Allergic responses to Aspergillus species exacerbate asthma and cystic fibrosis. The natural defense against live Aspergillus fumigatus spores or conidia depends on the recruitment and activation of mononuclear and polymorphonuclear leukocytes, events that are dependent on chemotactic cytokines. In this study, we explored the relative contribution of the monocyte chemoattractant protein-1 receptor, CCR2, in the pulmonary response to A. fumigatus conidia. Following sensitization to soluble A. fumigatus Ags, mice lacking CCR2 due to targeted deletion were markedly more susceptible to the injurious effects of an intrapulmonary challenge with live conidia compared with mice that expressed CCR2 or CCR2+/+. CCR2−/− mice exhibited a major defect in the recruitment of polymorphonuclear cells, but these mice also had significantly more eosinophils and lymphocytes in bronchoalveolar lavage samples. CCR2−/− mice also had significant increases in serum levels of total IgE and whole lung levels of IL-5, IL-13, eotaxin, and RANTES compared with CCR2+/+ mice. Airway inflammation, hyper-responsiveness to spasmogens, and subepithelial fibrosis were significantly enhanced in CCR2−/− mice compared with CCR2+/+ mice after the conidia challenge. Thus, these findings demonstrate that CCR2 plays an important role in the immune response against A. fumigatus, thereby limiting the allergic airway inflammatory and remodeling responses to this fungus.

Allergic responses to Asperigillus species are a major cause of human morbidity and have been implicated in the pathogenesis of asthma (1) and cystic fibrosis (2). Allergic responses to Aspergillus involve a number of immunologic abnormalities including elevated IgE, enhanced Th2 cytokines such as IL-4, IL-5, and IL-13 (3), eosinophilic inflammation, and profound airway remodeling (4, 5). As such, the elucidation of the mechanism of allergic responses to Asperigillus species is of clinical interest.

At least four groups of chemotactic cytokines or chemokines distinguished by the C, CC, CXC, or CXXXC motifs have been shown to have roles in diverse events such as host defense, inflammation, angiogenesis, hemopoiesis, and leukocyte chemotaxis (6). All chemokines activate a wide array of immune and nonimmune cells via unique G protein-coupled receptors. However, the CCRs have garnered recent attention because of the unique roles some of these receptors exert in the binding of HIV-1 to target cells (7). CCRs have also received attention in fungus-induced diseases as it has been shown that CCR1 and CCR5, receptors for macrophage inflammatory protein-1 α and RANTES, were necessary for the containment of Asperigillus fumigatus (8) and Cryptococcus neoformans (9), respectively. However, the role of CCR2, a major CCR involved in the recruitment and activation of mononuclear (10, 11) and polymorphonuclear (12) cells, has not been previously examined during pulmonary responses to fungus. The primary agonist of CCR2 is monocyte chemoattractant protein (MCP)3-1 although other lesser agonists of this receptor include MCP-2, 3, 4, and 5 (13). MCP-1 has been implicated in a wide array of diseases including atherosclerosis, fibrosis, and granulomatous lung disease (13).

Using a recently developed chronic model of allergic airway disease characterized by profound airway inflammation, hyperresponsiveness, and remodeling due to the intrapulmonary introduction of A. fumigatus conidia into A. fumigatus-sensitized mice (14), we addressed the following question: Does CCR2 modulate the allergic response of the host in this model? In this study, the airway inflammatory, resistance, and remodeling events in normal mice (CCR2+/+) were compared with those of mice with a targeted disruption of CCR2 (CCR2−/−). This study showed that CCR2−/− mice failed to adequately clear A. fumigatus Ags from their lungs and, as a consequence, experienced an exuberant inflammatory and remodeling response.

Breeding pairs of CCR2+/+ and CCR2−/− mice were originally provided by Dr. Israel Charo (Gladstone Institute, University of California, San Francisco, CA), and a breeding colony containing both mouse genotypes was maintained under specific pathogen-free conditions in the University Laboratory of Animal Medicine facility. As previously described, CCR2−/− mice were born at the expected Mendelian ratios and showed no evidence of abnormal growth patterns (15). CCR2+/+ and CCR2−/− mice were generated from mating homozygous mice of the same genetic background (C57BL/6 × 129Sv) and were intercrossed for five to seven generations. Prior approval for mouse usage in this study was obtained from the University Laboratory of Animal Medicine facility. Sensitization of mice to a commercially available preparation of soluble A. fumigatus Ags was performed as previously described in detail (14). Briefly, mice received an i.p. and s.c. injection of soluble A. fumigatus Ags dissolved in IFA. Two weeks after systemic sensitization, each mouse received a weekly intranasal challenge with A. fumigatus Ag to localize the allergic responsiveness to the airways. One week after the third intranasal challenge, each mouse received 5.0 × 106 A. fumigatus conidia suspended in 30 μl of 0.1% Tween 80 via the intratracheal route. Nonsensitized mice received normal saline alone via the same routes and over the same time periods and received the same number of conidia. This dose of conidia has previously been shown to be nonlethal in normal mice (8). The sensitization status of each mouse was confirmed by the presence of IgE in serum (data not shown). A. fumigatus-sensitized CCR2+/+ and CCR2−/− mice received 5.0 × 106 A. fumigatus conidia suspended in 30 μl of 0.1% Tween 80 via the intratracheal route (14).

Immediately before and at days 3, 7, and 30 after the intratracheal A. fumigatus conidia challenge, bronchial hyperresponsiveness to methacholine (10 μg, i.v.) was assessed in a Buxco plethysmograph (Buxco, Troy, NY) as previously described (16). Sodium pentobarbital (0.04 mg/g of mouse body weight; Butler, Columbus, OH) was used to anesthetize mice before their intubation, and ventilation with a Harvard pump ventilator (Harvard Apparatus, Reno, NV) (16). Immediately following the assessment of airway hyper-responsiveness, a bronchoalveolar lavage (BAL) was performed using 1 ml of filter-sterilized normal saline. Approximately 500 μl of blood removed from each mouse was 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).

Macrophages, neutrophils, eosinophils, and lymphocytes were quantified in BAL samples cytospun (Shandon Scientific, Runcorn, U.K.) onto coded microscope slides as previously described. These slides were then subjected to 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-powered fields (1000×) per slide. A total of 1 × 106 BAL cells were cytospun onto each slide to compensate for differences in cell retrieval.

Murine MCP-1, MCP-3, IL-4, IFN-γ, IL-5, IL-13, RANTES, eotaxin, and macrophage-derived chemokine (MDC) protein levels were determined in 50-μl samples from whole lung homogenates using a standardized sandwich ELISA technique previously described in detail (17). Each ELISA was screened to ensure the specificity of each Ab used. 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. Serum levels of IgE were analyzed using complementary capture and detection Ab pairs for IgE (PharMingen, San Diego, CA) and an ELISA performed according to the manufacturer’s directions. Duplicate sera samples were diluted to 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 recombinant IgE (5–2000 pg/ml).

Whole lungs from nonsensitized and A. fumigatus-sensitized mice before and after A. fumigatus conidia challenge were fully inflated with 10% formalin, dissected, and placed in fresh 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 one of Gomori methanamine silver (GMS), Masson trichrome, and hematoxylin and eosin (H&E). Inflammatory infiltrates and structural alterations were examined around blood vessels and airways using light microscopy at a magnification of ×400.

Total lung collagen levels were determined using a previously described assay (18). 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 of 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 per lung were calculated from a standard curve of known hydroxyproline concentrations of 0–100 μg/ml.

All results are expressed as mean ± SEM. ANOVA and Dunnett’s test for multiple comparisons were used to determine statistical significance in both groups at various times after the conidia challenge; p < 0.05 was considered statistically significant.

The whole lung levels of MCP-1 and MCP-3 were measured before and at various times after an intrapulmonary challenge of A. fumigatus conidia in CCR2+/+ and CCR2−/− mice. As shown in Table I, the highest levels of both CCR2 agonists in CCR2+/+ mice were measured before the conidia challenge. At days 3 and 7 after the conidia challenge, MCP-1 and MCP-3 levels were marginally lower than starting levels, but the greatest decrease in these chemokines was observed at day 30 after the conidia challenge. However, it is important to note that levels of MCP-1 were ∼8-fold lower in nonsensitized mice compared with A. fumigatus-sensitized mice before the conidia challenge. In addition, MCP-3 levels in whole lung samples from nonsensitized CCR2+/+ mice were below the limit of detection of the ELISA (50 pg/ml). Overall, MCP-1 and MCP-3 levels were detected in greater quantities in CCR2−/− mice than in their wild type counterparts. Interestingly, levels of both chemokines in whole lung samples followed a downward trend during the course of the conidia challenge. One exception was observed at day 3 after conidia, when the highest levels of MCP-1 in whole lung samples were observed in CCR2−/− mice. Once again, levels of MCP-1 and MCP-3 were markedly lower in nonsensitized CCR2−/− mice compared with CCR2−/− before and after the conidia challenge. Taken together, these data suggested that lung levels of CCR2 agonists were markedly increased in A. fumigatus-sensitized mice compared with nonsensitized mice, and the presence of CCR2 was not necessary for the induction of MCP-1 and MCP-3.

Table I.

MCP-1 and MCP-3 levels in whole lung homogenates from CCR2+/+ and CCR2−/− mice before and after the A. fumigatus conidia challenge

Mouse exposure to A. fumigatusCCR2+/+ MiceCCR2−/− Mice
MCP-1 (ng/ml)MCP-3 (ng/ml)MCP-1 (ng/ml)MCP-3 (ng/ml)
Nonsensitized 0.30 ± 0.07 ND 0.50 ± 0.05 ND 
Sensitized     
Prior to conidia 3.9 ± 0.35 0.5 ± 0.15 5.5 ± 0.50a 0.8 ± 0.10 
Day 3 after conidia 1.9 ± 0.10 0.3 ± 0.05 7.0 ± 0.07a 0.7 ± 0.15 
Day 7 after conidia 2.9 ± 0.17 0.4 ± 0.7 0.3 ± 0.07a 0.7 ± 0.15 
Day 30 after conidia 0.3 ± 0.04 0.3 ± 0.07 0.3 ± 0.07 0.5 ± 0.08 
Mouse exposure to A. fumigatusCCR2+/+ MiceCCR2−/− Mice
MCP-1 (ng/ml)MCP-3 (ng/ml)MCP-1 (ng/ml)MCP-3 (ng/ml)
Nonsensitized 0.30 ± 0.07 ND 0.50 ± 0.05 ND 
Sensitized     
Prior to conidia 3.9 ± 0.35 0.5 ± 0.15 5.5 ± 0.50a 0.8 ± 0.10 
Day 3 after conidia 1.9 ± 0.10 0.3 ± 0.05 7.0 ± 0.07a 0.7 ± 0.15 
Day 7 after conidia 2.9 ± 0.17 0.4 ± 0.7 0.3 ± 0.07a 0.7 ± 0.15 
Day 30 after conidia 0.3 ± 0.04 0.3 ± 0.07 0.3 ± 0.07 0.5 ± 0.08 
a

, p < 0.001; denotes significant differences in chemokine levels in CCR2−/− mice compared to CCR2+/+ controls at the same time point.

Increased IgE is a hallmark of hypersensitivity to A. fumigatus (19, 20). Measurement of serum IgE levels in this study revealed a marked difference in the generation of IgE by the two groups after their challenge with A. fumigatus conidia (Fig. 1). CCR2−/− mice had significantly greater total serum IgE levels compared with CCR2+/+ mice before conidia challenge. In both groups, peak IgE levels were measured at day 3 after conidia, but ∼2.5-fold far greater levels of IgE were evident in CCR2−/− mice compared with the CCR2+/+ group at this time (Fig. 1). A similar difference between the groups was noted at day 7, but IgE levels were ∼40-fold greater in the CCR2−/− group compared with the other group at day 30 after conidia. Thus, these results suggested that the introduction of A. fumigatus conidia into A. fumigatus-sensitized CCR2−/− mice greatly augmented the IgE response to this fungus.

FIGURE 1.

Serum IgE levels in A. fumigatus-sensitized CCR2+/+ and CCR2−/− mice before and at various times after A. fumigatus conidia challenge. Total IgE was measured using a specific ELISA as described in Materials and Methods. Data are expressed as mean ± SEM; n = 5/group/time point. ∗, p ≤ 0.05 compared with values measured in CCR2+/+ mice at the same time after the conidia challenge.

FIGURE 1.

Serum IgE levels in A. fumigatus-sensitized CCR2+/+ and CCR2−/− mice before and at various times after A. fumigatus conidia challenge. Total IgE was measured using a specific ELISA as described in Materials and Methods. Data are expressed as mean ± SEM; n = 5/group/time point. ∗, p ≤ 0.05 compared with values measured in CCR2+/+ mice at the same time after the conidia challenge.

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Previous studies have shown that CCR2−/− mice have significant defects in monocyte recruitment. Specifically, these mice did not exhibit monocyte recruitment into the peritoneal cavity in response to thioglycollate (10, 15), nor did these mice clear the intracellular bacteria, Listeria monocytogenes (11). In this study, the absence of CCR2 during an intrapulmonary challenge of A. fumigatus conidia in A. fumigatus-sensitized mice had a minor effect on recruitment of mononuclear cells into the airways as determined by BAL cell counts (Fig. 2,A). Although significantly greater numbers of macrophages were present in BAL samples from CCR2+/+ mice compared with CCR2−/− mice at day 3 after the conidia challenge, no differences between the two groups of mice were observed at any other time. Recruited neutrophils are a critical part of the first line of defense against A. fumigatus conidia because of their ability to kill conidia and destroy Aspergillus hyphae (21). Surprisingly, neutrophil recruitment into the airways of CCR2−/− mice was the most adversely affected following the intrapulmonary conidia challenge (Fig. 2 B). In marked contrast to CCR2+/+ mice, these polymorphonuclear cells were largely absent from BAL samples from CCR2−/− mice on day 3 after the conidia challenge. Thus, these data suggested that the recruitment of macrophages into the airways was only modestly affected by the absence of CCR2, whereas neutrophil recruitment into the airways was severely depressed in CCR2−/− mice following an A. fumigatus conidia challenge.

FIGURE 2.

Macrophage (A) and neutrophil (B) counts in BAL samples from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after A. fumigatus conidia challenge. BAL cells were dispersed onto microscope slides using a cytospin; macrophages and neutrophils were differentially stained with Wright-Giesma stain. A minimum of 15 high-powered fields or 300 cells were examined in each cytospin. Values are expressed as mean ± SEM. ∗, p ≤ 0.05 compared with values measured in CCR2−/− mice at the same time after the conidia challenge. A total of 1 × 106 BAL cells were cytospun onto each slide to compensate for differences in cell retrieval from each mouse.

FIGURE 2.

Macrophage (A) and neutrophil (B) counts in BAL samples from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after A. fumigatus conidia challenge. BAL cells were dispersed onto microscope slides using a cytospin; macrophages and neutrophils were differentially stained with Wright-Giesma stain. A minimum of 15 high-powered fields or 300 cells were examined in each cytospin. Values are expressed as mean ± SEM. ∗, p ≤ 0.05 compared with values measured in CCR2−/− mice at the same time after the conidia challenge. A total of 1 × 106 BAL cells were cytospun onto each slide to compensate for differences in cell retrieval from each mouse.

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The survival of A. fumigatus-sensitized CCR2−/− mice was not adversely affected following an intrapulmonary challenge with 5.0 × 106 conidia (data not shown). Although this observation suggested that CCR2−/− mice were not developing invasive pulmonary aspergillosis disease (22), it did not negate the possibility that A. fumigatus conidia or cell wall components persisted in the lungs of CCR2−/− mice. Therefore, GMS-stained whole lung sections from CCR2−/− and CCR2+/+ mice were examined histologically using GMS staining at various times before and after the conidia challenge. The GMS stain appears black in the presence of polysaccharide components from the fungus cell wall. No qualitative difference in GMS staining was observed between A. fumigatus-sensitized CCR2+/+ and CCR2−/− mice before the conidia challenge (not shown). At day 3 after conidia, GMS-positive cells were present in lung sections from CCR2+/+ mice (Fig. 3,A), although intact conidia were rarely detected. In contrast, whole conidia were apparent in numerous cells in the lungs of CCR2−/− mice at this time (Fig. 3,B). At day 7 after conidia, few GMS-positive cells were detected in CCR2+/+ mice (Fig. 3,C), but GMS staining was prominent in numerous mononuclear cells in the lungs of CCR2−/− mice (Fig. 3,D). A profound difference between the two groups of mice was also apparent when GMS-stained whole lung sections were examined at day 30 after the conidia challenge. CCR2+/+ mice completely lacked GMS-positive cells at this time (Fig. 3,E), whereas CCR2−/− mice showed clear evidence of increased GMS staining associated with mononuclear cells at days 30 postconidia (Fig. 3 F). Interestingly, the majority of the GMS staining at day 30 after conidia in CCR2−/− mice was associated with dense collections of mononuclear cells. However, the invasive or hyphal forms of A. fumigatus were not observed in lung sections from either group of mice at any time after the conidia challenge. Thus, a histological survey suggested that CCR2 was necessary for the clearance of A. fumigatus conidia and cell wall components from the airways of challenged mice.

FIGURE 3.

GMS-stained whole lung sections from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice at various times after A. fumigatus conidia challenge. The GMS stain appears black in the presence of intact conidia and/or the polysaccharide components of the conidia wall and can be seen in the macrophages from CCR2−/− mice. The photomicrographs shown are representative of lungs removed from CCR2+/+ mice at days 3 (A), 7 (C), and 30 (E) postconidia, and whole lung sections from CCR2−/− mice at days 3 (B), 7 (D), and 30 (F). Original magnification was ×400 for each photomicrograph.

FIGURE 3.

GMS-stained whole lung sections from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice at various times after A. fumigatus conidia challenge. The GMS stain appears black in the presence of intact conidia and/or the polysaccharide components of the conidia wall and can be seen in the macrophages from CCR2−/− mice. The photomicrographs shown are representative of lungs removed from CCR2+/+ mice at days 3 (A), 7 (C), and 30 (E) postconidia, and whole lung sections from CCR2−/− mice at days 3 (B), 7 (D), and 30 (F). Original magnification was ×400 for each photomicrograph.

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Eosinophils and T cells have been implicated in the protracted asthmatic responses observed in a large subset of patients with hypersensitivity to A. fumigatus Ags (5, 20). Eosinophilic inflammation around the airways poses a particularly serious problem because of the disruptive proteolytic enzymes released by these cells upon degranulation (1). Quantification of eosinophils and lymphocytes in BAL samples revealed marked differences between the two groups following conidia challenge (Fig. 4, A and B). No eosinophils were observed in the BAL of either group before the conidia challenge, but 3 days later significantly greater numbers of eosinophils were observed in BAL from CCR2−/− mice compared with BAL from CCR2+/+ mice (Fig. 4,A). At later time points, the eosinophil numbers in the BAL from CCR2−/− mice were similar to those quantified in similar samples from CCR2+/+ mice. Clinical and experimental data suggest that a T cell response directed toward IL-4 and IL-5 is the major predisposing factor for the development of allergic responses against Aspergillus in the lung (1). Lymphocyte counts in BAL samples were also markedly different between CCR2+/+ and CCR2−/− mice following the conidia challenge (Fig. 4 B). At days 3 and 7 after conidia, ∼3-fold more lymphocytes were present in CCR2−/− BAL compared with CCR2+/+ BAL. Thus, the introduction of conidia into previously sensitized CCR2−/− mice significantly augmented the movement of eosinophils and lymphocytes into the airways.

FIGURE 4.

Eosinophil (A) and lymphocyte (B) counts in BAL samples from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after A. fumigatus conidia challenge. BAL cells were dispersed onto microscope slides by cytospin; eosinophils and lymphocytes (predominately T cells) were differentially stained with Wright-Giesma stain. A minimum of 15 high-powered fields or 300 cells were examined in each cytospin. Values are expressed as mean ± SEM. ∗, p ≤ 0.05 compared with values measured in CCR2+/+ mice at the same time after the conidia challenge. A total of 1 × 106 BAL cells were cytospun onto each slide to compensate for differences in cell retrieval from each mouse.

FIGURE 4.

Eosinophil (A) and lymphocyte (B) counts in BAL samples from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after A. fumigatus conidia challenge. BAL cells were dispersed onto microscope slides by cytospin; eosinophils and lymphocytes (predominately T cells) were differentially stained with Wright-Giesma stain. A minimum of 15 high-powered fields or 300 cells were examined in each cytospin. Values are expressed as mean ± SEM. ∗, p ≤ 0.05 compared with values measured in CCR2+/+ mice at the same time after the conidia challenge. A total of 1 × 106 BAL cells were cytospun onto each slide to compensate for differences in cell retrieval from each mouse.

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Given the major changes in eosinophil and lymphocyte numbers in the BAL samples from CCR2−/− mice, we next examined whether the lung levels of cytokine and chemokines that regulate the activation and/or recruitment of these cell types were altered. IL-4 and IFN-γ are classically described as Th2 and Th1 cytokines, respectively (23). Temporal changes in IL-4 and IFN-γ levels in whole lung homogenates from both groups of mice are shown in Table II, and both cytokines were detected at similar levels in both groups before and at various times after the conidia challenge. It was of note that IL-4 levels were equivalent in whole lung samples from CCR2+/+ and CCR2−/− mice for the duration of the conidia challenge period, but whole lung levels of IFN-γ were markedly lower at day 30 after the conidia challenge compared with the earlier times after conidia. Thus, these data suggested that IL-4 and IFN-γ probably had minor roles in the exacerbation of the Aspergillus-induced allergic disease in CCR2−/− mice.

Table II.

IL-4 and IFN-γ levels in whole lung samples from CCR2+/+ and CCR2−/− mice before and after the A. fumigatus conidia challenge

Mouse exposure to A. fumigatusCCR2+/+ MiceCCR2−/− Mice
IL-4 (ng/ml)IFN-γ (ng/ml)IL-4 (ng/ml)IFN-γ (ng/ml)
Nonsensitized ND ND ND ND 
Sensitized     
Prior to conidia 3.8 ± 0.10 12.8 ± 0.15 3.6 ± 0.02 13.5 ± 0.50 
Day 3 after conidia 3.8 ± 0.10 10.6 ± 0.65 3.0 ± 0.02 12.0 ± 0.58 
Day 7 after conidia 4.0 ± 0.02 12.4 ± 1.30 4.0 ± 0.03 11.0 ± 1.33 
Day 30 after conidia 3.5 ± 0.02 0.9 ± 0.10 3.5 ± 0.05 0.3 ± 0.10 
Mouse exposure to A. fumigatusCCR2+/+ MiceCCR2−/− Mice
IL-4 (ng/ml)IFN-γ (ng/ml)IL-4 (ng/ml)IFN-γ (ng/ml)
Nonsensitized ND ND ND ND 
Sensitized     
Prior to conidia 3.8 ± 0.10 12.8 ± 0.15 3.6 ± 0.02 13.5 ± 0.50 
Day 3 after conidia 3.8 ± 0.10 10.6 ± 0.65 3.0 ± 0.02 12.0 ± 0.58 
Day 7 after conidia 4.0 ± 0.02 12.4 ± 1.30 4.0 ± 0.03 11.0 ± 1.33 
Day 30 after conidia 3.5 ± 0.02 0.9 ± 0.10 3.5 ± 0.05 0.3 ± 0.10 

IL-5 modulates the activation and survival of eosinophils (24), whereas IL-13 has been identified as a major mediator of airway hyperresponsiveness, eosinophil recruitment, mucus overproduction (25), and subepithelial fibrosis (26). Both before and at day 3 after the conidia challenge, IL-5 levels were significantly elevated in CCR2−/− mice compared with similar samples from CCR2+/+ mice (Fig. 5,A). IL-13 was present at similar levels in both groups of mice before and at days 3 and 7 after conidia (Fig. 5 B). However, at day 30 after conidia, whole lung homogenates from CCR2−/− mice contained significantly greater levels of IL-13 compared with those detected in whole lung from wild-type mice at this time.

FIGURE 5.

IL-5 (A) and IL-13 (B) levels in whole lung homogenates from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after an intrapulmonary A. fumigatus conidia challenge. Cytokine levels were measured using a specific ELISA as described in Materials and Methods. Values are expressed as mean ± SEM; n = 5 mice/group/time point. ∗, p ≤ 0.05 compared with values measured in the CCR2+/+ group at the same time after the conidia challenge.

FIGURE 5.

IL-5 (A) and IL-13 (B) levels in whole lung homogenates from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after an intrapulmonary A. fumigatus conidia challenge. Cytokine levels were measured using a specific ELISA as described in Materials and Methods. Values are expressed as mean ± SEM; n = 5 mice/group/time point. ∗, p ≤ 0.05 compared with values measured in the CCR2+/+ group at the same time after the conidia challenge.

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Eotaxin and RANTES are potent chemotactic agents for eosinophils during allergic airway inflammation, and RANTES is also a potent chemoattractant of lymphocytes under similar conditions (27). More recently, MDC was found to regulate the migration and accumulation of leukocytes during the development of airway hyperreactivity in acute allergic airway disease (28). Whole lung levels of eotaxin, RANTES, and MDC are shown in Fig. 6. Eotaxin levels were also significantly elevated in lung from CCR2−/− mice compared with CCR2+/+ mice before conidia (Fig. 6,A). In addition, RANTES levels were significantly elevated at day 3 after the conidia challenge when statistically compared with RANTES levels in CCR2+/+ mice at the same times after conidia (Fig. 6,B). Finally, the whole lung levels of MDC were significantly higher in CCR2−/− mice compared with CCR2+/+ mice at days 3 and 30 after the conidia challenge (Fig. 6 C). Thus, these data demonstrated that the generation of cytokines and chemokines with major stimulatory and chemotactic effects on eosinophils and lymphocytes were significantly enhanced in CCR2−/− mice following conidia challenge.

FIGURE 6.

Eotaxin (A), RANTES (B), and MDC (C) levels in whole lung homogenates from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after an A. fumigatus conidia challenge. Chemokine levels were measured using a specific ELISA as described in Materials and Methods. Values are expressed as mean ± SEM; n = 5 mice/group/time point. ∗, p ≤ 0.05 compared with values measured in the CCR2+/+ group at the same time after the conidia challenge.

FIGURE 6.

Eotaxin (A), RANTES (B), and MDC (C) levels in whole lung homogenates from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after an A. fumigatus conidia challenge. Chemokine levels were measured using a specific ELISA as described in Materials and Methods. Values are expressed as mean ± SEM; n = 5 mice/group/time point. ∗, p ≤ 0.05 compared with values measured in the CCR2+/+ group at the same time after the conidia challenge.

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Increased airway resistance represents a key reversible hallmark of clinical asthma, and in animal models of asthma an exaggerated bronchoconstrictor response is observed in response to a bronchial smooth muscle spasmogen that has little physiological consequence in a nonsensitized animal (29). We have previously observed that challenging mice previously sensitized to soluble A. fumigatus Ags with the conidia markedly augments airway hyperreactivity to methacholine challenge for up to 30 days (14). In this study, airway hyperresponsiveness to a methacholine challenge was measured in A. fumigatus-sensitized CCR2+/+ and CCR2−/− mice before and at various times after conidia (Fig. 7). The dose of 10 μg of methacholine had a negligible (i.e., less than a 2-fold increase) effect on the airway resistance response in nonsensitized mice (Fig. 7, dashed line). Immediately before conidia challenge, both groups of mice exhibited a similar 5-fold increase in airway resistance. However, at days 3, 7, and 30 after conidia, airway resistance changes after a methacholine challenge were significantly greater in CCR2−/− mice compared with airway responses measured in CCR2+/+ mice measured at the same time. Thus, the airway responsiveness to the smooth muscle spasmogen methacholine was clearly exacerbated in CCR2−/− mice compared with CCR2+/+ mice at all times after the conidia challenge.

FIGURE 7.

Airway hyper-responsiveness in CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after an A. fumigatus conidia challenge. Both groups of mice were sensitized to soluble A. fumigatus Ags before the conidia challenge. Changes in airway resistance or hyperresponsiveness (units = cm H2O/ml/s) were monitored at each time point by the i.v. injection of methacholine. The dose of 10 μg of methacholine had a negligible (i.e., less than a 2-fold increase) effect on the airway resistance response in nonsensitized mice (dashed line). Values are expressed as mean ± SEM; n = 5/group/time point. ∗, p ≤ 0.05 compared with the baseline airway resistance.

FIGURE 7.

Airway hyper-responsiveness in CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after an A. fumigatus conidia challenge. Both groups of mice were sensitized to soluble A. fumigatus Ags before the conidia challenge. Changes in airway resistance or hyperresponsiveness (units = cm H2O/ml/s) were monitored at each time point by the i.v. injection of methacholine. The dose of 10 μg of methacholine had a negligible (i.e., less than a 2-fold increase) effect on the airway resistance response in nonsensitized mice (dashed line). Values are expressed as mean ± SEM; n = 5/group/time point. ∗, p ≤ 0.05 compared with the baseline airway resistance.

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Another feature of an intrapulmonary conidia challenge in A. fumigatus-sensitized mice is the increase in peribronchial fibrosis (14). The presence of fibrosis in CCR2+/+ and CCR2−/− mice was first assessed using a histological method. As illustrated in Fig. 8,A, Masson trichrome staining, which highlights the presence of collagen, appears to be less prominent around large airways from CCR2+/+ mice compared with CCR2−/− mice shown in Fig. 8B at day 30 after the conidia challenge. Severe peribronchial inflammation and goblet cell hyperplasia are also prominent features of chronic allergic airway diseased due to Aspergillus. Examination of H&E-stained lung sections from CCR2+/+ mice (Fig. 8,C) revealed goblet cells in the epithelium but little evidence of peribronchial inflammation. Goblet cells and a prominent peribronchial and perivascular accumulation of mononuclear cells were evident in CCR2−/− mice (Fig. 8,D) at day 30 after the conidia challenge. In addition, numerous mononuclear cells containing A. fumigatus (stained intensely green) were also observed in close apposition to the inflamed airways in CCR2−/− mice at this time (Fig. 8 D).

FIGURE 8.

Masson trichrome- (A and B) and H&E-stained (C and D) whole lung sections from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice at day 30 after an A. fumigatus conidia challenge. Both CCR2+/+ (A) and CCR2−/− (B) mice showed evidence of peribronchial fibrosis (light blue staining) at 30 days after the conidia challenge. Airways in CCR2+/+ mice generally lacked any evidence of peribronchial inflammation although goblet cells were prominent in the airways of these mice (C). Peribronchial and vascular lymphocytic and eosinophilic inflammation was observed in CCR2−/− mice at day 30 after the conidia challenge (D). Note the presence of A. fumigatus (greenish-brown colored material) in mononuclear cells (arrow) adjacent to the inflamed airway depicted in (D). The presence of A. fumigatus was not observed in mononuclear cells present in the lungs of CCR2+/+ mice. Original magnification was ×200 for each photomicrograph.

FIGURE 8.

Masson trichrome- (A and B) and H&E-stained (C and D) whole lung sections from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice at day 30 after an A. fumigatus conidia challenge. Both CCR2+/+ (A) and CCR2−/− (B) mice showed evidence of peribronchial fibrosis (light blue staining) at 30 days after the conidia challenge. Airways in CCR2+/+ mice generally lacked any evidence of peribronchial inflammation although goblet cells were prominent in the airways of these mice (C). Peribronchial and vascular lymphocytic and eosinophilic inflammation was observed in CCR2−/− mice at day 30 after the conidia challenge (D). Note the presence of A. fumigatus (greenish-brown colored material) in mononuclear cells (arrow) adjacent to the inflamed airway depicted in (D). The presence of A. fumigatus was not observed in mononuclear cells present in the lungs of CCR2+/+ mice. Original magnification was ×200 for each photomicrograph.

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To confirm that subepithelial fibrosis was augmented in CCR2−/− mice, hydroxyproline levels were determined. Hydroxyproline is a major component of collagen, and thus its measurement supplies a quantitative way of measuring fibrosis in the lung. At all times after the conidia challenge, hydroxyproline levels in whole lung homogenates from CCR2−/− mice was significantly higher than those measured in similar samples from CCR2+/+ mice (Fig. 9).

FIGURE 9.

Hydroxyproline levels in whole lung homogenates from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after A. fumigatus conidia challenge. Hydroxyproline levels were measured as described in Materials and Methods. Values are expressed as mean ± SEM; n = 5/group/time point. ∗, p ≤ 0.05 compared with the values measured in CCR2+/+ mice at the same time after the conidia challenge.

FIGURE 9.

Hydroxyproline levels in whole lung homogenates from A. fumigatus-sensitized CCR2 wild-type (+/+) and CCR2 knockout (−/−) mice before and at various times after A. fumigatus conidia challenge. Hydroxyproline levels were measured as described in Materials and Methods. Values are expressed as mean ± SEM; n = 5/group/time point. ∗, p ≤ 0.05 compared with the values measured in CCR2+/+ mice at the same time after the conidia challenge.

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Allergic responses to A. fumigatus have been implicated in the pathogenesis of lung diseases such as asthma and cystic fibrosis (1, 30, 31). In this study, we demonstrate that CCR2 is required to modulate the allergic airway response to A. fumigatus conidia in A. fumigatus-sensitized mice. In particular, CCR2 appears to have an integral role in the clearance of A. fumigatus from the airways of these mice. A. fumigatus-sensitized CCR2−/− mice exhibited markedly diminished neutrophil recruitment into the airways following a conidia challenge. In addition, CCR2−/− mice exhibited marked increases in eosinophil and lymphocyte recruitment and specific activating and chemotactic factors including IL-5, IL-13, eotaxin, RANTES, and MDC. Finally, in contrast to CCR2+/+ mice, CCR2−/− mice exhibited persistent airway inflammation, hyperresponsiveness, and subepithelial fibrosis. Thus, the introduction of A. fumigatus conidia into A. fumigatus-sensitized mice lacking the expression of CCR2 resulted in the exacerbation of many of the features of this allergic pulmonary disease.

As revealed by in vivo gene-targeting technology, CCR2 is a major chemokine receptor that is required for macrophage recruitment and host defense against bacterial pathogens (10, 11, 15). MCP-1 appears to be the primary ligand that facilitates these CCR2-dependent functions (32). Given the requirement of CCR2 for the recruitment of macrophages in models of peritonitis, it was surprising to observe in this study that macrophage accumulation in the airways of CCR2−/− mice challenged with conidia was only modestly impaired. However, the mononuclear cells in the lungs of CCR2−/− mice were apparently unable to clear the polysaccharide components of A. fumigatus revealed by GMS staining. These findings were consistent with the previous findings of Kurihara et al. (11) who observed that CCR2−/− mice failed to clear the intracellular bacteria, Listeria monocytogenes. Why macrophages from CCR2−/− mice are unable to remove A. fumigatus from the airways is not presently apparent, but studies are presently directed at this dilemma. Conidia clearance, 30 days after challenge by macrophages, was also impaired in naive CCR2−/− mice; however, not to the same degree as A. fumigatus-sensitized mice. This observation would suggest that the primary phenotypic defect in CCR2−/− mice is the inability to efficiently clear conidia, whereas in the sensitized group, persistence of Ag leads to an exacerbated inflammatory response.

A. fumigatus poses the greatest danger in individuals with prolonged neutropenia or defects in neutrophil function, because the neutrophil is critical for the destruction of A. fumigatus conidia and hyphae (33). The absence of neutrophils in the airways of CCR2−/− mice was another striking feature of the allergic airway disease induced by Aspergillus in CCR2−/− mice. This defect was not a consequence of lowered circulating neutrophil numbers, as previous studies have shown that CCR2−/− mice have a normal compliment of circulating polymorphonuclear cells (15). The paucity of neutrophils may have been due to decreases in KC levels or a direct consequence of neutrophils lacking the ability to respond to MCP-1, which will promote the chemotaxis of neutrophils during acute (34) and chronic inflammatory conditions (12). Thus, these findings suggest that CCR2 has a major role in the recruitment of neutrophils during an intrapulmonary challenge with A. fumigatus conidia, and the paucity of neutrophils may explain the persistence of A. fumigatus in the airways of CCR2−/− mice.

Individuals that have been sensitized to A. fumigatus demonstrate significantly increased levels of T cell-derived IL-4 and IL-5, with low but significantly elevated levels of IFN-γ (35). Experimental studies in mice have revealed that IL-4 and IL-5 have unique roles in the development of lung pathology due to Aspergillus. Accordingly, IL-4−/− mice exhibited similar lung inflammation as their wild-type counterparts but completely lacked airway hyperresponsiveness to bronchial spasmogens (36). In another murine study of acute aspergillosis, eosinophilia resulted from the induction of IL-5 by soluble A. fumigatus Ags (37). Although no apparent difference in whole lung levels of IL-4 or IFN-γ was observed between CCR2−/− and CCR2+/+ mice before or after the conidia challenge, CCR2−/− mice had significantly greater whole lung levels of IL-5 before and at days 3 and 7 after the conidia challenge. The presence of IL-5 combined with increased eotaxin, RANTES, and MDC levels may explain the enhanced movement of eosinophils and lymphocytes into the BAL and the retention of these cells around the airways of CCR2−/− mice. In addition, all three chemokines have been shown to exert major effects on airway hyperresponsiveness in an OVA-induced model of allergic airway inflammation (27). Overall, these findings demonstrate that allergic responses to A. fumigatus, characterized by increased eosinophils and lymphocyte recruitment and augmented IL-5, eotaxin, RANTES, and MDC levels, are significantly enhanced in CCR2−/− mice.

Subepithelial fibrosis is another clinical feature of Aspergillus-induced airway disease and asthma that has been attributed to the eosinophilic and lymphocytic inflammation that persists around the airways of allergic individuals (1). Many of the previous studies in murine models of allergic aspergillosis have addressed the airway inflammation and hyperresponsiveness during allergic responses to soluble Aspergillus Ags, revealing roles for cytokines (38, 39, 40) and chemokines (16) in these processes. However, identification of the soluble factor(s) that contribute to all of the features of chronic allergic airway disease has been hampered by the lack of a model that recapitulates all of these clinical features. This study suggested that a number of cytokines and chemokines contributed to the augmented allergic responsiveness of CCR2−/− mice toward A. fumigatus conidia. However, of particular interest in this study was the finding that IL-13 and a C-C chemokine it strongly induces, namely MDC (41), remained significantly elevated in whole lung homogenates from CCR2−/− mice at day 30 after the conidia challenge, when profound airway inflammation, hyperresponsiveness, and peribronchial fibrosis were noted. These findings are important because of the prominent role that IL-13 has on a multitude of features associated with chronic asthma or allergic airway disease, including mononuclear and eosinophilic inflammatory recruitment, goblet cell metaplasia, eotaxin production, airway hyperresponsiveness, and subepithelial fibrosis (25, 26). Immunoneutralization of MDC during acute allergic airway inflammation was previously shown to prevent airway hyperreactivity and eosinophilia, suggesting that this chemokine was essential for the retention of leukocytes in the lung (28). Further studies are required to identify whether IL-13 and MDC are major participants in airway inflammation, hyperresponsiveness, and remodeling that follow an intrapulmonary conidia challenge in CCR2−/− and CCR2+/+ mice.

In conclusion, CCR2−/− mice are significantly more susceptible to the allergic consequences of an intrapulmonary A. fumigatus conidia challenge. The inability of these mice to completely clear antigenic components of A. fumigatus due to defects in macrophage activation and neutrophil recruitment appeared to account for the exacerbation of the chronic allergic airway disease induced by Aspergillus. Further studies are required to ascertain whether CCR2 agonists would aid in the rapid clearance of fungus from the airways of A. fumigatus-sensitized mice and thereby reduce the intensity of the allergic airway disease associated with this model.

1

This study was supported by an American Lung Association Research Grant (to C.M.H.) and National Institutes of Health Grants K08 HL04220-01 (to B.M.), HL35276 (to S.L.K.), HL31963 (to S.L.K.), and AI36302 (to S.L.K.).

3

Abbreviations used in this paper: MCP, monocyte chemoattractant protein; BAL, bronchoalveolar lavage; MDC, macrophage-derived chemokine; GMS, Gomori methanamine silver; H&E, hematoxylin and eosin.

1
Kauffman, H. F., J. F. Tomee, T. S. van der Werf, J. G. de Monchy, G. K. Koeter.
1995
. Review of fungus-induced asthmatic reactions.
Am. J. Respir. Crit. Care Med.
151
:
2109
2
Schonheyder, H., T. Jensen, N. Hoiby, C. Koch.
1988
. Clinical and serological survey of pulmonary aspergillosis in patients with cystic fibrosis.
Int. Arch. Allergy Appl. Immunol.
85
:
472
3
Skov, M., L. K. Poulsen, C. Koch.
1999
. Increased antigen-specific Th-2 response in allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis.
Pediatr. Pulmonol.
27
:
74
4
Knutsen, A. P., K. R. Mueller, A. D. Levine, B. Chouhan, P. S. Hutcheson, R. G. Slavin.
1994
. Asp f I CD4+ TH2-like T-cell lines in allergic bronchopulmonary aspergillosis.
J Allergy Clin. Immunol.
94
:
215
5
Greenberger, P. A., R. Patterson.
1987
. Allergic bronchopulmonary aspergillosis: model of bronchopulmonary disease with defined serologic, radiologic, pathologic and clinical findings from asthma to fatal destructive lung disease.
Chest
91
:
165S
6
Kunkel, S. L., N. Lukacs, R. M. Strieter.
1995
. Chemokines and their role in human disease.
Agents Actions Suppl.
46
:
11
7
Michael, N. L., L. G. Louie, A. L. Rohrbaugh, K. A. Schultz, D. E. Dayhoff, C. E. Wang, H. W. Sheppard.
1997
. The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression.
Nat. Med.
3
:
1160
8
Gao, J. L., T. A. Wynn, Y. Chang, E. J. Lee, H. E. Broxmeyer, S. Cooper, H. L. Tiffany, H. Westphal, J. Kwon-Chung, P. M. Murphy.
1997
. Impaired host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine balance in mice lacking CC chemokine receptor 1.
J. Exp. Med.
185
:
1959
9
Huffnagle, G. B., L. K. McNeil, R. A. McDonald, J. W. Murphy, G. B. Toews, N. Maeda, W. A. Kuziel.
1999
. The role of CCR5 in organ-specific and innate immunity to Cryptococcus neoformans.
J. Immunol.
163
:
4642
10
Kuziel, W. A., S. J. Morgan, T. C. Dawson, S. Griffin, O. Smithies, K. Ley, N. Maeda.
1997
. Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2.
Proc. Natl. Acad. Sci. USA
94
:
12053
11
Kurihara, T., G. Warr, J. Loy, R. Bravo.
1997
. Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor.
J. Exp. Med.
186
:
1757
12
Johnston, B., A. R. Burns, M. Suematsu, T. B. Issekutz, R. C. Woodman, P. Kubes.
1999
. Chronic inflammation upregulates chemokine receptors and induces neutrophil migration to monocyte chemoattractant protein-1.
J. Clin. Invest.
103
:
1269
13
Taub, D. D..
1996
. Chemokine-leukocyte interactions: the voodoo that they do so well.
Cytokine Growth Factor Rev.
7
:
355
14
Hogaboam, C. M., K. Blease, B. Mehrad, M. L. Steinhauser, T. J. Standiford, S. L. Kunkel, N. W. Lukacs.
2000
. Chronic airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis during allergic airway disease induced by Aspergillus fumigatus.
Am. J. Pathol.
156
:
723
15
Boring, L., J. Gosling, S. W. Chensue, S. L. Kunkel, R. V. Farese, Jr, H. E. Broxmeyer, I. F. Charo.
1997
. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice.
J. Clin. Invest.
100
:
2552
16
Hogaboam, C. M., C. S. Gallinat, D. D. Taub, R. M. Strieter, S. L. Kunkel, N. W. Lukacs.
1999
. Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis.
J. Immunol.
162
:
6071
17
Evanoff, H., M. D. Burdick, S. A. Moore, S. L. Kunkel, R. M. Strieter.
1992
. A sensitive ELISA for the detection of human monocyte chemoattractant protein-1 (MCP-1).
Immunol. Invest.
21
:
39
18
Keane, M. P., J. A. Belperio, T. A. Moore, B. B. Moore, D. A. Arenberg, R. E. Smith, M. D. Burdick, S. L. Kunkel, R. M. Strieter.
1999
. Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis.
J. Immunol.
162
:
5511
19
Murali, P. S., V. P. Kurup, N. K. Bansal, J. N. Fink, P. A. Greenberger.
1998
. IgE down regulation and cytokine induction by Aspergillus antigens in human allergic bronchopulmonary aspergillosis.
J. Lab. Clin. Med.
131
:
228
20
Cockrill, B. A., C. A. Hales.
1999
. Allergic bronchopulmonary aspergillosis.
Annu. Rev. Med.
50
:
303
21
Schaffner, A., H. Douglas, A. Braude.
1982
. Selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus: observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes.
J. Clin. Invest.
69
:
617
22
Mehrad, B., R. M. Strieter, T. J. Standiford.
1999
. Role of TNFα in pulmonary host defense in murine invasive aspergillosis.
J. Immunol.
162
:
1633
23
Mosmann, T. R., R. L. Coffman.
1989
. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties.
Am. Rev. Immunol.
7
:
145
24
Rothenberg, M. E., J. Petersen, R. L. Stevens, D. S. Silberstein, D. T. McKenzie, K. F. Austen, W. F. Owen.
1989
. IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibody-dependent cytotoxicity.
J. Immunol.
143
:
2311
25
Grunig, G., M. Warnock, A. E. Wakil, R. Venkayya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, D. B. Corry.
1998
. Requirement for IL-13 independently of IL-4 in experimental asthma.
Science
282
:
2261
26
Zhu, Z., R. J. Homer, Z. Wang, Q. Chen, G. P. Geba, J. Wang, Y. Zhang, J. A. Elias.
1999
. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production.
J. Clin. Invest.
103
:
779
27
Gonzalo, J. A., C. M. Lloyd, D. Wen, J. P. Albar, T. N. Wells, A. Proudfoot, A. C. Martinez, M. Dorf, T. Bjerke, A. J. Coyle, J. C. Gutierrez-Ramos.
1998
. The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness.
J. Exp. Med.
188
:
157
28
Gonzalo, J. A., Y. Pan, C. M. Lloyd, G. Q. Jia, G. Yu, B. Dussault, C. A. Powers, A. E. Proudfoot, A. J. Coyle, D. Gearing, J. C. Gutierrez-Ramos.
1999
. Mouse monocyte-derived chemokine is involved in airway hyperreactivity and lung inflammation.
J. Immunol.
163
:
403
29
Drazen, J. M., T. Takebayashi, N. C. Long, G. T. De Sanctis, S. A. Shore.
1999
. Animal models of asthma and chronic bronchitis.
Clin. Exp. Allergy
29
: (Suppl. 2):
37
30
Henderson, A. H., M. P. English, R. J. Vecht.
1968
. Pulmonary aspergillosis: a survey of its occurrence in patients with chronic lung disease and a discussion of the significance of diagnostic tests.
Thorax
25
:
513
31
Laufer, P. J., N. Fink, W. T. Bruns, G. F. Unger, J. H. Kalbfleisch, P. A. Greenberger, R. Patterson.
1984
. Allergic bronchopulmonary aspergillosis in cystic fibrosis.
J. Allergy Clin. Immunol.
73
:
44
32
Lu, B., B. J. Rutledge, L. Gu, J. Fiorillo, N. W. Lukacs, S. L. Kunkel, R. North, C. Gerard, B. J. Rollins.
1998
. Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice.
J. Exp. Med.
187
:
601
33
Elstad, M. R..
1991
. Aspergillosis and lung defenses.
Semin. Respir. Infect.
6
:
27
34
Tessier, P. A., P. Cattaruzzi, S. R. McColl.
1996
. Inhibition of lymphocyte adhesion to cytokine-activated synovial fibroblasts by glucocorticoids involves the attenuation of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 gene expression.
Arthritis Rheum.
39
:
226
35
Walker, C., W. Bauer, R. K. Braun, G. Menz, P. Braun, F. Schwarz, T. T. Hansel, B. Villiger.
1994
. Activated T cells and cytokines in bronchoalveolar lavages from patients with various lung diseases associated with eosinophilia.
Am. J. Respir. Crit. Care Med.
150
:
1038
36
Kurup, V. P., J. Q. Xia, D. A. Rickaby, C. A. Dawson, H. Choi, J. N. Fink.
1999
. Aspergillus fumigatus antigen exposure results in pulmonary airway resistance in wild-type but not in IL-4 knockout mice.
Clin. Immunol.
90
:
404
37
Murali, P. S., A. Kumar, H. Choi, N. K. Banasal, J. N. Fink, V. P. Kurup.
1993
. Aspergillus fumigatus antigen induced eosinophilia in mice is abrogated by anti-IL-5 antibody.
J. Leukocyte Biol.
53
:
264
38
Kurup, V. P., J. Guo, P. S. Murali, H. Choi, J. N. Fink.
1997
. Immunopathologic responses to Aspergillus antigen in interleukin-4 knockout mice.
J. Lab. Clin. Med.
130
:
567
39
Grunig, G., D. B. Corry, M. W. Leach, B. W. Seymour, V. P. Kurup, D. M. Rennick.
1997
. Interleukin-10 is a natural suppressor of cytokine production and inflammation in a murine model of allergic bronchopulmonary aspergillosis.
J. Exp. Med.
185
:
1089
40
Chu, H. W., J. M. Wang, M. Boutet, M. Laviolette.
1996
. Tumor necrosis factor-α and interleukin-1 α expression in a murine model of allergic bronchopulmonary aspergillosis.
Lab. Anim. Sci.
46
:
42
41
>Andrew, D. P., M. S. Chang, J. McNinch, S. T. Wathen, M. Rihanek, J. Tseng, J. P. Spellberg, C. G. Elias, 3rd..
1998
. STCP-1 (MDC) CC chemokine acts specifically on chronically activated Th2 lymphocytes and is produced by monocytes on stimulation with Th2 cytokines IL-4 and IL-13.
J. Immunol.
161
:
5027