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*
Department of Pathology and
Division of Pulmonary and Critical Care, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109;
Department of Pathology, Veteran Affairs Medical Center, Ann Arbor, MI 48105; and
§
Department of Medicine, Children’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and RANTES were significantly elevated after A.
fumigatus-sensitized mice received an intrapulmonary challenge
with A. fumigatus spores or conidia, the present study
addressed the role of their receptor, C-C chemokine receptor 1 (CCR1),
in this model. A. fumigatus-sensitized CCR1 wild-type
(+/+) and CCR1 knockout (-/-) mice exhibited similar increases in
serum IgE and polymorphonuclear leukocyte numbers in the
bronchoalveolar lavage. Airway hyperresponsiveness was prominent in
both groups of mice at 30 days after an intrapulmonary challenge with
A. fumigatus spores or conidia. However, whole lung
levels of IFN-
were significantly higher whereas IL-4, IL-13, and
Th2-inducible chemokines such as C10, eotaxin, and macrophage-derived
chemokine were significantly lower in whole lung samples from
CCR1-/- mice compared with CCR1+/+ mice at 30
days after the conidia challenge. Likewise, significantly fewer goblet
cells and less subepithelial fibrosis were observed around large
airways in CCR1-/- mice at the same time after the
conidia challenge. Thus, these findings demonstrate that CCR1 is a
major contributor to the airway remodeling responses that arise from
A. fumigatus-induced allergic airway
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines are one common link between the immune and nonimmune cells
that contribute to asthmatic and allergic airway diseases. In addition
to the Th2 cytokines, chemotactic cytokines or chemokines have been
shown to be present and/or involved during these diseases
(11). Monocyte chemoattractant protein-1
(MCP-1),3 RANTES, and
macrophage-inflammatory protein 1 (MIP-1) are major examples of
C-C chemokines detected in bronchoalveolar lavage (BAL) isolates from
asthmatics (12, 13). All three chemokines exert major
effects on the recruitment of eosinophilic leukocytes into the airways
during experimental allergic disease (14, 15, 16). In
addition, neutralization of MCP-1 and RANTES markedly attenuates airway
hyperresponsiveness, whereas anti-MIP-1 Ab treatment only
marginally reduced this parameter during OVA-induced allergic airway
disease (15). Nevertheless, because most C-C chemokine
receptors have overlapping chemokine and leukocyte specificity, the
absence of one chemokine can often be compensated for by other
chemokines during disease processes (17). This fact has
directed research toward identifying chemokine receptors that are
potential targets in the treatment of asthmatic and allergic
disease (18). Interest in the role of C-C chemokine
receptor 1 (CCR1) during allergic airway disease stems from the fact
that CCR1 binds MIP-1, RANTES, and MCP-3 and is expressed on a
number of leukocytes including neutrophils, monocytes, lymphocytes, and
eosinophils. All three chemokine ligands have been shown to have
prominent effects on the recruitment of these leukocytes during
allergic airway disease (11). In addition, MIP-1 has
been shown to be involved in the pulmonary fibrotic response
(19) due to its chemotactic effects on mononuclear
phagocyte accumulation after bleomycin challenge (20).
Nonetheless, the role of CCR1 on the progression of the airway
remodeling response during chronic allergic airway disease has not been
previously examined.
Thus, the aim of the present study was to determine whether CCR1 had a distinct role in the development of chronic allergic airway disease due to A. fumigatus conidia. In the present study, we examined the response of A. fumigatus-sensitized CCR1-/- mice to an intrapulmonary challenge with live A. fumigatus conidia. CCR1-/- mice showed similar changes in leukocyte recruitment into the airways and airway hyperresponsiveness compared with their wild-type counterparts at all times after the conidia challenge. However, CCR1-/- mice showed markedly less histological and biochemical evidence of goblet cell hyperplasia and airway fibrosis compared with their wild-type controls. These findings suggest that CCR1 has a major role in the airway remodeling response during chronic allergic airway disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs of mice with a targeted disruption of the CCR1 gene (CCR1-/-; C57BL/6 (B6) x 129Sv intercrossed 10–20 generations) were provided by Dr. Craig J. Gerard. A breeding colony of these mice was maintained under specific pathogen-free conditions at the University Laboratory Animal Medicine facility at the University of Michigan Medical School. As previously described (21), CCR1-/- mice showed no overt evidence of abnormal breeding or growth patterns while maintained in the University Laboratory Animal Medicine facility. Age-matched, wild-type control (CCR1+/+) B6 x 129Sv (intercrossed 10–20 generations) were obtained from Dr. Sergio Lira (Schering-Plough Research Institute, Kenilworth, NJ) or purchased from The Jackson Laboratory (Bar Harbor, ME).
A novel model of chronic allergic aspergillosis
We have recently described a novel model of chronic allergic airway disease induced by A. fumigatus that exhibits airway inflammation, hyperresponsiveness, and remodeling (22). CCR1-/- and CCR1+/+ mice were similarly sensitized to a commercially available preparation of soluble A. fumigatus Ags, and the sensitization status of each mouse was confirmed by the presence of total IgE in serum (23). Seven days after the third intranasal challenge, each mouse received 5.0 x 106 A. fumigatus conidia suspended in 30 µl 0.1% Tween 80 via the intratracheal route (22).
Measurement of bronchial hyperresponsiveness
Immediately before and at days 3, 7, 21, and 30 after an
intratracheal A. fumigatus conidia challenge, bronchial
hyperresponsiveness was assessed in a Buxco plethysmograph (Buxco,
Troy, NY) as previously described (22, 23). Sodium
pentobarbital (Butler, Columbus, OH, 0.04 mg/g of mouse body weight)
was used to anesthetize mice before their intubation, and ventilation
was with a Harvard pump ventilator (Harvard Apparatus, Reno, NV)
(23). Once baseline airway resistance was established, 10
µg of methacholine were introduced into each mouse via a cannulated
tail vein, and airway hyperresponsiveness was monitored for 
3 min.
The peak increase in airway resistance was then recorded. The 10-µg
dose of methacholine was used because it elicited <2-fold increases in
airway hyperresponsiveness in nonsensitized mice. After the assessment
of airway hyperresponsiveness, a BAL was performed using 1 ml
filter-sterilized normal saline. Approximately 500 µl of blood
removed from each mouse were centrifuged at 15,000 rpm for 10 min to
yield serum. Finally, whole lungs were dissected from each mouse
and snap frozen in liquid N2 or fixed in 10%
formalin for histological analysis (see below).
Preparation of cDNA and RT-PCR amplification
Total RNA samples were prepared from whole lung samples removed from CCR1+/+ mice at days 3, 7, 21, and 30 after conidia challenge (24). RNA from specific samples was reverse transcribed into cDNA utilizing a BRL reverse transcription kit and oligo(dT)12–18 primer. The amplification solution contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), and 2.5 mM MgCl2. Specific oligonucleotide primers were added (200 ng/sample) to the buffer, along with 5 µl reverse transcribed cDNA sample. The following oligonucleotide primers were used. CCR1 primer sequences: sense, 5'-GACCAGCATCTACCTGTTCA-3'; antisense, 5'-GCAGAAACAAATACACTCAG-3', 587-bp product. ß-actin primer sequences: sense, 5'-GCTCGGCCGTGGTGGTGAAGC-3'; antisense, 5'-GTGGGGCGCCCCAGGCACCA-3', 450-bp product.
The cDNA was amplified using the following cycling parameters. The mixture was first incubated for 4 min at 94°C and then cycled 35 times at 94°C for 45 s, 55°C for 45 s, and elongated at 72°C for 45 s. After amplification, the samples were separated on a 2% agarose gel containing 0.3 µg/ml ethidium bromide, and bands were visualized and photographed using a translucent UV source.
Morphometric analysis of leukocyte accumulation in BAL samples
Neutrophils, macrophages, eosinophils, and lymphocytes were quantified in BAL samples cytospun (Shandon Scientific, Runcorn, U.K.) onto coded microscope slides. Each slide was stained with a Wright-Giemsa differential stain, and the average number of each cell type was determined after counting a total of 300 cells in 10–20 high power fields (x1000) per slide. A total of 1 x 106 BAL cells were cytospun onto each slide to compensate for differences in cell retrieval.
ELISA analysis
Murine MIP-1, RANTES, MCP-3, IFN-, IL-4, IL-5, IL-13, C10,
MDC, and eotaxin protein levels were determined in 50-µl samples from
whole lung homogenates using a standardized sandwich ELISA technique
previously described in detail (25). Serum IgE was also
determined using an ELISA technique. Each ELISA was screened to ensure
Ab specificity and recombinant murine cytokines, chemokines, and IgE
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.
Whole lung histological analysis
Whole lungs from A. fumigatus-sensitized CCR1-/- and CCR1+/+ mice before and at various times after A. fumigatus conidia challenge were fully inflated with 10% formalin, dissected, and placed in fresh 10% formalin for 24 h. Routine histological techniques were used to paraffin embed the entire lung, and 5-µm sections of whole lung were stained with Gomori methanamine silver (GMS), periodic acid-Schiff reagent (PAS), or Masson trichrome. Morphological evaluations of inflammatory infiltrates and structural alterations were determined around blood vessels and airways using light microscopy at a magnification of x1000.
Hydroxyproline assay
Hydroxyproline concentrations were determined using a previously described assay (26). Processed whole lung samples were added in triplicate to 96-well plates and then incubated at room temperature for 20 min before the addition of 100 µl Ehrlich’s solution (Aldrich, Milwaukee, WI). These samples were subsequently incubated for 15 min at 65°C and cooled to room temperature before the 96-well plate was read at 550 nm in an ELISA plate scanner. Hydroxyproline concentrations were calculated from a standard curve of known hydroxyproline concentrations of 0–100 µg/ml.
Statistical analysis
All results are expressed as mean ± SEM (SE). Student’s t test or ANOVA and a Student-Newman-Keuls multiple comparison test were used to determine statistical significance between CCR1+/+ and CCR1-/- mice at various times after the conidia challenge; p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and RANTES are significantly elevated in
CCR1-/- mice during chronic fungal allergic airway
disease
We first examined changes in immunoreactive levels of MIP-1,
RANTES, and MCP-3 in whole lung homogenates from CCR1+/+
mice. As illustrated in Fig. 1
A, before the conidia
challenge, whole lung concentrations of MIP-1 were at the limit of
ELISA detection at 50 pg/ml. Compared with MIP-1 levels measured
before the conidia challenge, significantly higher levels of this
chemokine were detected in whole lung samples from CCR1+/+
mice at days 21 and 30 after the conidia challenge. Similarly, RANTES
levels in whole lung homogenates were also significantly elevated, but
only at day 7 after the conidia challenge (Fig. 1B). MCP-3
levels in whole lung homogenates did not reach concentrations that were
detectable by ELISA at any time before or after the conidia challenge
(data not shown). Thus, these initial observations suggested that the
pulmonary exposure of A. fumigatus-sensitized mice to live
A. fumigatus conidia significantly increased the pulmonary
concentrations of CCR1 ligands such as MIP-1 and RANTES.
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RT-PCR analysis of CCR1 mRNA in whole lung samples was conducted
at various times after the introduction of A. fumigatus
conidia into CCR1+/+ mice. CCR1 mRNA was detected at days
7, 21, and 30 after the conidia challenge, but the greatest expression
(based on densitometry analysis) of CCR1 mRNA was observed at day 30
after the conidia challenge (Fig. 2). Thus,
these data suggested that CCR1 mRNA expression was altered during the
course of the A. fumigatus conidia challenge, and the
greatest level of CCR1 mRNA was present at day 30 after conidia.
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With the evidence that MIP-1 and CCR1 were increased after the
intrapulmonary challenge of CCR1+/+ mice with A.
fumigatus conidia, we next compared the nature of the airway
response in CCR1+/+ mice to that in mice-lacking CCR1
through gene targeting (27). Serum IgE levels are shown in
Fig. 3, and at no time before or after the
conidia challenge were statistical differences in IgE levels detected
between the two groups of mice. These data suggested that the
development of an IgE response to soluble A.
fumigatus Ags was not dependent on the presence of CCR1.
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Previous studies suggested that function and chemotaxis of
peripheral neutrophils to MIP-1 was greatly impaired in
CCR1-/- mice (27). As a consequence,
CCR1-/- mice were markedly more susceptible to invasive
aspergillosis when A. fumigatus conidia were introduced
systemically into these mice (27). However, previous
studies have not addressed the consequences of an intrapulmonary
challenge of A. fumigatus conidia in CCR1-/-
mice. In the present study, no lethality was observed in A.
fumigatus-sensitized CCR1-/- or CCR1+/+
mice that received an intrapulmonary challenge with A.
fumigatus conidia (data not shown), presumably because of a marked
influx of neutrophils into the lungs of both groups of mice (Fig. 4B). Eosinophil (Fig. 4A) numbers in BAL samples were similar in
CCR1-/- and CCR1+/+ mice at all times after
the conidia challenge. Lymphocyte (Fig. 4C) and macrophage
(Fig. 4D) numbers in the BAL of CCR1-/- mice
were significantly greater at day 30 after conidia than were the
quantities of the same cells in the BAL of CCR1+/+ mice. A
second experiment with CCR1-/- and CCR1+/+
sensitized and challenged mice (n = 4/group) revealed
similar changes in BAL leukocyte numbers after the A.
fumigatus conidia challenge (data not shown). Taken together,
these data suggested that CCR1 has a minor role in the chemotaxis of
polymorphonuclear cells but modulates the chronic recruitment of
lymphocytes and macrophages into the lungs of mice experiencing
prolonged allergic airway disease.
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Airway hyperresponsiveness as revealed by a nonspecific
bronchoconstrictor such as methacholine is a hallmark of the allergic
airway response to A. fumigatus Ags. We have previously
observed that mice sensitized to A. fumigatus before an
intrapulmonary challenge exhibited airway hyperresponsiveness that
persisted for up to 30 days after the conidia challenge. As shown in
Fig. 5, CCR1-/- and
CCR1+/+ mice showed pronounced, 4- to 6-fold, increases in
airway resistance at all times after the conidia challenge compared
with airway resistance measured in both groups before the conidia
challenge. A second experiment with CCR1-/- and
CCR1+/+ sensitized and challenged mice (n =
4/group) revealed similar changes in airway hyperresponsiveness, but no
differences were observed between these groups at any time after
conidia challenge (data not shown). These data suggested that CCR1 is
not necessary for the development of airway hyperresponsiveness during
chronic fungal allergic airway disease.
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in CCR1-/-
mice
The cytokine balance in various inflammatory responses in
CCR1-/- mice is skewed toward Th1 cytokine production
(27, 28). We next examined whether a similar skewing was
present in our chronic model of allergic airway disease. Changes in
whole lung levels of IFN- shown in Fig. 6 were similar in CCR1-/- and
CCR1+/+ mice at days 3, 7, and 21 after the conidia
challenge. However, at day 30 after conidia, significantly greater
levels of IFN- were present in whole lung samples from
CCR1-/- mice compared with similar samples from
CCR1+/+ mice. A second experiment with
CCR1-/- and CCR1+/+ sensitized and challenged
mice revealed a similar significant difference in whole lung IFN- at
30 days between these groups (n = 4) after the conidia
challenge (not shown). These data suggested that CCR1-/-
mice had greater pulmonary concentrations of IFN- than their
wild-type counterparts, but this increase was observed only at day 30
after conidia.
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Our previous studies showed that the introduction of live A.
fumigatus conidia into A. fumigatus-sensitized mice
resulted in a marked increase in the production of Th2 cytokines such
as IL-4 and IL-13. The absence of CCR1 markedly affected the production
of Th2 cytokines in whole lung homogenates as illustrated in Fig. 7. Significantly lower whole lung
concentrations of IL-4 were observed in CCR1-/- mice at
days 21 and 30 after conidia compared with those in CCR1+/+
mice at similar times (Fig. 7A). Likewise, whole lung
concentrations of IL-13 in CCR1-/- mice were
significantly lower at day 21 after the conidia challenge compared
again with the wild-type group at this time (Fig. 7B).
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). All three chemokines have been shown to
exert prominent effects in acute models of allergic airway disease
(16, 23, 29). Immunoreactive levels of C10 (Fig. 8A), MDC (Fig. 8B), and eotaxin (Fig. 8C) in CCR1-/- mice were lower than levels
measured in CCR1+/+ mice at all times after the conidia
challenge, but differences in chemokine levels between the two groups
only reached statistical significance at day 30 after conidia. A second
experiment with CCR1-/- and CCR1+/+
sensitized and challenged mice revealed a similar significant decrease
in whole lung concentrations of Th2 cytokines and chemokines in
CCR1-/- mice (n = 4) compared with their
wild-type controls at 30 days after the conidia challenge (not shown).
Thus, these data demonstrated that the Th2 cytokine response in
A. fumigatus-sensitized CCR1-/- mice was
significantly diminished at days 21 and 30 after conidia challenge.
Likewise, the decreased Th2 cytokine levels in CCR1-/-
mice at day 30 after the conidia challenge was also associated with
decreased levels of Th2-inducible chemokines.
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Given the major differences in cytokines and chemokines measured
at day 30 after the conidia challenge, a histological analysis of whole
lung sections from CCR1+/+ and CCR1-/- mice
at this time was performed and is summarized in Fig. 9. GMS staining is used to reveal the
presence of fungal elements in tissue, and the application of this
stain to whole lung tissue sections showed that alveolar macrophages
were positive for fungal Ags in CCR1+/+ mice at day 30
after the conidia challenge (Fig. 9A). Similar staining was
absent in CCR1-/- mice at this time (Fig. 9B).
The staining of goblet cells in whole lung sections with the PAS stain
revealed that markedly more mucus-containing cells were present in the
airways of CCR1+/+ mice (Fig. 9C) compared with
CCR1-/- mice (Fig. 9D) at day 30 after the
conidia challenge. In addition, Masson trichrome staining of both
groups of mice suggested that markedly more collagen was present around
the airways of CCR1+/+ mice (Fig. 9E) compared
with CCR1-/- mice (Fig. 9F) at day 30 after
the conidia challenge. These histological findings at 30 days after the
conidia challenge were confirmed in a second experiment with
CCR1-/- and CCR1+/+ sensitized and challenged
mice (not shown). Taken together, these data suggested that airway
remodeling due to chronic allergic airway disease was dependent on the
presence of CCR1.
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). Similar
levels of hydroxyproline were measured in whole lung homogenates from
CCR1-/- and CCR1+/+ mice before and at days
3, 7, and 21 after conidia. In contrast, at day 30 after the conidia
challenge, CCR1+/+ mice had significantly greater levels of
hydroxyproline in whole lung homogenates compared with
CCR1-/- mice at the same time. These findings were
confirmed in a second experiment with CCR1-/- and
CCR1+/+ sensitized and challenged mice (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Temporal examination of chemokines in whole lung homogenates from
A. fumigatus-sensitized and challenged CCR1 wild-type (+/+)
mice revealed that RANTES and MIP-1 levels were significantly
elevated above baseline (i.e., before the conidia challenge) at various
times after the conidia challenge. RT-PCR analysis also showed that
CCR1 mRNA was markedly increased in the lungs of CCR1+/+
mice at day 30 after the conidia challenge. Therefore, the present
study addressed the role of CCR1 in the airway-inflammatory and
remodeling events during chronic allergic aspergillosis. After
intrapulmonary exposure to live A. fumigatus conidia,
A. fumigatus-sensitized mice lacking CCR1
(CCR1-/-) showed similar changes in polymorphonuclear
leukocyte accumulation and airway hyperresponsiveness compared with
wild-type CCR1+/+ mice. However, the cytokine profile in
CCR1-/- mice was skewed away from a Th2 cytokine and
chemokine profile in favor of a Th1 cytokine profile characterized by
increased IFN-. As revealed by GMS staining, A. fumigatus
was detected in the lungs of CCR1+/+ mice, but not
CCR1-/- mice, at day 30 after the conidia challenge. In
addition, chronic airway remodeling features such as goblet cell
hyperplasia and peribronchial fibrosis that were present in
CCR1+/+ mice were absent in the airways of
CCR1-/- mice.
Previous studies by Gao et al. (27) suggested that
nonsensitized CCR1-/- mice were markedly more susceptible
than nonsensitized CCR1+/+ mice to the lethal effects of
5.0 x 106 A. fumigatus conidia
delivered i.v. The inability of CCR1-/- mice to control
the growth of A. fumigatus in various organs was attributed
to a defect in neutrophil recruitment from the periphery into infected
organs (27). Contrary to these former observations, we did
not observe any lethality in A. fumigatus-sensitized
CCR1-/- mice given an intrapulmonary challenge of
5.0 x 106 live conidia. GMS-staining of
histological lung sections showed that there was also no evidence of
invasive disease and that Aspergillus Ag did not persist in
the lungs of A. fumigatus-sensitized CCR1-/-
mice in contrast with CCR1+/+ mice. Sensitized
CCR1-/- mice presumably survived after the intrapulmonary
challenge with conidia because the TNF--dependent innate antifungal
response initiated by alveolar macrophages remained intact in these
mice (30, 31, 32). Indeed, LPS-challenged mononuclear cells
from CCR1-/- mice have been shown to generate
significantly greater amounts of TNF- compared with similar cells
from CCR1+/+ mice (28). BAL samples from
A. fumigatus-sensitized CCR1-/- mice contained
significantly more macrophages than similar samples from their
wild-type counterparts at day 30 after the conidia challenge. Topham et
al. (28) also demonstrated increased macrophage
trafficking, somewhat similar to the results in the present study; in
contrast, this was associated with increased severity of renal damage.
Increased severity of airway inflammation in the present study may be
associated with the increase in macrophage numbers because conidia
clearance has been shown to be dependent on macrophage phagocytosis. In
addition, neutrophil recruitment into the airways of A.
fumigatus-sensitized CCR1-/- mice was not inhibited
at any time after the conidia challenge. These last findings are
consistent with our preliminary data that CCR2 (C. Hogaboam,
unpublished observations) and CXCR2 facilitate neutrophil recruitment
in the absence of CCR1 (B. Mehrad, unpublished observations). In
contrast, Gerard et al. (21) demonstrated decreased
neutrophil-mediated lung damage in CCR1-/- animals;
however, this model of secondary respiratory distress syndrome in
response to acute pancreatitis suggests a differential role of CCR1 in
the development of lung inflammation between these two models. Taken
together, the present findings suggest that CCR1 expression in
sensitized mice is not necessary for the pulmonary immune response
against live A. fumigatus conidia. Future studies will
address the pulmonary remodeling outcome after intrapulmonary challenge
with A. fumigatus conidia in nonsensitized
CCR1-/- and CCR1+/+ mice.
Intermittent reversible airway hyperresponsiveness and obstruction
characterizes human asthma. In animal models of asthma, airway
hyperresponsiveness is an exaggerated bronchoconstrictor response that
would have little physiological consequence in a nonsensitized mouse
(33). Because airway resistance can be examined in murine
models of allergic airway disease using stimulus-independent
bronchoconstrictors, a number of studies have examined the role of
various cytokines in this response. Unlike RANTES (15) and
MCP-3 (34), the immunoneutralization of MIP-1 did not
appear to markedly affect the development of airway hyperresponsiveness
in an acute model of OVA-induced allergic airway disease
(15). Inasmuch as Topham et al. (28) recently
demonstrated that MIP-1 is the major functional ligand for mouse
CCR1, our finding that the lack of CCR1 did not prevent the development
of airway hyperresponsiveness during chronic allergic airway disease is
not unexpected. Although RANTES and MCP-3 have been described as
ligands for mouse CCR1, these ligands appear to function more
effectively through CCR5 and CCR3, respectively (28). The
unencumbered development of airway hyperreactivity in
CCR1-/- mice probably reflects the fact that
polymorphonuclear leukocyte recruitment into the airways of
CCR1-/- was similar or enhanced compared with
CCR1+/+ mice at all times after the conidia challenge. Once
again, this finding was not unexpected in light of other studies
showing that the lack of CCR1 did not markedly diminish the recruitment
of polymorphonuclear leukocytes into the inflamed kidney
(28). Thus, the lack of CCR1 during chronic allergic
aspergillosis did not prevent the development or persistence of airway
hyperresponsiveness.
Irreversible airway remodeling is an insidious result of chronic allergic inflammation in atopic asthma, which involves the thickening of the airway wall due to increased goblet cell numbers in the epithelium and subepithelial collagen deposition (3, 35). Although airway remodeling contributes significantly to the airflow obstruction, airway edema, and mucus hypersecretion observed in asthmatics, considerable interest and debate surrounds the impact of airway remodeling on the hyperresponsiveness of the airway to contractile provocation (36). Some studies have indicated that the severity of asthma is directly related to the intensity of the airway remodeling response (37) (38). However, data proving a causal relationship between airway remodeling and asthma severity remain elusive. Other studies suggest that although increased subepithelial fibrosis is evident in asthmatic airways, it was not an indicator of the differences in severity of asthma (39). The results from the present study support the latter observation because a marked divergence between these two events in our model was most apparent at day 30 after the conidia challenge. Although CCR1-/- and CCR1+/+ mice had airway inflammation and hyperresponsiveness at all times after the conidia challenge, CCR1-/- mice did not exhibit the airway remodeling present in the wild-type mice.
The absence of airway remodeling in the airways of the
CCR1-/- mouse, particularly at day 30 after the conidia
challenge, may be partly explained by the increased presence of IFN-
concomitant with decreased Th2 cytokines and Th2-inducible chemokines
in these mice. Previous studies have demonstrated that Th1 cytokines or
Th1 cells can be used to balance the aggressive Th2 response associated
with asthma and allergy (40), thereby attenuating many of
the features of experimental allergic airway disease (41, 42). Interestingly, the modulatory effects of Th1 cells
introduced into mice with Th2 cell-mediated allergic airway disease
were not observed until after 9 days had elapsed (42).
Goblet cell hyperplasia and subepithelial fibrosis associated with the
bronchi of asthmatics appear to be triggered by cytokines and growth
factors secreted by IL-4-, IL-5-, and IL-13-activated immune and
nonimmune cells (43). Th2 cytokines have also been shown
to directly activate goblet cells (42, 44) and fibroblasts
(45, 46). The significance of IL-13 in airway remodeling
was recently highlighted in an IL-13-transgenic mouse that exhibited
mucus cell metaplasia, and airway fibrosis and obstruction
(47). Th2 cytokines also induce chemokines such as C10,
MDC, and eotaxin that contribute in unique ways to the development of
acute allergic airway disease (11). An explanation for the
divergence and modulation of cytokine production in
CCR1-/- mice experiencing chronic fungal allergic airway
disease is not presently known, but our findings are consistent with
those recently published pertaining to the development of renal injury
in these mice (28). In this prior study, the lack of CCR1
was not associated with defects in polymorphonuclear leukocyte
recruitment but rather in the skewing of the cytokine production in
favor of Th1 cytokines. Similar to the results shown in the present
study, Gao et al. (27) measured increased IFN- and
decreased IL-4 concentrations in CCR1-/- mice compared
with controls after Schistosoma mansoni i.v. injection. In
this study, CCR1-/- mice exhibited a reduction in the
size of lung granulomas compared with CCR1+/+ littermates,
similar to the reduction of fibrosis in the lungs of A.
fumigatus-challenged CCR1-/- mice in the present
study. Ongoing studies will address the precise role of CCR1 on the
cytokine synthetic capacity of Th1 and Th2 cells from our model of
fungal allergic airway disease.
In conclusion, the data from the present study show that CCR1 does not regulate polymorphonuclear leukocyte recruitment into the airways or airway hyperresponsiveness due to experimental fungal asthma. Instead this chemokine receptor appeared to have a prominent role in the regulation of Th2 cytokines and chemokines that have been implicated in the allergic airway remodeling response. Recognizing that more research is required to identify factors that promote the remodeling of airways during asthmatic and allergic airway diseases (35, 38), it is conceivable that therapy directed against CCR1 may prove clinically useful in the prevention of airway remodeling in these diseases.
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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 Road, Ann Arbor, MI 48109-0602.
3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; CCR1, chemokine receptor 1; MIP-1, macrophage-inflammatory protein-1; BAL, bronchoalveolar lavage; GMS, Gomori methanamine silver; PAS, periodic acid-Schiff.
Received for publication February 15, 2000. Accepted for publication May 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in pulmonary host defense in murine invasive aspergillosis. J. Immunol. 162:1633. regulate allergic airway inflammation and mucus production. J. Exp. Med. 190:1309.This article has been cited by other articles:
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  M. A. Schaller, L. E. Kallal, and N. W. Lukacs A Key Role for CC Chemokine Receptor 1 in T-Cell-Mediated Respiratory Inflammation Am. J. Pathol., February 1, 2008; 172(2): 386 - 394. [Abstract] [Full Text] [PDF]
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 P. Joubert, S. Lajoie-Kadoch, M. Welman, S. Dragon, S. Letuvee, B. Tolloczko, A. J. Halayko, A. S. Gounni, K. Maghni, and Q. Hamid Expression and Regulation of CCR1 by Airway Smooth Muscle Cells in Asthma J. Immunol., January 15, 2008; 180(2): 1268 - 1275. [Abstract] [Full Text] [PDF]
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S. R. Beaty, C. E. Rose Jr., and S.-s. J. Sung Diverse and Potent Chemokine Production by Lung CD11bhigh Dendritic Cells in Homeostasis and in Allergic Lung Inflammation J. Immunol., February 1, 2007; 178(3): 1882 - 1895. [Abstract] [Full Text] [PDF]
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 A. L. Mora, E. Torres-Gonzalez, M. Rojas, C. Corredor, J. Ritzenthaler, J. Xu, J. Roman, K. Brigham, and A. Stecenko Activation of Alveolar Macrophages via the Alternative Pathway in Herpesvirus-Induced Lung Fibrosis Am. J. Respir. Cell Mol. Biol., October 1, 2006; 35(4): 466 - 473. [Abstract] [Full Text] [PDF]
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 D. W. Denning, B. R. O'Driscoll, C. M. Hogaboam, P. Bowyer, and R. M. Niven The link between fungi and severe asthma: a summary of the evidence. Eur. Respir. J., March 1, 2006; 27(3): 615 - 626. [Abstract] [Full Text] [PDF]
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S. J. Park, M. T. Wiekowski, S. A. Lira, and B. Mehrad Neutrophils Regulate Airway Responses in a Model of Fungal Allergic Airways Disease J. Immunol., February 15, 2006; 176(4): 2538 - 2545. [Abstract] [Full Text] [PDF]
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 S. Gupta, S. Schulz-Maronde, C. Kutzleb, R. Richter, W.-G. Forssmann, A. Kapp, U. Forssmann, and J. Elsner Cloning, expression, and functional characterization of cynomolgus monkey (Macaca fascicularis) CC chemokine receptor 1 J. Leukoc. Biol., November 1, 2005; 78(5): 1175 - 1184. [Abstract] [Full Text] [PDF]
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K. Chen, Y. Wei, A. Alter, G. C. Sharp, and H. Braley-Mullen Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis J. Leukoc. Biol., September 1, 2005; 78(3): 716 - 724. [Abstract] [Full Text] [PDF]
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S. Arora, Y. Hernandez, J. R. Erb-Downward, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Role of IFN-{gamma} in Regulating T2 Immunity and the Development of Alternatively Activated Macrophages during Allergic Bronchopulmonary Mycosis J. Immunol., May 15, 2005; 174(10): 6346 - 6356. [Abstract] [Full Text] [PDF]
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C. M. Hogaboam, K. Takahashi, R. A. B. Ezekowitz, S. L. Kunkel, and J. M. Schuh Mannose-binding lectin deficiency alters the development of fungal asthma: effects on airway response, inflammation, and cytokine profile J. Leukoc. Biol., May 1, 2004; 75(5): 805 - 814. [Abstract] [Full Text] [PDF]
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B. Ma, Z. Zhu, R. J. Homer, C. Gerard, R. Strieter, and J. A. Elias The C10/CCL6 Chemokine and CCR1 Play Critical Roles in the Pathogenesis of IL-13-Induced Inflammation and Remodeling J. Immunol., February 1, 2004; 172(3): 1872 - 1881. [Abstract] [Full Text] [PDF]
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 J. M. Schuh, K. Blease, S. L. Kunkel, and C. M. Hogaboam Eotaxin/CCL11 is involved in acute, but not chronic, allergic airway responses to Aspergillus fumigatus Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L198 - L204. [Abstract] [Full Text] [PDF]
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Z. Zhu, B. Ma, T. Zheng, R. J. Homer, C. G. Lee, I. F. Charo, P. Noble, and J. A. Elias IL-13-Induced Chemokine Responses in the Lung: Role of CCR2 in the Pathogenesis of IL-13-Induced Inflammation and Remodeling J. Immunol., March 15, 2002; 168(6): 2953 - 2962. [Abstract] [Full Text] [PDF]
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K. Blease, J. M. Schuh, C. Jakubzick, N. W. Lukacs, S. L. Kunkel, B. H. Joshi, R. K. Puri, M. H. Kaplan, and C. M. Hogaboam Stat6-Deficient Mice Develop Airway Hyperresponsiveness and Peribronchial Fibrosis during Chronic Fungal Asthma Am. J. Pathol., February 1, 2002; 160(2): 481 - 490. [Abstract] [Full Text] [PDF]
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K. Blease, C. Jakubzick, J. M. Schuh, B. H. Joshi, R. K. Puri, and C. M. Hogaboam IL-13 Fusion Cytotoxin Ameliorates Chronic Fungal-Induced Allergic Airway Disease in Mice J. Immunol., December 1, 2001; 167(11): 6583 - 6592. [Abstract] [Full Text] [PDF]
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 S. W. Chensue Molecular Machinations: Chemokine Signals in Host-Pathogen Interactions Clin. Microbiol. Rev., October 1, 2001; 14(4): 821 - 835. [Abstract] [Full Text] [PDF]
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 D. D'AMBROSIO, M. MARIANI, P. PANINA-BORDIGNON, and F. SINIGAGLIA Chemokines and Their Receptors Guiding T Lymphocyte Recruitment in Lung Inflammation Am. J. Respir. Crit. Care Med., October 1, 2001; 164(7): 1266 - 1275. [Full Text] [PDF]
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T. Chtanova, R. A. Kemp, A. P. R. Sutherland, F. Ronchese, and C. R. Mackay Gene Microarrays Reveal Extensive Differential Gene Expression in Both CD4+ and CD8+ Type 1 and Type 2 T Cells J. Immunol., September 15, 2001; 167(6): 3057 - 3063. [Abstract] [Full Text] [PDF]
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K. Blease, C. Jakubzick, J. Westwick, N. Lukacs, S. L. Kunkel, and C. M. Hogaboam Therapeutic Effect of IL-13 Immunoneutralization During Chronic Experimental Fungal Asthma J. Immunol., April 15, 2001; 166(8): 5219 - 5224. [Abstract] [Full Text] [PDF]
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K. Blease, B. Mehrad, N. W. Lukacs, S. L. Kunkel, T. J. Standiford, and C. M. Hogaboam Antifungal and Airway Remodeling Roles for Murine Monocyte Chemoattractant Protein-1/CCL2 During Pulmonary Exposure to Asperigillus fumigatus Conidia J. Immunol., February 1, 2001; 166(3): 1832 - 1842. [Abstract] [Full Text] [PDF]
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0.05 compared with levels measured in
CCR1+/+ mice before the conidia challenge. **,
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