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Departments of
*
Pathology and
Internal Medicine, Division of Pulmonary and Critical Care, University of Michigan Medical School, Ann Arbor, MI 48109; and
National Institute of Aging, National Institutes of Health, Baltimore, MD 21224
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. From in vitro studies, it was
evident that major pulmonary sources of C10 may have included alveolar
macrophages, lung fibroblasts, and vascular smooth muscle cells.
Experimental ABPA was associated with severe peribronchial
eosinophilia, bronchial hyperresponsiveness, and augmented IL-13 and
IgE levels. The immunoneutralization of C10 with polyclonal
anti-C10 antiserum 2 h before the intratracheal A.
fumigatus challenge significantly reduced the airway
inflammation and hyperresponsiveness in this model of ABPA, but had no
effect on IL-10 nor IgE levels. Taken together, these data suggest that
C10 has a unique role in the progression of experimental
ABPA. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite advances in the characterization of immune events in
experimental ABPA, a major deficit in knowledge persists regarding the
role of chemotactic cytokines in airway allergic responses to
Aspergillus fungus. Clinical observations in asthmatic
patients show that C-C chemokines such as monocyte-chemoattractant
protein-1 (MCP-1), RANTES, eotaxin, and macrophage-inflammatory
protein-1 (MIP-1) are elevated in allergic asthmatic patients
before and following Ag challenge (14, 15, 16, 17). These findings have
supplied the impetus for the exploration of the role of these and other
chemokines in experimental allergic airway responses to a number of
small protein allergens (18, 19, 20). Surprisingly, functional redundancy
among the chemokines involved in the allergic airway response appears
to be minor due in large part to the orchestrated timing and the
tissue-specific localization of chemokine production (18, 21).
Growing evidence also suggests that certain chemokines possess shadow
or modulating roles within immune responses (22). Originally identified
in GM-CSF-activated bone marrow cells (23), MIP-related protein 1 or
C10 is a chemokine that is postulated to fit this role. C10 has a
genomic structure that includes an additional exon, making it unique
from other chemokines (24), and this additional exon is necessary for a
significant portion of the biologic activity of this molecule (25). C10
is chemotactic for B cells and CD4+ T cells (26), and is
highly homologous to human chemokines such as MIP-1
(27), CCF18
(28), HCC-1, and HCC-2 (29). These human chemokines all possess similar
affinity for CC chemokine receptor 1 and promote T cell and monocyte
chemotaxis. Previous studies indicated that unlike numerous other
chemokines, C10 is IL-4- but not LPS-inducible in macrophages and
requires de novo protein synthesis that delays its appearance for at
least 24 h after cell activation (26). Thus, the present study
was directed at elucidating the role of C10 in the development of
allergic airway inflammation and hyperresponsiveness to A.
fumigatus challenge.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free, female CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained under specific pathogen-free conditions before and during experiments. Sensitization of mice to soluble A. fumigatus Ags was achieved using a previously described procedure (30, 31). Briefly, all mice received a total of 10 µg of A. fumigatus crude Ag (Greer Laboratories, Lenoir, NC) dissolved in 0.2 ml of IFA (Sigma, St. Louis, MO). One-half of this preparation was then deposited in the peritoneal cavity, and the remainder was delivered s.c. Two weeks later, mice received a total of 20 µg of A. fumigatus Ags dissolved in normal saline via the intranasal route. Four days after the intranasal challenge, mice received 20 µg of A. fumigatus Ag dissolved in normal saline via the intratracheal route. In additional groups of A. fumigatus-sensitized mice, 0.5 ml of polyclonal anti-C10 antiserum or normal rabbit serum was delivered into the peritoneal cavity of these mice 2 h before the intratracheal Ag challenge. Mouse lung responsiveness to i.v. methacholine administration and a number of additional parameters of allergic airway inflammation were examined at various times after the A. fumigatus intratracheal challenge. Prior approval for mouse usage in these studies was obtained from University Laboratory Animal Medicine (ULAM) facility at the University of Michigan Medical School (Ann Arbor, MI).
Anti-C10 Ab generation
Polyclonal anti-C10 antiserum was generated by the multiple
site immunization of a New Zealand White rabbit using an
Escherichia coli-expressed C10 protein (25, 26). The
resulting anti-C10 antiserum was purified over a protein A affinity
column, and 10 µl of rabbit anti-C10 antiserum neutralized
approximately 20 ng of C10 in vitro (data not shown). This antiserum
was titrated by direct ELISA, and no cross-reactivity with the
following recombinant murine cytokines and chemokines: IL-1ß,
TNF-, IL-4, IFN-
, IL-10, IL-6, MIP-1, MCP-1, MIP-1ß,
RANTES, KC, eotaxin, MIP-2, and MARC (MCP-3). For this and other
methods described below, all recombinant murine cytokines and
chemokines were obtained from R&D Systems (Minneapolis, MN), Genzyme
(Cambridge, MA), or Pepro Tech (Rocky Hill, NJ).
Cell isolation and culture
Alveolar macrophages were isolated from bronchoalveolar lavage
(BAL) samples taken from nonsensitized CBA/J mice. BAL samples were
obtained through the multiple intratracheal introduction of 1 ml of PBS
containing 50 mM EDTA. MC-9 mast cells (ATCC CRL 8306) represent a
mature, nontransformed mast cell line, and these cells were maintained
in culture with IL-3 supplementation and DMEM growth medium containing
1% (v/v) antibiotic-antimycotic and 15% (v/v) FBS (DMEM-15).
Fibroblasts and vascular smooth muscle cells (VSMCs) from nonsensitized
CBA/J mice were grown out from lung using techniques previously
described in detail elsewhere (32, 33). Briefly, pulmonary fibroblasts
were grown out from mechanically dispersed whole lungs in 175-ml tissue
culture flasks containing DMEM-15. VSMCs were grown from explants of
large pulmonary vessels in 60-mm tissue culture plates in the presence
of DMEM-15. After a minimum of three passages, homogenous populations
of fibroblasts and VSMCs were transferred to six-well tissue culture
plates. Before an experiment, lung fibroblasts and VSMCs were stained
for actin, desmin, and -naphthyl acetate esterase. After the
third passage, lung fibroblasts stained weakly for actin,
suggesting a myofibroblast-type phenotype, and cultures of these cells
were found to be completely free of -naphthyl acetate
esterase-positive cells such as macrophages (data not shown). In
contrast, VSMCs stained strongly for actin (i.e., 
95% positive),
and confluent cultures of these cells exhibited a "hill and valley"
appearance that is typical of cultured smooth muscle cells, and these
cultures of VSMCs were devoid of macrophages. Lung fibroblasts and
VSMCs were used in these experiments up to the sixth passage.
Cell culture protocols
Preparations of alveolar macrophages were typically greater than
95% pure, and these cells were suspended in RPMI 1640 containing 10%
FBS (RPMI-10) at 1 x 106 cells/well of a six-well
tissue culture plate. Individual wells were then exposed to RPMI-10
alone or to IL-1ß, TNF-, IL-4, IFN-, or IL-10 at 10 ng/ml in
RPMI-10 for 24 h. MC-9 mast cells at a density of 2 x
105 cells/well in six-well tissue culture plates were left
untreated or received 10 µg/ml of compound 48/80, a potent
nonspecific mast cell activator (34). Each well in a six-well tissue
culture plate was seeded with approximately 1 x 106
fibroblasts or VSMCs. Twenty-four hours later, the DMEM growth medium
was replaced with RPMI containing 10% FBS (RPMI-10) containing
IL-1ß, TNF-, IL-4, IFN-, or IL-10 at 10 ng/ml. Cytokine
combinations of IL-1ß + IL-4, IL-1ß + IFN-, IL-1ß + IL-10,
TNF- + IL-4, TNF- + IFN-, or TNF- + IL-10 (10 ng/ml of
each) were also added to other wells. Twenty-four hours after the
addition of cytokines to cultured fibroblasts and VSMCs, cell-free
supernatants were removed for the measurement of C10 levels by ELISA.
Eosinophil chemotaxis
A modified Boyden chamber technique was used to quantify eosinophil chemotactic responses to C10 and eotaxin, a potent and selective eosinophil chemoattractant (35). The eosinophils used in this experiment were isolated from mice chronically infected with Schistosoma mansoni (these mice were obtained from the National Institute of Health, Bethesda, MD). Briefly, 1 ml of thioglycolate broth was delivered by i.p. injection into each mouse, and 48 h later the peritoneal cavity of these mice was thoroughly lavaged with normal saline. The collected cells were suspended in RPMI 1640 containing 5% FBS, 2-ME (10 µM), sodium pyruvate (2 mM), L-glutamine (20 mM), penicillin (100 U), and streptomycin (100 mg/ml). Eosinophils were adhesion purified in 175-ml tissue culture flasks for 1 h to yield preparations that contained approximately 85% eosinophilic granulocytes. Contaminating cells were primarily lymphocytes. These cells were suspended at 1 x 107 cells/ml of RPMI-10, and a 100-µl aliquot of this suspension was placed in individual wells of a 24-well microchemotaxis chamber. The upper wells were separated from lower wells containing 10–100 ng/ml of C10 or eotaxin by a 3-µm-pore-size polycarbonate filter (Nucleopore, Pleasanton, CA). The microchemotaxis chambers were incubated for 4 h at 37°C in a 5% CO2 incubator, after which the filters were fixed in 4% paraformaldehyde, stained in hematoxylin and eosin, and mounted onto a glass microscope slide for light microscopy visualization. Migrated eosinophils were subsequently quantified in at least twenty x400 fields of view.
Assessment of bronchial hyperresponsiveness
Bronchial hyperresponsiveness was assessed in a Buxco
plethysmograph (Buxco, Troy, NY) specifically designed for the low
tidal volumes of mice, as described previously (36). Sodium
pentobarbital (Butler, Columbus, OH; 0.04 mg/g of mouse body
weight)-anesthetized mice were intubated and constantly ventilated
using a Harvard pump ventilator (Harvard Apparatus, Reno, NV). The
following ventilation parameters were employed for each mouse: tidal
volume = 0.25 ml, breathing frequency = 120/min, and positive
end-expiratory pressure 
3 cm H2O. Within the sealed
plethysmograph mouse chamber, transpulmonary pressure (i.e., 
tracheal pressure - mouse chamber pressure) and inspiratory
volume or flow were monitored online by an adjacent computer. Airway
resistance was continuously calculated online by a specialized computer
software program (Buxco), and this measurement was the result of the
division of the transpulmonary pressure by the change in inspiratory
volume. Following a baseline period, mice received 10 µg of
methacholine by tail vein injection, and airway responsiveness to this
nonselective bronchoconstrictor was again calculated online.
Nonsensitized mice exhibited little change in airway resistance
following a similar challenge with methacholine. At the conclusion of
the assessment of airway responsiveness, a BAL was performed on each
mouse. The cell-free supernatant from each BAL sample was frozen at
-20°C before chemokine and cytokine ELISA. Whole lungs were then
dissected from the thoracic cavity, and snap frozen in liquid
N2 or prepared for histologic analysis.
Quantification of leukocytes in BAL
Cells suspended in the BAL were pelleted onto glass slides by cytocentrifugation and subjected to Diff-Quik (Baxter, McGraw Park, IL) staining, and polymorphonuclear and mononuclear cells were then quantified by light microscopy at x200 magnification.
Chemokine and cytokine ELISA analysis of BAL and whole lung
Murine eotaxin, MCP-1, C10, KC, MIP-2, MARC (mouse MCP-3),
MIP-1, IL-13, and IL-10 were determined in 50-µl supernatant
samples from cell-free BAL samples or whole lung homogenates using a
standardized sandwich ELISA (37). Briefly, Nunc-immuno ELISA plates
(MaxiSorp) were coated with the appropriate cytokine capture polyclonal
Ab at a dilution of 1–5 µg/ml of coating buffer (0.6 M NaCl; 0.26 M
H3BO4; 0.08 M NaOH; pH 9.6) for 16 h at
4°C. The rabbit polyclonal Abs directed against murine eotaxin, C10,
KC, IL-10, and IL-13 were purchased from R&D Systems. The rabbit
polyclonal Abs directed against murine MCP-1, MIP-2, and MARC were
purified from the sera of immunized rabbits, as previously described
(37). These rabbit polyclonal Abs were used for capture and detection
in the ELISA system, and the specificity of each was confirmed by
running a panel of recombinant cytokines and chemokines through each
ELISA (see panel listed above). The unbound capture Ab was washed away,
and each plate was blocked with 2% BSA-PBS for 90 min at 37°C. Each
ELISA plate was then washed with PBS Tween-20 (0.05%; v/v), and
50-µl samples either undiluted or diluted 1/10 were added to
duplicate wells and incubated for 1 h at 37°C. Following the
incubation period, the ELISA plates were then thoroughly washed and the
appropriate biotinylated polyclonal rabbit anti-cytokine Ab (3.5
µg/ml) was added. The polyclonal Abs were biotinylated using an
EZ-Link system from Pierce (Rockford, IL). After washing the plates 30
min later, streptavidin-peroxidase (Bio-Rad Laboratories, Richmond, CA)
was added to each well for 30 min, and each plate was thoroughly washed
again. Chromagen substrate (Bio-Rad Laboratories) was added, and
optical readings at 492 nm were obtained using an ELISA plate scanner.
Recombinant murine cytokines and chemokines were used to generate the
standard curves from which the concentrations present in the samples
were derived. The limit of ELISA detection for each cytokine was
consistently above 50 pg/ml.
Lung histologic analysis
Whole lungs from A. fumigatus-sensitized mice were fully inflated by intratracheal perfusion with 4% paraformaldehyde before dissection from the thoracic cavity and placement in fresh paraformaldehyde for 24 h. Routine histologic techniques were used to paraffin-embed this tissue, and 5-µm sections of whole lung were counterstained with Mayer’s hematoxylin (Mayer & Myles Laboratories, Coopersburg, PA) for the visualization and identification of eosinophils. Inflammatory infiltrates and other histologic changes were monitored around blood vessels and airways using light microscopy at a magnification of x200.
Determination of systemic IgE
Sera from A. fumigatus-sensitized mice were analyzed for total IgE before and following intratracheal A. fumigatus challenge. Complementary capture and detection Ab pairs for mouse IgE were obtained from PharMingen (San Diego, CA), and the IgE ELISA was performed according to the enclosed directions. Duplicate sera samples were diluted 1/100, IgE levels in each were calculated from OD readings at 492 nm, and IgE concentrations were calculated from a standard curve generated using rIgE (5–2000 pg/ml).
Data statistical analysis
All results are expressed as mean ± SEM (SE). All test conditions were completed in duplicate wells of a tissue culture plate. A Student’s t test was used to determine statistical significance between the control and anti-C10 groups; p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although C-C chemokines have previously been shown to exert
significant roles during airway allergic responses to a number of small
protein Ags, the expression of chemokines during experimental ABPA was
previously uncharacterized. The results from ELISA analysis of BAL
cell-free supernatants from our experimental model of ABPA for the
presence of C-C chemokines (i.e., MCP-1, eotaxin, MIP-1, and MARC),
CXC chemokines (i.e., MIP-2 and KC), and C10 are shown in Fig. 1
. Major elevations above baseline values
(measured in BAL from sensitized mice before intratracheal A.
fumigatus challenge) were observed in BAL levels of C10, MCP-1,
and eotaxin, but C10 was increased the greatest to approximately 30
ng/ml by 48 h after intratracheal challenge (Fig. 1). By
comparison, MCP-1 and eotaxin levels reached 5 and 1 ng/ml,
respectively, over the same period of time (Fig. 1). At 72 h after
A. fumigatus challenges, BAL levels of all of the chemokines
examined had returned to baseline values or were not detected by ELISA.
These findings suggested that the allergic airway responses to A.
fumigatus Ag involved profound changes in C10 generation,
particularly in the 48-h period after A. fumigatus Ag
challenge.
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Given the very dramatic elevations in C10 observed in the ABPA
mice following A. fumigatus challenge, the putative cellular
sources of C10 in the lung were next explored. Consistent with previous
studies showing that C10 is an IL-4-inducible chemokine in isolated
peritoneal macrophages (26), isolated alveolar macrophages from
nonsensitized mice were a constitutive source of C10, and IL-4 was the
most potent inducer of C10 release by these cells (Table I). Another immune cell, the MC-9 mast
cell, was a much poorer constitutive source of C10 (0.38 ± 0.02
ng/ml), and 48/80 activation of these mast cells did not markedly
augment their release of C10 (0.44 ± 0.02 ng/ml) above the
constitutive level. Our previous studies suggested that structural
cells in the lung such as lung fibroblasts and smooth muscle might also
generate C10 (38). Thus, the putative contribution of fibroblasts and
VSMCs to the overall C10 production within the lung was also examined.
As shown in Fig. 2, constitutive and
cytokine-inducible C10 production was detected in 24-h cultures of both
structural cell types. In VSMCs, only IL-1ß, either alone or in
combination with IL-4, IFN-, or IL-10, enhanced C10 levels
approximately 3-fold above levels detected in untreated (i.e., control)
cultures (Fig. 2). The presence of either IL-1ß, TNF-, or IL-10 in
cultures of pulmonary fibroblasts enhanced C10 production at least
2-fold above control levels (Fig. 2). C10 levels were also enhanced
between 3- and 5-fold (above control level) in cultures of pulmonary
fibroblasts that were incubated with similar combinations of cytokines
for 24 h. Taken together, these data indicated that alveolar
macrophages and pulmonary structural cells generated C10 in response to
inflammatory and immune cytokines.
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To evaluate the significance of changes in C10 generation within
the allergic airways, C10 was immunoneutralized in A.
fumigatus-sensitized mice 2 h before intratracheal challenge
with A. fumigatus Ag. C10 levels in BAL samples from both
treatment groups were markedly greater than levels measured in the
whole homogenates, consistent with the observation noted above that
alveolar macrophages were a major source of C10 particularly following
exposure to IL-4 (see Table I
). The successful immunoneutralization of
C10 using an i.p. injection of anti-C10 antiserum was confirmed in
BAL (Fig. 3) and in whole lung
homogenates (Fig. 3) removed before and 24 and 48 h after
intratracheal A. fumigatus Ag challenge. However, in the
lung homogenates at 48 h after A. fumigatus challenge,
C10 levels were not different between the two treatment groups.
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). Immunoneutralization of C10 in
A. fumigatus-sensitized mice reversed the increase in airway
hyperresponsiveness at 24 and 48 h after intratracheal challenge.
In addition, airway hyperresponsiveness to methacholine at 48 h
post-A. fumigatus challenge was significantly lower than
responses measured in allergic mice that received normal rabbit serum
(Fig. 4).
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The directed migration or chemotaxis of leukocytes down a
chemokine concentration gradient is a classical hallmark of chemokine
activation (39). The chemotaxis of eosinophils following chemokine
activation is limited to a select group of C-C chemokines that until
now did not include C10. However, the present experiments showed that
eosinophils from the peritoneal cavity of mice chronically infected
with S. mansoni exhibited marked chemotactic responses to
100 ng/ml of C10 (Table II).
Nevertheless, C10 was not as potent as similar concentrations of
eotaxin in the chemotaxis of eosinophils (Table II).
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As depicted in Fig. 5, the presence
of eosinophils in the BAL of allergic mice that received anti-C10
antiserum was significantly reduced at 24 and 48 h after A.
fumigatus challenge compared with allergic mice that were
pretreated with normal serum. Also at the 24-h timepoint after A.
fumigatus challenge, significantly fewer lymphocytes and
macrophages were present in cytospins of BAL (Fig. 5). However,
macrophage counts in the BAL of anti-C10 antiserum-pretreated mice
were significantly elevated at 48 h after Ag challenge compared
with BAL counts from mice that were pretreated with normal serum.
Neutrophil counts were not different between the two treatment groups.
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, A and B), at
24 h (Fig. 6, C and D), and at 48 h
(Fig. 6, E and F) after this challenge. Little
inflammation was present in either the normal serum (Fig. 6A) or anti-C10 antiserum (Fig. 6B) treatment
groups before the intratracheal challenge of these
Aspergillus-sensitized mice with A. fumigatus Ag.
After A. fumigatus challenge, dramatic histologic changes in
the lungs of ABPA mice included major inflammatory infiltrates
surrounding various sized airways and blood vessels at 24 h (Fig. 6C) and at 48 h (Fig. 6E) after A.
fumigatus Ag challenge. The inflammatory infiltrate around the
airways of these mice that received normal serum was composed of
abundant eosinophils, accompanied by smaller quantities of lymphocytes,
macrophages, and neutrophils. Conversely, a clear paucity of
inflammatory cells surrounding large airways was observed when ABPA
mice received anti-C10 antiserum, most notably at the 24-h (Fig. 6D) and 48-h (Fig. 6F) timepoints after A.
fumigatus challenge. Thus, these data suggested that C10 has a
major effect on the recruitment or movement of eosinophils and other
leukocytes into the airways during experimental ABPA.
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While the previous data suggested that C10 directly affected the
recruitment of leukocytes to the allergic airway, we next examined the
possibility that changes in recruitment were partly the consequence of
C10 modulating the generation of other chemotactic cytokines within the
lung. To test this postulate, specific ELISA examined MCP-1 and eotaxin
levels in BAL and whole lung homogenates. These C-C chemokines were
also elevated with C10 in the BAL from A.
fumigatus-challenged mice (see Fig. 1), and both have been shown
to be major participants in other eosinophil-mediated airway allergic
models (36, 40). Immunoneutralization of C10 in ABPA mice significantly
inhibited the levels of eotaxin and MCP-1 present in BAL and whole lung
homogenates before and at 24 h after intratracheal A.
fumigatus challenge (Fig. 7).
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Because IL-13 (41) and IL-10 (42) have been ascribed modulatory
roles in the Th2-type cytokine response, changes in both cytokines were
examined in the ABPA model. Interestingly, recent data have revealed
that IL-10 appears to suppress the production of Th1 and Th2 cytokines,
and the bronchial inflammation associated with experimental ABPA (13).
The experimental model of ABPA examined in the present study was
characterized by a robust increase in IL-13 levels within whole lung
homogenates to 75 ng/ml at 24 h and approximately 100 ng/ml at
48 h after A. fumigatus challenge. (Fig. 8). Immunoneutralization of C10
significantly reduced IL-13 levels at the 48-h timepoint. In contrast,
at 24 and 48 h after Aspergillus challenge, IL-10
levels were reduced significantly by greater than 50% in both
treatment groups (Fig. 8).
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A hallmark of clinical ABPA is increased systemic IgE levels (43).
Similar to the clinical condition, the murine model of ABPA used in the
present study showed similar dramatic changes in serum levels of IgE
(Fig. 9). Although lower levels of IgE
were observed before and at 48 h after Aspergillus
intratracheal challenge, these changes did not reach statistical
significance. These findings are interesting when considering previous
studies showing that allergic responses to A. fumigatus
proceed even in the absence of IgE (12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sources of C10 include a diverse array of cells from macrophage (26) to trigeminal ganglia (44). Based on our previous studies of a Th2-type pulmonary granulomatous response in the lung (38), we postulated that a number of cell types in the lung might also be capable of generating C10. Accordingly, alveolar macrophages, VSMCs, and fibroblasts from lung were constitutive generators of C10, and all of these cells augmented C10 production following exposure to cytokines. Coinciding with a previous report (26), IL-4 was a potent stimulator of C10 production by alveolar macrophages. However, in cultures of pulmonary fibroblasts and VSMCs, IL-1ß was the most potent stimulant of C10 production. This finding is notable because of prior observations showing that IL-1 is rapidly induced following intrapulmonary A. fumigatus challenge in mice (45). Thus, pulmonary resident cells following exposure to inflammatory or immune cytokines are capable of augmenting C10 synthesis, and this response possibly explains the major changes in C10 observed during A. fumigatus-induced airway inflammation.
The enhanced generation of C10 following A. fumigatus
challenge appears to provide an important signal for the recruitment of
inflammatory leukocytes into the lungs. The infiltration of eosinophils
and T cells into the lungs is a major feature of allergic airway
inflammation, but the precise role of these leukocytes in allergic
disease is still being explored. Although the profound suppression of
eosinophilia using anti-IL-5 (10), anti-MIP-1, or
anti-RANTES Abs (36) did not reduce the airway hyperresponsiveness
in allergic mice, eosinophils are a significant source of cationic and
major basic proteins that are toxic to the airway epithelium (19), and
these cells are involved in tissue remodeling during chronic pulmonary
inflammation (46). As mentioned above, CD4+ T cells,
particularly those that express Th2 cytokines such as IL-4 and IL-5,
are abundant and appear to contribute to the airway inflammation and
hyperresponsiveness associated with many experimental allergic
responses in the lungs, including ABPA (7, 47). Indeed, clear evidence
shows that the attenuation of T cell recruitment to the allergic lung
through anti-MCP-1 (36, 40) or anti-MCP-3 (48) Ab treatments
results in a pronounced decrease in airway inflammation and
hyperresponsiveness. Based on previous studies demonstrating that C10
is a chemoattractant for CD4+ T cells (26) and our present
data showing that C10 is chemotactic for eosinophils, the
immunoneutralization of C10 during experimental ABPA presumably
attenuates the movement of both cell types into the allergen-challenged
lungs. In addition, the absence of recruited leukocytes in the BAL and
airways of anti-C10 Ab-treated mice may also explain, in part, the
decreased C10 levels measured in these mice. Thus, the
immunoneutralization of C10 during the allergic response to A.
fumigatus abolishes the recruitment of key immune effector
cells, and stems the augmentation of C10 synthesis within the lung.
Other observations in the ABPA model during the suppression of endogenous C10 activity indicated that C10 possibly modulates the levels of other mediators previously shown to be present during or to contribute to the development of allergic responses. MCP-1 and eotaxin are two examples of a proallergic mediator (36, 40, 49). Although the diminution of MCP-1 and eotaxin levels observed in the present study may reflect the reduction in recruited leukocytes in the lungs, pulmonary resident cells are also excellent sources of both chemokines (32, 50). Furthermore, the lack of endogenous C10 may have directly affected the ability of these cells to generate MCP-1 and eotaxin. IL-13 is a product of T cells and alveolar macrophages (51) that induces Ig production, B cell proliferation, and monocyte differentiation. In addition, IL-13 and IL-4 share the same cell activation pathway (52), which may explain why allergic responses to A. fumigatus develop normally in mice genetically deficient in IL-4 (8). Consistent with the decrease in lymphocyte recruitment in anti-C10 antiserum-treated mice, less IL-13 was also detected in whole lung homogenates from these mice. IL-10 appears to exert a prominent role during pulmonary responses to A. fumigatus that relates to the ability of this cytokine to balance the Th1 and Th2 cytokine responses in this model (13). The presence of IL-10 also suppresses the oxidative burst response, but enhances the phagocytic activity of mononuclear cells exposed to Aspergillus (53). In the present study, IL-10 levels in whole lung homogenates were significantly attenuated as the allergic response progressed, but additional changes in IL-10 were not observed in the ABPA model when C10 was inhibited. The relative impact of reduced pulmonary IL-10 during the progression of the allergic response to A. fumigatus is not presently apparent, and warrants further study. In addition, the mechanism through which C10 modulates the synthesis of MCP-1, eotaxin, and IL-13 within the allergic lungs deserves further attention.
In patients with clinical ABPA, elevations of IgE correlate both with allergic inflammation of the airways and with bronchial hyperresponsiveness (2). Whereas a central role for IgE in the pathogenesis of the eosinophilic inflammation as well as in the obstructive airway physiology associated with ABPA has been suggested, experimental ABPA proceeds normally in IgE-deficient mice (12). The present data demonstrate that although C10 effectively abolishes many of the features of experimental ABPA, this chemokine does not appear to affect serum levels of IgE. Therefore, these findings suggest that C10 may not participate in the sensitization process to A. fumigatus Ags.
Chemokines have been shown to exert defined effects on leukocyte recruitment to the allergic airways and the development of airway hyperresponsiveness in experimental models (36, 40, 54). The role of chemokines in the pathogenesis of ABPA is less well defined, but the results from the present study show that the C10 chemokine exerts a prominent role in the development of lung inflammation and bronchial hyperresponsiveness. Further examination of the role of these types of chemokines in clinical and experimental ABPA may be warranted.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cory M. Hogaboam, Department of Pathology, University of Michigan Medical School, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. E-mail address:
3 Abbreviations used in this paper: ABPA, allergic bronchopulmonary aspergillosis; BAL, bronchoalveolar lavage; MCP, monocyte-chemoattractant protein; MIP, macrophage-inflammatory protein; VSMC, vascular smooth muscle cell.
Received for publication November 30, 1998. Accepted for publication February 22, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0.05 compared with the corresponding control mice that received
normal rabbit serum before A. fumigatus Ag
challenge.
, p = 0.034 compared with the corresponding
control mice that received normal rabbit serum before A.
fumigatus Ag challenge.
