and/or cytolysis by a subset of recruited eosinophils is
unlikely, implying that low levels of EPO may be actively released by
yet a third independent mechanism (e.g., de novo gene expression and
secretion by activated eosinophils recruited to the airways). However,
it is noteworthy that the free mouse EPO in the BAL may be
artifactual, resulting as a consequence of hydrodynamic shear of a
fraction of the eosinophils present during the recovery of BAL.
Regardless of the resolution of this quandary, the presence of only
nominal levels of 3-bromotyrosine in the proteins recovered from the
BAL of OVA-challenged mice (i.e., >90% decrease relative to asthma
patients) highlights a significant difference between humans and mice.
Although this difference may simply reflect species differences in the
turnover of oxidized proteins or the kinetics of EPO release, a more
likely explanation is that release of EPO by mouse eosinophils
recruited to the BAL is minimal, and/or concurrent events necessary to
elicit EPO effector function (e.g., generation of reduced oxygen
species by eosinophil respiratory burst) may be significantly
reduced.
The lack of phenotypic consequences associated with the loss of either
EPO or MBP-1 suggest that neither of the two most abundant ESGPs in the
mouse contribute to the pathologies observed in this model. However,
these knockout mice do not rule out that other eosinophil-associated
effector functions (e.g., Ag presentation (68, 69) or the
release of cysteinyl leukotrienes (70, 71)) lead to
allergen-induced pathologies. Nonetheless, the implicit conclusion
drawn from these studies is that mouse and human eosinophil effector
functions are different, and the lack of degranulation in the mouse is
a manifestation of this difference. This hypothesis is exemplified in
the photographs of Fig. 10 which
exemplify the absence of eosinophil degranulation in mouse models of
Ag-mediated inflammation relative to the release of ESGPs in asthma
patients. The images presented are compelling examples encapsulating
the literature at large. The lung sections presented in Fig. 10 clearly
show that human eosinophils recruited to the lungs of asthma patients
display dramatic evidence of degranulation and extracellular matrix
deposition of ESGPs such as human MBP. In contrast, peroxidase activity
(i.e., the generation of nitrotyrosine) and MBP-1 each remain localized
within recruited mouse eosinophils. The question that arises is,
"What is the basis of this observed difference?" In particular, is
the OVA model an inadequate representation of the inflammatory
reactions occurring in asthma patients, or in other words, are there
costimulatory signals missing in the mouse models preventing eosinophil
degranulation? The inability of isolated mouse eosinophils either to
undergo degranulation or generate significant amounts of
3-bromotyrosine after exposure to PMA (data not shown) suggests that
the OVA models are not at issue. Instead, the difference in the mouse
models and asthma patients apparently lies within the eosinophil
itself. This would imply that selective evolutionary pressures on mouse
vs human eosinophils during the 75–100 million years since the
divergence of the mammalian orders Rodentia and Primata
(72) have led to species differences in
eosinophil-mediated activities. Specifically, it was (is) selectively
advantageous for rodent and/or primate eosinophils to adopt different
modes of executing effector functions that become apparent within the
context of the artificial circumstances surrounding an OVA
sensitization/challenge model system (i.e., eosinophil activities may
contribute to pathology in mouse OVA models but not through an
extensive release of ESGPs as occurs in asthma patients). However,
a logical extension of this hypothesis is the convergence of eosinophil
effector functions in mice and humans with regard to evolutionarily old
defensive mechanisms (i.e., pathways predating the radiation of extant
mammalian orders) such as eosinophil-mediated host defenses against
parasites (73). The observations of Pearlman and
colleagues (74) demonstrating the deposition of
extracellular MBP on the surface of airway epithelial cells in the
lungs of mice responding to an active infestation of the parasite
Onchocerca volvulus imply that, under specific
circumstances, mouse eosinophils are capable of releasing ESGPs (i.e.,
degranulation) reminiscent of their human counterparts. Thus, although
the necessity of degranulation to eosinophil effector function has
remained critical, the mechanisms and/or circumstances required to
elicit this event have evolved differently among mammalian orders. The
absence of ESGP release in OVA mouse models, exemplified by attenuated
levels of EPO-mediated protein oxidation relative to asthma patients,
and the apparent lack of OVA-mediated pathologies in EPO and
MBP-1 knockout mice is likely a reflection of this
evolutionary difference in eosinophil effector function(s).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Nancy A. or James J. Lee, Department of Biochemistry and Molecular Biology, Samuel C. Johnson Medical Research Buliding, Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, AZ 85259. E-mail addresses: nlee@mayo.edu or jlee{at}mayo.edu
3 Abbreviations used in this paper: EPO, eosinophil peroxidase; BAL, bronchoalveolar lavage; MBP, major basic protein; ESGP, eosinophil secondary granule protein; P, primer; ES, embryonic stem; MCA, Mesocestoides corti Ag; DAB, diaminobenzidine; PAS, periodic acid-Schiff reagent; AHR, airway hyperresponsiveness; PENH, enhanced pause; cfegs, clusters of free eosinophil granules.
Received for publication December 26, 2000. Accepted for publication June 1, 2001.
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300
cells. The data represent mean values ± SEM (*,
p < 0.05).
