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on Human Eosinophils: I. Loss of Membrane IL-5 Receptor on Airway Eosinophils and Increased Soluble IL-5 Receptor in the Airway After Allergen Challenge1
* Allergy and Immunology and
Pulmonary and Critical Care Sections of Department of Medicine, and Departments of
Biomolecular Chemistry and
Pediatrics, University of Wisconsin, Madison, WI 53792; and
¶ Department of Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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on bronchoalveolar lavage (BAL) eosinophils; 2) the
responsiveness of these cells to exogenous IL-5; and 3) the presence of
soluble (s)IL-5R in BAL fluid. To accomplish these goals, blood and
BAL eosinophils were obtained from atopic subjects 48 h after
segmental bronchoprovocation with Ag. There was a striking reduction in
mIL-5R on airway eosinophils compared with circulating cells.
Furthermore, sIL-5R concentrations were elevated in BAL fluid, but
steady state levels of sIL-5R mRNA were not increased in BAL
compared with blood eosinophils. Finally, BAL eosinophils were
refractory to IL-5 for ex vivo degranulation, suggesting that the
reduction in mIL-5R on BAL eosinophils may regulate IL-5-mediated
eosinophil functions. Together, the loss of mIL-5R, the presence of
sIL-5R, and the blunted functional response (degranulation) of
eosinophils to IL-5 suggest that when eosinophils are recruited to the
airway, regulation of their functions becomes IL-5 independent. These
observations provide a potential explanation for the inability of
anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled
Ag, despite a reduction in eosinophil
recruitment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-5 is unquestionably an important factor in eosinophil maturation,
differentiation, survival, and activation (1). In mouse
models of Ag-induced airway inflammation, the significance of IL-5 to
eosinophilopoiesis in the bone marrow, release of eosinophils into the
circulation, and their recruitment to the airway in response to Ag
sensitization has been clearly demonstrated in mice made deficient in
IL-5 or IL-5R through genetic deficiency (7, 8), and by
administration of anti-IL-5 Abs (9) or antisense
oligodeoxynucleotides for IL-5R (10). Studies in humans
have demonstrated that ex vivo exposure of peripheral blood eosinophils
to IL-5 can induce eosinophil activation, as indicated by enhanced
integrin-mediated adhesion, expression of membrane receptors,
chemotactic responses, release of eosinophil-derived cytokines and
mediators (11), and extracellular signal-regulated kinase
(ERK) activation (12). The characteristics of these
IL-5-stimulated blood eosinophils are mimicked in vivo by BAL
eosinophils recruited following airway Ag challenge. For example,
compared with cells in the circulation, airway eosinophils have
heightened release of toxic oxygen species (13), enhanced
ex vivo survival (13), increased activation of the
mitogen-activated protein kinases ERK1 and ERK2 (14), and
a greater expression of activation markers (15). The
contribution of IL-5 to these features in vivo and the localization of
potential IL-5-mediated eosinophil activation events await further
characterization.
Regulation of IL-5-mediated eosinophil activation is even less
well characterized. Because of the potential release of toxic granules
when eosinophils are activated, it is likely that compensatory
mechanisms have developed to limit IL-5-mediated eosinophil activation.
Several mechanisms can be envisioned, including IL-5-specific
desensitization of its receptor or the presence of neutralizing factors
such as soluble (s)IL-5R. Ex vivo exposure of blood eosinophils to
IL-5, GM-CSF, or IL-3 leads to decreased expression of IL-5R mRNA
(16). This effect would presumably result in lower
membrane (m)IL-5R on the cell surface; however, this has not been
documented. rsIL-5R has been produced as a potential antagonist for
IL-5-mediated functions (17). In vitro studies have
demonstrated that rsIL-5R prevents the association of IL-5 with
mIL-5R (18), and thus acts as an antagonist to inhibit
IL-5-mediated signal transduction (18), survival
(18), and maturation/differentiation of eosinophils
(19). In asthma, mIL-5R and sIL-5R mRNA-positive
cells are present in the bronchial mucosa. Airway obstruction is
associated with increased numbers of mIL-5R
mRNA+ cells and decreased numbers of cells
expressing sIL-5R mRNA (20). Consequently, it has been
suggested that sIL-5R may have a protective role in asthma. However,
to date, the presence and biological importance of sIL-5R in the
airway or any human fluids have not been explored.
The following studies were performed to compare mIL-5R expression in
human airway and circulating eosinophils and to explore the possibility
that the IL-5 antagonist, sIL-5R, is present in the airway following
airway Ag challenge. To accomplish these goals, airway eosinophilia was
induced by segmental bronchoprovocation with Ag (SBP-Ag) in atopic
subjects. Eosinophil cell surface expression of mIL-5R was
determined by flow cytometric analysis; steady state levels of soluble
and membrane IL-5R transcripts were determined by RT-PCR with
Southern blot analysis; and the concentration of sIL-5R in BAL fluid
was evaluated by ELISA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine levels of IL-5 and sIL-5R, BAL fluid was
obtained from 25 atopic subjects before and after SBP-Ag. Subject
characteristics are provided in Table I
.
In addition, in subjects 14–25, BAL eosinophils were obtained for
analysis of mIL-5R and mGM-CSFR by flow cytometry. Abs became
available for the 
-chain (c) after the completion of the IL-5R
studies; therefore, an additional six subjects were recruited for
analysis of membrane expression of c in relation to mIL-5R and
mGM-CSFR. Studies were approved by the University of Wisconsin
Health Sciences Human Subjects Committee, and informed consent was
obtained from all subjects before participation.
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Analysis of BAL fluid
BAL cells were recovered from the lavage fluid by centrifugation at 400 x g for 10 min at 4°C, then washed twice with HBSS containing 2% newborn calf serum (NCS). Total BAL cell numbers were determined by hemacytometer using Turk’s counting solution containing acetic acid and methylene blue. For differential cell counts, cytospin preparations of BAL cells were stained with the Giemsa’s-based Diff-Quik stain (Baxter Scientific Products, McGaw Park, IL). BAL cells were used for flow cytometric analysis and to obtain purified eosinophils. BAL fluids were stored at -70°C until analyzed.
Protein measurements
A sensitive two-step sandwich-type ELISA was developed to
measure sIL-5R in 20x concentrated BAL fluids. Half-area well ELISA
plates (Corning, Corning, NY) were coated overnight at 4°C
with a predetermined optimal concentration of purified monoclonal
anti-human IL-5R (clone A17; BD PharMingen, San Diego, CA).
Nonspecific binding sites were blocked with 10% dialyzed NCS. Test
samples were incubated overnight at 4°C on Ab-coated plates, and
sIL-5R was detected with a biotinylated goat anti-human IL-5R
polyclonal Ab (R&D Systems, Minneapolis, MN). Streptavidin conjugated
to a HRP polymer (POLY-HRP-40; Research Diagnostics, Flanders, NJ) was
used to increase assay sensitivity. A one-component substrate,
3,3',5'5'-tetramethylbenzidine (Kirkegaard & Perry Laboratories,
Gaithersburg, MD), was used for color development, and the reaction was
stopped by addition of 0.18 M sulfuric acid. OD at 450 nm was
determined with a Dynatech MR500 microplate reader, and data were
analyzed with Biolink Software (Dynatech Laboratories, Chantilly, VA).
The concentration of sIL-5R in BAL fluids was calculated by
comparison with a standard curve generated with known amounts of human
rsIL-5R (Sigma-Aldrich, St. Louis, MO). The assay sensitivity for
sIL-5R was 
12 pg/ml. IL-5 was also measured by a two-step
ELISA, as previously described (6). The coating Abs and
biotinylated detection Abs were purchased from BD PharMingen. The
sensitivity for IL-5 was 3 pg/ml.
Eosinophil purification
BAL eosinophils were purified by a modified Percoll gradient. BAL cells (50 x 106/5 ml HBSS without calcium + 2% NCS) were carefully layered over a 1.085/1.100 g/ml Percoll gradient and centrifuged for 20 min at 700 x g. Eosinophils were collected from the 1.085/1.100 g/ml interface. In selected experiments, BAL eosinophils were compared with purified blood eosinophils, which were isolated by negative selection using immunomagnetic beads (23). For both purified BAL and blood eosinophils, viability was >97% and purity was >98%.
Eosinophil-derived neurotoxin (EDN) analysis
Isolated peripheral blood and BAL eosinophils (1 x 106/ml) were activated with 1 ng/ml of IL-5 or GM-CSF in medium containing HBSS supplemented with 0.03% gelatin (HBSS/gel; Sigma-Aldrich) for 4 h at 37°C. The cell-free supernatant fluids were frozen at -20°C until EDN was measured by RIA (24). The sensitivity for EDN was 2 ng/ml. Total cellular EDN was determined from parallel cultures to which an equal volume of 1% Triton X-100 in 0.1 N HCl was added.
Flow cytometric analysis
For initial analysis of cell surface receptors, eosinophils were
stained in whole blood (100 µl) and nonpurified BAL preparations
(1 x 105 cells). Ab included PE-conjugated
mAb to IL-5R (CD125; BD PharMingen), the c (CD131; eBioscience,
San Diego, CA), and FITC anti-GM-CSFR (CD116; BD PharMingen).
For analysis, 10,000 events were collected using a BD Biosciences
FACScan II, and data analyses were performed using CellQuest software
(BD Biosciences). An electronic eosinophil region (R1) within the
forward and side scatter plot was established by backgating on
FITC-CD45 bright, PE-autofluorescent cells (25). The
majority of cells within this forward and side scatter gate are
eosinophils. Nonetheless, all test samples contained a FITC- or
PE-labeled anti-CD16 and anti-CD14 cocktail, which allowed for
further electronic exclusion of any contaminating neutrophils and
monocytes, respectively. To determine the percentage of positive cells,
dot blots were created based on R1, and a tight electronic gate (R2)
was set to encompass only the eosinophils in the isotype control
sample. A larger region, R3, was drawn to include the isotype control
and all positive eosinophils. Cells were considered positive if there
was an electronic shift out of the R2 isotype control region and into
R3. Thus, the percentage of positive eosinophils was determined as
(1 - (R2/R3)) x 100.
RT-PCR for detection of IL-5R mRNA
Total RNA was extracted from eosinophil cell pellets using a
one-step phenol/chloroform extraction reagent (Tri Reagent;
Sigma-Aldrich). The total RNA was treated with DNase (RQ1 RNase-free
DNase; Promega, Madison, WI) to degrade DNA, and cDNA was synthesized,
as previously described (26). PCR was performed by
transferring 4 µl of cDNA to a 650 µl thin-walled PCR tube along
with 2.5 U platinum Taq (Invitrogen Life
Technologies, Carlsbad, CA), 5 µl 10x PCR buffer, 0. 01 µM
dNTPs, 50 mM MgCl2, and 0.2 µM of primer in a
final volume of 50 µl. A forward primer specific for mIL-5R,
position 1033–1056, and two different reverse primers, sIL-5R at
position 1279–1298 and mIL-5R at position 1542–1564, were
constructed using published sequences (17). Primers for
amplification of GAPDH mRNA were as previously described
(26). Upstream and downstream primers were separated by
introns so that any genomic DNA amplified by these procedures could be
discriminated from cDNA based on size. The predicted size of cDNA
fragments was 266 bp for sIL-5R and 527 bp for mIL-5R. PCR was
conducted with the following protocol: 94°C for 2 min, then 24 cycles
of 96°C for 30 s, 60°C for 30 s, and 72°C for 30
s. The number of PCR cycles (24) was optimized to maintain
a linear relationship between mRNA and PCR products. Controls included
in each PCR run included samples containing reagents with no cells and
samples that had not been transcribed. A DNA probe (205 bp, position
1033–1237) for both forms of IL-5R was synthesized and labeled with
HRP, and PCR products were detected by Southern blot analysis (ECL
system; Amersham, Piscataway, NJ).
GAPDH mRNA was analyzed in parallel with IL-5R mRNA to assess
consistency of RNA preparations. GAPDH mRNA levels were quantitated by
a competitive PCR (cPCR) ELISA. cDNA competitors consisting of nonsense
DNA flanked by GAPDH-specific primers were added to test samples in
graded quantities to compete with native cDNA for primer binding
(27). cPCR was then performed with a biotinylated
forward primer to end label the PCR product for analysis by ELISA. To
perform the DNA ELISA, the biotinylated PCR product was denatured and
added to a 96-well streptavidin-coated plate. PCR products were
detected by hybridization with a fluorescein-labeled probe
(28), followed by incubation with an anti-fluorescein
Ab conjugated to aequorin (Aqualite; Chemicon, Temecula, CA)
(29). Upon addition of calcium ions, blue light is emitted
and detected in a microplate luminometer. The concentration of the
sample is calculated by plotting the sample/competitor signal ratio
against the concentration of the competitor: when the ratio equals one,
then the concentrations are equal. The RT-cPCR ELISA was found to be
linear and reproducible, with a coefficient of variation of
10%.
The relative amount of IL-5R mRNA was normalized to GAPDH. The band
intensity of the IL-5R mRNA species was determined from the Southern
blots and given as arbitrary units. These units were then divided by
the amount of GAPDH mRNA calculated from the RT-cPCR ELISA to give the
ratio of sIL-5R/GAPDH or mIL-5R/GAPDH.
Statistical analysis
Statistical analysis was performed using the SigmaStat software package (Jandel Scientific Software, San Rafael, CA). Data are expressed as medians with 25 and 75 interquartiles (or the mean ± SEM for normally distributed data). The Wilcoxon signed rank test (or a paired t test for normally distributed data) was used to compare different time points to 0 h or to compare data obtained at baseline and 48 h after SBP-Ag. Correlations were made using Spearman rank order correlation test. A p value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on BAL eosinophils following
airway Ag challenge
Forty-eight hours after SBP-Ag challenge, both eosinophil
percentage (Table II) and number in BAL
fluids were markedly increased (54.4 ± 3.8% and 131.5 ±
29.9 x 104/ml BAL fluid, mean ± SE)
compared with baseline (1.2 ± 0.4% and 0.01 ± 0.00 x
104/ml BAL fluid, n = 25,
p < 0.001). The expression of mIL-5R on peripheral
blood (0 and 48 h after Ag challenge) and BAL (48 h) eosinophils
was determined by flow cytometry. Analysis of mGM-CSFR was also
performed because IL-5 and GM-CSF have overlapping functions and the
c subunit of their receptors is identical. Both the percentage of
mIL-5R-positive eosinophils (Fig. 1A) and the amount of cell
surface mIL-5R (Fig. 1, B and C) were
significantly diminished on BAL (48 h) compared with peripheral blood
eosinophils obtained either before (0 h) or 48 h after Ag
challenge. In addition, c, which was expressed on 100% of
circulating eosinophils both at 0 and 48 h after Ag challenge, was
markedly reduced on BAL eosinophils (Fig. 1, D–F). mGM-CSFR was expressed on nearly 100%
of peripheral blood and BAL eosinophils (Fig. 1G), and, in
contrast to mIL-5R and c, the relative intensity of mGM-CSFR
staining was significantly augmented on BAL compared with peripheral
blood eosinophils (Fig. 1, H and I). BAL
eosinophils could not be evaluated at baseline due to the small numbers
of available cells.
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in BAL fluid and the relationships between
BAL fluid levels of sIL-5R, IL-5, and eosinophil numbers 48 h
after Ag challenge
Concomitant with the reduction of cell surface mIL-5R (Fig. 1
A), BAL fluid concentrations of sIL-5R were
significantly elevated 48 h after Ag challenge when compared with
baseline values (Fig. 2A).
Moreover, there was a strong positive correlation (Spearman’s
correlation coefficient (rs) = 0.741,
p < 0.001) between the levels of sIL-5R in BAL
fluid and the number of BAL eosinophils 48 h after Ag
challenge (Fig. 2B). The levels of sIL-5R also correlated
(rs = 0.731, p < 0.001)
with IL-5 concentrations in BAL fluid (Fig. 2C).
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and IL-5 in BAL
fluid and flow cytometric assay for detection of cell surface
mIL-5R
Because IL-5 is present in BAL fluid following Ag challenge
(6), it was necessary to determine whether IL-5 interferes
with the ability to accurately measure mIL-5R and sIL-5R. For the
sIL-5R ELISA, addition of high concentrations of IL-5 (10 ng/ml) to
sIL-5R standard had no effect on sIL-5R detection (Fig. 3A), suggesting that IL-5 in
BAL fluid did not interfere with measurement of sIL-5R. Furthermore,
a high concentration of sIL-5R (10 ng/ml) had no effect on the
detection of IL-5 standard in the IL-5 ELISA (Fig. 3B). For
flow cytometric analysis, it was also necessary to exclude the
possibility that prior binding of endogenous IL-5 in BAL fluid to
airway eosinophils blocked the detection of mIL-5R. To evaluate this
possibility, increasing concentrations of rIL-5 were added to
peripheral blood eosinophils before staining of mIL-5R. Even at
concentrations of 10 ng/ml IL-5, there was no significant effect on
detection of mIL-5R by flow cytometric analysis (Fig. 3C).
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mRNA in BAL and peripheral blood eosinophils
The regulation of IL-5R and IL-5R mRNA in human blood
eosinophils by the IL-5-family cytokines was previously reported by
Wang et al. (16). However, the expression of mRNA for
these receptor subunits in airway eosinophils has not been studied.
Because there were not sufficient numbers of BAL eosinophils present in
the airway before Ag challenge, mRNA levels in purified BAL eosinophils
obtained 48 h after Ag challenge were compared with peripheral
blood eosinophils obtained from the same subject immediately before the
post-Ag BAL. sIL-5R and mIL-5R mRNA were measured by RT-PCR, and
the identity of PCR products was confirmed by Southern blot analysis
using sIL-5R- and mIL-5R-specific probes. The housekeeping gene,
GAPDH, was determined by an RT-PCR competitive ELISA-type assay. No
enhanced expression of IL-5R mRNA was discernible in any of the four
BAL samples relative to the expression in the peripheral blood samples
(Fig. 4). In fact, when the levels of
IL-5R mRNA were expressed as a ratio to GAPDH (as indicated by the
number below each blot), there was a noticeable decrease in both
sIL-5R and mIL-5R in BAL compared with blood eosinophils in two
of the four subjects.
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Eosinophil degranulation was used to assess the potential
functional significance of the reduction of mIL-5R on BAL
eosinophils. Isolated blood or BAL eosinophils were cultured with
medium alone or 1 ng/ml IL-5 or GM-CSF, and released EDN was
determined. Blood eosinophils showed significant cytokine-induced EDN
release in response to either IL-5 or GM-CSF (Fig. 5A). In contrast, BAL
eosinophils responded to GM-CSF, but were refractory to stimulation by
IL-5 (Fig. 5B). These data indicate that a reduction in
mIL-5R expression, which is seen on BAL eosinophils, is associated
with a decrease in the ability of these cells to respond to IL-5 with
the release of EDN, i.e., there was a demonstrable functional correlate
for the observed change in receptor expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and c were markedly reduced on airway
eosinophils compared with circulating eosinophils. Furthermore,
sIL-5R protein was detected, and the concentrations were
significantly elevated in BAL fluid 48 h after Ag challenge.
Although the precise role of mIL-5R and sIL-5R in the regulation
of airway eosinophil functions is unknown, we speculate that these
events control IL-5-mediated activation of eosinophils. In this regard,
we have demonstrated that, in contrast to their circulating
counterparts, BAL eosinophils obtained following SBP-Ag do not release
EDN when exposed ex vivo to IL-5. Taken together, these observations
further support the notion that the primary function of IL-5 is
eosinophil hemopoiesis and release from the bone marrow (30, 31). Moreover, the decrease in mIL-5R expression on BAL
eosinophils and the lack of responsiveness to IL-5 suggest a switch to
IL-5-independent cell function once this cell is in the airway.
The decrease in the expression of mIL-5R on human airway eosinophils
compared with their circulating counterparts is consistent with the
observations by Tomaki et al. (9), who showed that
mIL-5R was detectable by immunohistochemistry on bone marrow, but
not BAL eosinophils following airway allergen challenge in a murine
model of allergen-induced eosinophilic airway inflammation. The
decrease in mIL-5R expression could occur via several possible
mechanisms. First, the receptor could undergo proteasome degradation.
Recent studies in an IL-5-responsive erythroleukemia line (TF-1) have
shown that IL-5 induces proteasome-mediated cleavage of the c
cytoplasmic tail, followed by receptor internalization and degradation
in the lysosome (32). Second, the receptor could be
internalized via lysosomal-mediated mechanisms and be either degraded
or recycled to the cell surface. For example, loss of the chemokine
receptor CCR3 involves internalization with both degradation and
limited recycling of receptors to the cell surface (33, 34). Finally, the ectodomain of mIL-5R could be shed from the
cell surface through a proteolytic process to give rise to a soluble
form of the receptor. A number of integral membrane proteins are known
to be enzymatically cleaved from the cell surface, including cytokine
receptors (TNF-, IL-6, TGF-1), Ig receptors (FcR
III, FcRII,
FcR
), and adhesion molecules (VCAM-1, CD14, L-selectin)
(35). Based on the observations that the sIL-5R protein
was detected in BAL fluid and that levels increased following Ag
challenge in the absence of increased steady state levels of sIL-5R
mRNA, we speculate that the presence of sIL-5R in BAL fluid may
result from cleavage of the mIL-5R ectodomain from the cell surface
and examine this possibility in the accompanying manuscript (40).
The demonstration of sIL-5R in human biological fluids is a novel
and potentially important finding toward an understanding of eosinophil
function in allergic inflammation. The significance and functional
activity of this protein remain to be determined, as other soluble
receptors have been shown to inhibit or enhance cytokine function
(36). It has been suggested that sIL-5R may function as
an IL-5 antagonist. This is based on the observation that rsIL-5R
binds with high affinity to IL-5 (37), and is a potent in
vitro antagonist for IL-5-mediated signal transduction
(18) and differentiation of eosinophil progenitor cells
(19). In addition, Yasruel et al. (20) have
reported that the presence of sIL-5R mRNA-positive cells in the
bronchial mucosa from asthmatic subjects correlated with an improvement
in pulmonary function, whereas the presence of mIL-5R mRNA-positive
cells was associated with airflow obstruction. Based on these
observations, it is tempting to speculate that sIL-5R may serve a
protective role in IL-5-mediated airway diseases. However, definitive
studies await purification of the protein from BAL fluid.
We have presented compelling evidence that IL-5-mediated eosinophil
activation in the airway is controlled at the level of IL-5R
expression. We recognize, however, that there are certain limitations
to our studies of human airway eosinophils. First, we cannot ascertain
where the switch to IL-5 unresponsiveness may occur. This is due, in
part, to the lack of sufficient numbers of BAL eosinophils for analysis
at baseline, and the inability to study the functional capacity of
eosinophils in the airway mucosa. Second, we have yet to identify the
IL-5-independent factor(s) that controls eosinophil activation in the
airway. The role of GM-CSF in this regard is not entirely clear.
Although airway eosinophils retain some degree of responsiveness to
GM-CSF, as demonstrated by EDN release, the expression of c is
markedly reduced on these cells, and the degree of responsiveness to
exogenous GM-CSF is significantly less than that of circulating
eosinophils. Whether the differences between blood and BAL eosinophils
are due to reduced signaling by GM-CSFR or reflects other differences
between these cells is not yet known.
In conclusion, we propose that, following airway Ag challenge, IL-5 is
primarily responsible for the release of mature eosinophils from the
bone marrow and their subsequent recruitment to the airway. This is
based on the high expression of mIL-5R on circulating, but not
airway eosinophils, and is consistent with a number of animal studies
showing IL-5-induced eosinophilopoiesis and recruitment to the airway.
Within the airway, expression of mIL-5R on eosinophils is
attenuated, and sIL-5R is released into the BAL fluid. As a result,
the response of the airway eosinophil to IL-5 is ablated, and
additional factors may be required for further eosinophil activation.
Thus, we postulate that within the airway, the regulation of eosinophil
functions may be switched from an IL-5-dependent to IL-5-independent
mechanism(s). Although these conclusions are, at present, speculative,
we propose that this paradigm may begin to explain why recent studies
with an anti-IL-5 mAb treatment of mild asthmatic patients reduced
circulating and sputum eosinophils, but did not inhibit the airway
response to inhaled allergen (38). In support of this
possibility, Kay and colleagues (39) have recently
reported that a significant proportion of eosinophils is retained
within the airway mucosa following anti-IL-5 treatment of patients
with asthma. Furthermore, anti-IL-5 had no effect on the detection
of eosinophil granule protein present in the bronchial mucosa. Taken
together with our observations that airway eosinophils (and presumably
tissue-dwelling cells) have reduced expression of mIL-5R and do not
degranulate to IL-5, it is not surprising that anti-IL-5 did not
modulate the airway response to Ag. This possibility emphasizes the
need to closely evaluate effects of allergic mediators in various
compartments, i.e., circulation, bronchial mucosa, and airway lumen,
during allergic inflammation, and suggests that effective treatment may
require selective elimination of multiple cytokine pathways.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Elizabeth A. (Becky) Kelly, Section of Pulmonary and Critical Care Medicine, 600 Highland Avenue, CSC K4/928, University of Wisconsin School of Medicine, Madison, WI 53792-9988. E-mail address: eak{at}medicine.wisc.edu
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; c, -chain; cPCR, competitive PCR; EDN, eosinophil-derived neurotoxin; ERK, extracellular signal-regulated kinase; m, membrane form; NCS, newborn calf serum; rs, Spearman’s correlation coefficient; s, soluble form; SBP, segmental bronchoprovocation.
Received for publication July 2, 2002. Accepted for publication October 1, 2002.
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) of 10 ng/ml IL-5.
B, Detection of IL-5R by ELISA in the presence (•) and
absence (
