Published online before print February 3, 2004
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* Department of Pathology, University of Michigan Medical School, Ann Arbor; and
Laboratory of Developmental Immunology, Massachusetts General Hospital for Children, Boston
1Correspondence: Department of Pathology, University of Michigan Medical School, Rm. 5214 Med Sci I, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail: janeschu{at}umich.edu
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levels were significantly increased at day 28 after conidia when compared with controls. However, histological evidence showed similar airway remodeling at day 28 after conidia (i.e., subepithelial fibrosis and goblet cell metaplasia) in the two groups of mice. Thus, these findings show that MBL-A is not required for mouse survival following exposure to A. fumigatus conidia, and this murine collectin isoform contributes to the development and maintenance of airway hyperresponsiveness but not chronic airway remodeling during chronic fungal asthma.
Key Words: infectious immunity-fungi • allergy • lung • inflammatory mediators
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Whereas one form of MBL is found in humans, mice contain two homologous isoforms, MBL-A and MBL-C [10 , 11 ]. MBL-C (which was originally identified as the liver form, although recent evidence indicates that both isoforms are generated by hepatocytes and are found in the circulation [12 ]) is found in greater abundance in mouse serum than MBL-A; both are typically found in microgram quantities, and the introduction of lipopolysaccharide into mice induces MBL-A but not MBL-C [13 ]. The recent generation of MBL-A null mice that were MBL-C-sufficient has facilitated the examination of the role of MBL-A in innate immune responses to bacteria and fungus. One study led to the interesting observation that murine MBL-A is involved in the regulation of cytokine generation during bacterial-induced septic peritonitis [14 ]. As a result of the ability of MBL-A to modulate the inflammatory response, MBL-A-deficient (MBL-A–/–) mice showed an enhanced survival response in this model of sepsis [14 ]. Thus, MBL is a physiological, significant molecule in the innate immune response.
The spores or conidia of A. fumigatus are a constant airborne challenge to humans and animals alike, particularly in developed countries [15 ]. This ubiquitous airborne saprophytic fungi is normally contained and eliminated by neutrophil and macrophage-driven innate immune events in the immunocompetent host by innate immune mechanisms [16 ]. A. fumigatus is major clinical concern in immunocompromised patients, as this fungus can grow to the point of fatal invasive aspergillosis. Nonimmunocompromised individuals can also be adversely affected by pulmonary exposure to this fungus, as those sensitized to this fungus are at risk for allergic bronchopulmonary aspergillosis, fungal asthma, or chronic necrotizing pulmonary aspergillosis (CNPA) [15 ]. Recently, it was shown that individuals deficient in MBL were susceptible to CNPA [17 ], but aside from this study, little in known about the in vivo role of MBL in the immune response to A. fumigatus.
Thus, in the present study, we investigated the role of MBL-A in pulmonary innate and allergic responses to A. fumigatus in MBL-A-sufficient (MBL-A+/+) and MBL-A–/– mice. Specific pathogen-free (SPF) MBL-A–/– mice did not succumb to an intravenous (i.v.) or intratracheal (i.t.) bolus challenge with live A. fumigatus conidia. In a well-established model of chronic fungal asthma [18 ], MBL-A–/– mice, in contrast with their wild-type counterparts, exhibited airway remodeling following the i.t. challenge of conidia. The allergic airway response to A. fumigatus conidia also differed between MBL-A+/+ and MBL-A–/– mice; airway hyperreactivity, but not airway remodeling, was attenuated in the deficient mice compared with their wild-type counterparts during the course of chronic fungal asthma.
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A. fumigatus culture and conidia challenge of nonsensitized MBL-A+/+ and MBL-A–/– mice
A. fumigatus American Type Culture Collection (Manassas, VA) strain 13073 was used in the present studies and was grown according to our previously published protocol [18
, 19
]. Conidia were extensively washed to remove all debris such as mycelium before quantification and injection into MBL-A+/+ and MBL-A–/– mice. In the present study, groups of five MBL-A+/+ mice were challenged i.t. with 5.0 x 106 conidia suspended in 30 µl 0.1% Tween-80 in phosphate-buffered saline (PBS). Groups of five MBL-A–/– mice received 1.0 x 106 or 5.0 x 106 conidia via the i.t. route. In separate experiments, MBL-A+/+ mice (n=5) received 5.0 x 105 conidia suspended in 500 µl 0.1% Tween-80 in PBS. MBL-A–/– mice also received conidia via the i.v. route; 5.0 x 105 or 1.0 x 105 conidia was administered to groups of five MBL-A–/– mice. Each experimental treatment was conducted once. Mouse survival was monitored in all groups of challenged mice. At day 28 after conidia challenge, whole lung tissue was removed from each surviving mouse to assess histological changes (see below).
Sensitization of MBL-A+/+ and MBL-A–/– mice to A. fumigatus and establishment of chronic fungal asthma
Age-matched, SPF male and female MBL-A+/+ and MBL-A–/– mice were maintained in a SPF facility for the duration of this study. Prior approval for mouse use in the development of this allergic fungal asthma model was obtained from the University Laboratory Animal Medicine facility at the University of Michigan Medical School. Sensitization of mice to a commercially available preparation of soluble A. fumigatus antigens (Greer Laboratories, Lenoir, NC) was performed as described previously [18
]. Briefly, mice received a total of 10 µg A. fumigatus antigens dissolved in 0.2 ml incomplete Freund’s adjuvant (Sigma Chemical Co., St. Louis, MO), distributed equally between an intraperitoneal (i.p.) and a subcutaneous injection. Two weeks later, and for 3 subsequent weeks, mice received a total of 20 µg A. fumigatus antigen, dissolved in normal saline, by intranasal (i.n.) installation. One week after the third i.n. challenge, each mouse received 5.0 x 106 live A. fumigatus conidia suspended in 30 µl 0.1% Tween-80 via the i.t. route.
Measurement of bronchial hyperresponsiveness
At days 4 and 28 after the i.t. A. fumigatus conidia challenge, bronchial hyperresponsiveness in A. fumigatus-sensitized MBL-A+/+ and MBL-A–/– mice was assessed in a BuxcoTM plethysmograph (Buxco, Troy, NY) [18
]. Mice were anesthetized with sodium pentobarbital (40 mg/ml i.p.; Butler Co., Columbus, OH) before their intubation and ventilation with a Harvard pump ventilator (Harvard Apparatus, Reno, NV). Airway resistance was calculated online via computer software (Buxco) and was determined by the division of the transpulmonary pressure by the change in inspiratory volume. Following a baseline period in the Buxco plethysmograph, anesthetized and intubated mice received a dose of 210 and 420 µg/kg methacholine by tail vein injection, and airway responsiveness to this nonselective bronchoconstrictor was again calculated. At day 4 when acute inflammation and airway hyperresponsiveness are most pronounced, the lower 210-µg dose of methacholine was used for the airway hyperresponsiveness calculation, and the 420-µg dose was used at day 28. At the conclusion of the assessment of airway hyperresponsiveness, 500 µl blood was collected from each mouse and transferred to a microcentrifuge tube. Sera were obtained after the sample was centrifuged at 15,000 g for 10 min. A bronchoalveolar lavage (BAL) was performed with 1 ml normal saline, and fluid was removed from cells by centrifugation. BAL cells were subsequently cytospun onto coded microscope slides for differential analysis (see below). Finally, whole lungs were dissected from each mouse and snap-frozen in liquid N2 or prepared for histological analysis.
Morphometric analysis of leukocyte accumulation in BAL samples
Neutrophils, macrophages, eosinophils, and lymphocytes were quantified in BAL samples cytospun onto coded microscope slides using a cytospin (Shandon Scientific, Runcorn, UK). Identification of each cell type was facilitated by differential staining with Wright-Giemsa differential stain and expressed as average cells per high-powered field (1000x).
Enzyme-linked immunosorbent assay (ELISA) analysis
Murine interleukin (IL)-4, IL-5, IL-13, interferon- (IFN-), tumor necrosis factor 
(TNF-), IL-10, IL-12, CXC chemokine ligand (CXCL)2, CXCL3, CXCL9, CCL3, CCL5, CCL11, and CCL22 protein levels were determined in 50 µl whole lung homogenates, using a standardized sandwich ELISA technique described previously in detail [20
]. Whole lungs were homogenized in 1 ml normal saline containing 1 mg protease inhibitor (CompleteTM, Boehringer Mannheim, Indianapolis, IN). 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 and chemokine was consistently below 50 pg/ml, and the ELISA was screened to ensure the specificity of each antibody used.
Whole lung histological analysis
Whole lungs from nonsensitized MBL-A+/+ and MBL-A–/– mice at day 28 after A. fumigatus conidia challenge and from A. fumigatus-sensitized MBL-A+/+ and MBL-A–/– mice at days 4 and 28 after conidia were fully inflated by i.t. perfusion with 4% paraformaldehyde. Lungs were then dissected and placed in fresh paraformaldehyde for 24 h. Routine histological techniques were used to paraffin-embed this tissue, and 5-µm sections of whole lung were stained with hematoxylin and eosin (H/E), periodic acid Schiff (PAS), and Gomori methanamine silver (GMS).
Statistical analysis
All results are expressed as mean ± SEM. A Student’s t-test was used to determine statistical significance between the MBL-A+/+ and MBL-A–/– groups at various times after conidia challenge; P < 0.05 was considered statistically significant.
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Nonsensitized MBL-A–/– mice exhibited peribronchial inflammation and goblet cell metaplasia at day 28 after i.v. challenge with A. fumigatus conidia
As the i.v. or i.t. administration of A. fumigatus conidia into nonsensitized MBL-A–/– mice did not adversely affect the survival of these mice, we next examined whether the presence of the conidia in these mice altered the histological appearance of various tissues at day 28 after the conidia challenge. In MBL-A+/+ and MBL-A–/–mice that received an i.v. bolus of 1.0–5.0 x 105 A. fumigatus conidia, we observed no evidence of tissue damage in kidney, lung, liver, and brain (not shown) nor was there any histological evidence that A. fumigatus was retained in any of the above tissues in MBL-A–/– mice (i.e., GMS staining was absent), and this observation was similar to that seen in MBL-A+/+ mice (GMS staining is not shown for either group of mice). However, the i.t. challenge of 5.0 x 106 A. fumigatus conidia into MBL-A–/– mice appeared to cause major changes to the architecture of airways in these mice. In contrast to MBL-A+/+ mice at day 28 after conidia (Fig. 1A
and 1C
), the lungs of MBL-A–/– mice exhibited marked peribronchial inflammation (Fig. 1B)
and goblet cell metaplasia (Fig. 1D)
. Thus, this histological analysis revealed that the i.t. administration of A. fumigatus in nonsensitized MBL-A–/– mice promoted the maintenance of airway inflammation and remodeling.
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Figure 1. Representative photomicrographs of H/E (A and B)- and PAS (C and D)-stained whole lung sections from nonsensitized MBL-A+/+ (A and C) and MBL-A–/– (B and D) mice at day 28 after conidia challenge. Both groups of mice (n=five/group) were injected i.t. with 5.0 x 106 conidia. The peribronchial inflammatory response was markedly enhanced in the MBL-A–/– group (B) compared with the MBL-A+/+ (A) group. Although PAS-stained goblet cells were absent in the MBL-A+/+ group (C), several goblet cells were evident in the airways of MBL-A–/– mice (D). Original magnification, 200x.
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Figure 2. Airway resistance in A. fumigatus-sensitized MBL-A+/+ and MBL-A–/– mice at days 4 (upper) and 28 (lower) after i.t. challenge with 5.0 x 106 A. fumigatus conidia. Hyperresponsiveness, as measured by changes in airway resistance (units=cm H2O/ml/s), was measured in MBL-A+/+ and MBL-A–/– mice at days 4 and 28 after conidia challenge. A baseline reading was taken for each animal before methacholine injection (represented as solid lines across methacholine-challenged response bars). At day 4, 210 µg/kg methacholine was used to assess the airway response, and 420 µg/kg methacholine was used at day 28. Values are expressed as mean ± SEM; n = five/group/time point. *, P  0.05; **, P 0.01, compared with methacholine-induced airway hyperresponsiveness in MBL-A+/+ mice.
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Figure 3. Leukocyte counts in BAL samples from A. fumigatus-sensitized MBL-A+/+ and MBL-A–/– mice at days 4 and 28 after i.t. challenge with 5.0 x 106 A. fumigatus conidia. BAL cells were dispersed onto microscope slides using a cytospin; neutrophils, eosinophils, lymphocytes, and macrophages were differentially stained with Wright-Giesma stain. A minimum of 20 high-powered fields was examined in each cytospin. Values are expressed as mean ± SEM of the total cells counted/high-powered field (1000x).
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at day 28 was significantly higher in the knockout mice compared with the wild-type controls (Fig. 4D)
. Although Th2 cytokines enhance allergic responses to Aspergillus, Th1 cytokines, particularly IFN-, are potent inhibitors of this response (reviewed in ref. [21
]). Other cytokines relevant to immune responses elicited by Aspergillus, including TNF- [28
], IL-10 [29
], and IL-12 [30
], were not affected by MBL-A deficiency at either time after conidia challenge in A. fumigatus-sensitized mice (data not shown). These findings demonstrate that MBL-A deficiency has a major effect on the early elaboration of Th2 cytokines in the lungs of A. fumigatus-sensitized mice during chronic fungal asthma, and this may have implications in the balance of Th1/Th2 cytokine production in the chronic stages of the disease.
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Figure 4. Whole lung cytokine levels in A. fumigatus-sensitized MBL-A+/+ and MBL-A–/– mice at days 4 and 28 after i.t. challenge with 5.0 x 106 conidia. Cell-free supernatants from whole lung samples by specific ELISA measured IL-13 (A), IL-4 (B), IL-5 (C), and IFN- (D). Specific cytokine levels were normalized to total protein in each sample. Values are expressed as mean ± SEM; n = five/group/time point. *, P 0.05; **, P 0.01; ***, P 0.001, compared with whole lung cytokine levels in MBL-A+/+ mice.
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Figure 5. Whole lung chemokine levels in A. fumigatus-sensitized MBL-A+/+ and MBL-A–/– mice at days 4 and 28 after i.t. challenge with 5.0 x 106 conidia. Cell-free supernatants from whole lung samples by specific ELISA measured CCL5, CCL22, and CXCL9. Specific levels were normalized to total protein in each sample. Values are expressed as mean ± SEM; n = five/group/time point. *, P 0.05, compared with whole lung chemokine levels in MBL-A+/+ mice. MIG, Monokine induced by IFN-; RANTES, regulated on activation, normal T expressed and secreted; MDC, macrophage-derived chemokine.
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Figure 6. Representative photomicrographs of GMS-stained whole lung sections from A. fumigatus-sensitized MBL-A+/+ (A and C) and MBL-A–/– (B and D) mice at days 4 (A and B) and 28 (C and D) after i.t. challenge with 5.0 x 106 conidia. The arrows highlight fungal material. In MBL-A+/+ mice, the majority of fungal material was observed in mononuclear cells at day 4 (A), and little fungal material was observed at day 28 (C) after conidia challenge. In MBL-A–/– mice, the majority of fungal material was visualized around pulmonary vessels. Original magnification, 200x.
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Figure 7. Representative photomicrographs of H/E-stained whole lung sections from A. fumigatus-sensitized MBL-A+/+ (A and C) and MBL-A–/– (B and D) mice at days 4 (A and B) and 28 (C and D) after i.t. challenge with 5.0 x 106 conidia. MBL-A–/– (B) mice exhibited markedly greater peribronchial inflammation compared with the MBL-A+/+ (A) group at day 4 after conidia. Original magnification, 200x.
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Figure 8. Representative photomicrographs of PAS-stained whole lung sections from A. fumigatus-sensitized MBL-A+/+ (A and C) and MBL-A–/– (B and D) mice at days 4 (A and B) and 28 (C and D) after i.t. challenge with 5.0 x 106 conidia. MBL-A–/– (B) mice exhibited markedly greater goblet cell metaplasia compared with the MBL-A+/+ (A) group at day 4 after conidia. Original magnification, 200x.
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Figure 9. Representative photomicrographs of trichrome Masson-stained whole lung sections from A. fumigatus-sensitized MBL-A+/+ (A) and MBL-A–/– (B) mice at day 28 after i.t. challenge with 5.0 x 106 conidia. No differences in peribronchial fibrosis were observed between the two groups. Original magnification, 200x.
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Through recognition of distinct PAMPs, MBL triggers complement fixation through a modified classical pathway that involves the activation of C4 and C2 via MBL-associated serine proteases to generate complement components that have C3-convertase activity (reviewed in ref. [1 ]). Considerable interest in MBL has emerged because of growing clinical evidence that MBL makes an important contribution to innate immunity, with its absence typically increasing individual susceptibility to infectious pathogens and/or affecting the course of underlying disease [7 , 9 ]. Although a strong association between MBL deficiency and susceptibility to bacterial and parasitic infections has been documented [9 ], far less is known about the importance of this collectin in antifungal responses. MBL appears to be important in containing A. fumigatus, given a recent clinical study that showed that decreased circulating concentrations of MBL were significantly associated with the presence of CNPA [17 ]. The advent of mice lacking one of the two MBL isoforms found in mice [14 , 42 ] allowed us to undertake a detailed examination of the role of MBL in containing a bolus challenge of A. fumigatus conidia in the context of nonimmunocompromised mice that lacked prior sensitization to A. fumigatus or mice that were systemically sensitized to soluble A. fumigatus antigens.
The mode of recognition of Aspergillus conidia by phagocytes has been studied previously and yielded the following: Prior opsonization of the spores with normal rabbit serum, rabbit anti-A. fumigatus serum, complement, or lung lavage fluid has no profound enhancing effect on the phagocytosis or killing of the spores [43 ]; optimal association between phagocyte and conidia was dependent on an active, alternative complement pathway [44 ]; conidia cell wall components can degrade bound C3 [45 , 46 ]; conidia pigmentation regulates C3 binding [47 ]; and bound MBL was able to promote C4 deposition in a concentration-dependent manner [4 ]. Although MBL-A–/– mice were not more susceptible to Candida albicans or Plasmodium yoelil compared with MBL-A+/+ [42 ], we nonetheless first assessed whether MBL-A–/– mice were similarly resistant to an i.t. or i.v. challenge with live A. fumigatus conidia. Our data, combined with the previous study in MBL-A–/– [42 ], suggest that the lack of MBL-A may not be sufficient to profoundly impair the antifungal innate immune response, given that MBL-C may fully compensate for the lack of A isoform. However, MBL-A deficiency did have an effect on the histological appearance of the lung, possibly suggesting that the clearance of A. fumigatus from the lungs of nonsensitized MBL-A–/– mice was not as efficient as that in their wild-type counterparts. The airway features most prominent in the MBL-A–/– group at day 28 after the i.t. introduction of 5.0 x 106 conidia were peribronchial inflammation and goblet cell hyperplasia. Both of these airway features are cardinal signs of persistence of fungal antigens with the lung, possibly in a manner similar to that we have observed in the context of A. fumigatus-sensitized mice [18 ]. One caveat to this hypothesis is that we did not observe GMS-stained material in the lungs of MBL-A–/– mice, but it is possible that this histological technique for fungal antigen detection may not be sufficiently sensitive to detect the minute amounts of fungal antigen that may be driving the airway inflammatory and remodeling events. Thus, the lack of MBL-A did not affect mortality following a bolus conidia challenge, but its absence did affect the lung in a manner reminiscent of Aspergillus-induced airway disease [21 ].
Asthma is a chronic, Th2, cytokine-dominated, inflammatory disease of the lung, resulting in airway hyperreactivity and remodeling. Th2 cytokines, particularly IL-4, IL-5, and IL-13, regulate the airway hyperresponsiveness, eosinophil activation, and mucus production. Although not historically viewed as important in the pathogenesis of asthma [48
], recent studies have shown that the complement anaphylatoxins C3a [49
50
51
] and C5a [52
] contribute to pulmonary allergy in experimental allergic airway responses. The major features of allergic airway disease that were significantly attenuated by the absence of complement or complement receptors were airway hyperresponsiveness, airway eosinophilia, and Th2 cytokine levels. In the present study, the results obtained from A. fumigatus-sensitized MBL-A–/– mice, challenged i.t. with conidia, shared similarities with those observed in the previous studies referenced above [48
]. At day 4 after conidia challenge in A. fumigatus-sensitized MBL-A–/– mice, we observed a profound decrease in airway hyperreactivity and whole lung Th2 cytokine levels compared with their wild-type counterparts. However, BAL eosinophilia was actually enhanced in the day-4 MBL-A–/– group. This finding is possibly explained by the significant increases in potent eosinophil chemoattractants CCL5 and CCL22 [33
, 35
] in the lungs of this group. Coinciding with a significant increase in whole lung levels of IFN-, airway hyperreactivity and BAL eosinophilia were significantly attenuated at day 28 after conidia challenge in the MBL-A–/– group compared with the MBL-A+/+ group. However, the absence of MBL-A in A. fumigatus-sensitized mice did not attenuate the goblet cell metaplasia and peribronchial fibrosis typically associated with chronic fungal asthma [18
]. This finding contrasts with that of Drouin and colleagues [51
] who found that C3a receptor-deficient mice had significantly reduced mucus production compared with their wild-type controls. Although we have observed a dissociation between airway responsiveness to methacholine challenge and airway remodeling (i.e., goblet cell metaplasia) during chronic fungal asthma [32
, 35
, 53
], it is possible that the presence of MBL-C in MBL-A–/– mice or inflammatory responses modulated by chemokines such as CCL5 [33
] and CCL22 [35
] may have promoted the airway remodeling features intensely evident at day 28 after conidia challenge in both groups of sensitized mice. Generation of mice deficient in both murine MBL isoforms may facilitate whether airway-remodeling events are driven by MBL-C.
In summary, the present study demonstrates that MBL-A has a nonredundant role in the context of murine innate and allergic pulmonary responses to A. fumigatus conidia. Although the mice examined in the present study were sufficient in MBL-C [14 ], it was clear that the absence of MBL-A modulated airway inflammatory and remodeling events in nonsensitized and A. fumigatus-sensitized mice. Taken together, these above findings recapitulate the earlier conclusions of Takahashi et al. [14 ] in which they demonstrated that the role of MBL-A appeared to extend beyond that of opsonin to modulator of inflammatory processes in the mouse.
Received July 14, 2003; revised December 4, 2003; accepted January 6, 2004.
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in pulmonary host defense in murine invasive aspergillosis J. Immunol. 162,1633-1640 and GM-CSF in neutrophil recruitment Med. Mycol. 37,183-194[CrossRef][Medline] interferon-independent pathway Infect. Immun. 67,3047-3050This article has been cited by other articles:
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