| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
,¶
* Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas-Houston,
Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, and Departments of
Medicine and
Immunology and
¶ Biology of Inflammation Center, Baylor University College of Medicine, Houston, TX 77030
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This classification of asthma has led to the concept that the immediate response after allergen challenge is mediated by mast cells, whereas T lymphocytes and eosinophils are the predominant effector cells in the late asthmatic reaction. The role of the mast cell becomes evident when challenging WBB6F1W/Wv mast cell-deficient mice with OVA (7). These mice fail to develop AHR even though lung eosinophil levels and serum IgE responses are comparable to wild-type responses. Research efforts have also demonstrated that CD4+ T cells, which produce a Th2 pattern of cytokines, play a pivotal role in the pathogenesis of this disease (8). Through the release of cytokines such as IL-4, IL-13, and IL-5, Th2 cells are thought to contribute to bronchial hyperreactivity and mucus hypersecretion as well as orchestrate the recruitment and activation of eosinophils (9, 10). Moreover, T cell-deficient, RAG-/- mice, which lack Th2 cells, fail to develop AHR and to recruit eosinophils into the airways after challenge with Ag (11). IL-4 also plays a prominent role in allergy by initiating B lymphocyte differentiation and production of IgE Abs (12, 13).
Traditionally, asthma was considered a simple type I hypersensitivity reaction, and, as such, the complement system was not considered important in its pathogenesis (14). However, data from mice deficient in the third component of the complement system (C3) revealed diminished AHR, lung eosinophil levels, and IL-4 production in Ag-challenged lungs as well as reduced Ag-specific IgE and IgG1 responses, and clearly demonstrated that these features of asthma are significantly attenuated in the absence of C3 (15). Evidence has also surfaced suggesting the importance of the complement-derived C3a anaphylatoxin in asthma with elevated levels of the C3a peptide being detected in the lungs of asthmatic patients (16, 17, 18, 19). Considering that C3 is the parent molecule of C3a and that known plant, fungal, and insect allergens can cleave C3 and release C3a (20), these findings suggest that the C3a anaphylatoxin may be a key participant in asthma pathogenesis.
As an anaphylatoxin, C3a triggers contraction of smooth muscle, increases permeability of small blood vessels, and regulates vasodilation (21, 22). However, many studies have described a much broader ability of C3a to regulate effector functions of white blood cells and pulmonary cell types typically associated with asthma. C3a stimulates release of histamine from basophils (23) and mast cells (24) and regulates synthesis of eosinophil cationic protein and adhesion to endothelial cells by eosinophils (25, 26, 27). C3a is also a chemotactic molecule for both eosinophils (28) and mast cells (29). Within the lung, the functional importance of C3a has been established in studies examining the inflammatory effects of intrabronchial instillation of C3a in guinea pig lungs. Instillation of this anaphylatoxin induces respiratory distress characterized by contraction of the smooth muscle walls in bronchioles and aggregation of leukocytes in pulmonary vessels (30). Moreover, synthetic peptides, based on the C-terminal sequence of C3a, can also mimic these properties (31), and the addition of inhibitors to carboxypeptidase N, a known regulator of C3a activity, potentiates the respiratory distress stimulated by instillation of C3a (32, 33).
The C3a peptide regulates these inflammatory functions by interacting with its receptor, C3aR, which belongs to the rhodopsin family of seven transmembrane G protein-coupled receptors (34). The C3aR is present on many cell types associated with asthma such as eosinophils (35), mast cells (36), T lymphocytes (37), and B lymphocytes (38), as well as lung bronchial epithelial and smooth muscle cells (39). Involvement of this receptor in rodent models of pulmonary allergy has also been demonstrated with reports showing that C3aR mediates AHR in C3aR-deficient mice (18) and immediate airway bronchoconstriction in C3aR-deficient guinea pigs (40). Furthermore, elevated expression of this receptor has been documented on bronchial smooth muscle cells in mouse lung challenged with Ag (39). Although these studies are significant and implicate the C3aR as a regulator of airway tone in asthma, the reported data have not addressed the role of the C3aR within the context of Th2 responses, IgE regulation, or mucus production, which are also major characteristics of asthma. Accordingly, to elucidate the role of C3a and its receptor in asthma, we have studied a murine model of pulmonary allergy using a mixed Ag preparation of Aspergillus fumigatus cell culture filtrate and OVA in mice deficient for the C3aR. Data from these mice reveal an attenuated allergic response very similar to C3-deficient animals with diminished AHR, eosinophil recruitment, Th2 cytokine production, and mucus secretion in the lung, as well as reduced Ag-specific IgE and IgG1 responses. These results demonstrate a requisite contribution of the C3aR to allergic lung disease and provide evidence that the C3aR has a novel role in Th2 development in pulmonary allergy.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The C3aR-deficient (C3aR-/-) mice used have been described previously (41). The C3aR-/- mice were backcrossed eight generations onto the C57BL/6 background, and their wild-type littermates (C3aR+/+) were used as controls. This work was conducted in accordance with institutional and National Institutes of Health guidelines and care.
Sensitization and challenge protocol
The Ag preparation is a mixture of A. fumigatus cell culture filtrate prepared free of living organisms as described (42) and OVA (Sigma-Aldrich, St. Louis, MO). OVA added to the preparation permitted detection of Ag-specific IgE, IgG1, and IgG2a levels after AHR measurements to ascertain immune responses during the course of disease. Two batches of A. fumigatus culture filtrate with equivalent activities (lot nos. 5323R1, 5325) were aliquoted at a concentration of 6 mg/ml and stored at -70°C for use in all the experiments described herein. The LPS content of the Ag extracts was <0.2 EU/100 µg of Ag when tested with the Limulus amebocyte lysate test (BioWhittaker, Walkersville, MD) (42). Four-week-old C3aR+/+ and C3aR-/- mice were sensitized i.p. with the mixed Ag preparation on days 1, 5, 9, and 13 followed by two intranasal (i.n.) challenges on days 17 and 19. In all cases, 25 µg of OVA and 35 µg of A. fumigatus culture filtrate were diluted in PBS and delivered in a total volume of 50 µl for the i.p. or i.n. administration. All mice receiving i.n. challenges were anesthetized with isofluorane and held upright until all of the Ag administered into their nostrils was inhaled. PBS controls shown for each experiment were C3aR+/+ or C3aR-/- mice that were sensitized with the Ag and then challenged with PBS.
AHR measurements
Airway responsiveness to i.v. acetylcholine (ACh) challenge was measured as previously described (43). In brief, mice were anesthetized i.p. with etomidate (Abbott Laboratories, Abbott Park, IL) at 20 µg/g body weight and locally with lidocaine (Sigma-Aldrich). The tracheas were surgically exposed, cannulated with a blunt-ended, 20-gauge angiocatheter, and connected to a rodent ventilator (Harvard Apparatus, Holliston, MA). Mice were ventilated with 100% oxygen at a rate of 150 breaths per min and a tidal volume of 9 µl/g. Following paralysis with pancuronium bromide (4 µg/g; Gensia Laboratories, Irvine CA), i.v. access was established using a 27-gauge needle placed into a tail vein, and mice were placed into a rodent plethysmograph capable of determining tidal volume, airflow, and transthoracic resistance continuously (Buxco Electronics, Sharon, CT). Airway responses were expressed as the provocative concentration of ACh, in micrograms per gram body weight, required to double baseline transthoracic resistance (PC200). Lower PC200 values represent greater airway reactivity.
Bronchoalveolar (BAL) lavage measurements
Airway inflammation was assessed by BAL. Lungs were lavaged three times with 1 ml of PBS. Between 2.2 and 2.4 ml of BAL fluid was recovered from each lung, and total cell numbers were determined using a hemacytometer. Differential cell counts were obtained from BAL cells spun onto slides with a cytocentrifuge (Thermo Shandon, Pittsburgh, PA) and treated with Wright-Giemsa stain (Sigma-Aldrich). Five-hundred cells were classified based on characteristic morphology revealed after staining. Absolute numbers of specific cell types (neutrophils, lymphocytes, macrophages, and eosinophils) were calculated from the recovered BAL volume, total cell count, and percent abundance of specific cells.
Lung histology
After AHR measurements, lungs were inflated with 0.5 ml of formalin (Sigma-Aldrich) and the trachea was tied off. The lungs were then removed from the chest cavity and placed in formalin overnight at 4°C. Tissues were embedded in paraffin, cut into 5-µm sections, and stained with H&E. Inflammatory infiltrates and lung architecture were assessed using light microscopy.
Measurement of lung mucin levels
For determination of lung mucin levels, BAL fluid was collected as described, and cells were removed by centrifugation. Lung mucin levels were measured as previously reported (44) using the mucin-binding lectin, jacalin (45). Wells of microtiter plates (Immulon IV; Fisher Scientific, Pittsburgh, PA) were precoated with serial 2-fold dilutions of mucin standard (Sigma-Aldrich) and BAL samples diluted in PBS for 2 h at 37°C and then blocked with 0.2% I-block (Applied Biosystems, Foster City, CA) in PBS containing 0.05% Tween 20. Plates were washed with PBS containing 0.05% Tween 20 and then incubated with 5 µg/ml biotinylated jacalin (Vector Laboratories, Burlingame, CA) diluted in PBS for 1 h at 37°C. Plates were washed and incubated with a 1/500 dilution of streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at room temperature. After a final wash, plates were developed with p-nitrophenyl phosphate (Sigma-Aldrich) diluted in alkaline buffer solution (Sigma-Aldrich), and ODs were measured at 405 nm.
Measurement of Th2 responses in the lung
Th2 responses in the lung were assayed for the presence of IL-4-
and IFN-
-producing cells by ELISPOT (R&D Systems, Minneapolis, MN)
as per the manufacturer’s instructions. Briefly, single-cell
suspensions from whole lung cells were distributed in duplicate
aliquots (106 cells in RPMI 1640 with 5% FBS and
antibiotics) into 96-well microtiter plates (Immulon IV; Fisher
Scientific) that had been precoated with polyclonal Abs against either
mouse IL-4 or mouse IFN- (R&D Systems). Serial 2-fold dilutions were
prepared, and the plates were incubated undisturbed for 8 h at
37°C. Wells were washed with PBS to remove cells and incubated with
biotinylated secondary polyclonal Abs against mouse IL-4 or mouse
IFN- (R&D Systems). After 1 h, wells were washed and incubated
for 1 h with 100 µl of streptavidin-conjugated alkaline
phosphatase (Jackson ImmunoResearch Laboratories) in PBS with 0.05%
Tween 20 and 5% FBS. Color was developed with
5-bromo-4-chloro-3-indolyl phosphate in 0.1 M
2-amino-2-methyl-1-propanol buffer (Sigma-Aldrich) suspended in 0.6%
agarose. After solidification of the agar, individual blue spots were
counted by inverted microscopy (9). For both ELISPOT
assays, cytokine-producing cells could be measured to 50 cells/ml.
Levels of IL-5 and IL-13 were determined in the BAL fluid to further assess the Th2 response in this model. BAL fluid was collected as previously described, and cells were removed by centrifugation. The BAL supernatants were then assayed for the presence of IL-5 and IL-13 by ELISA (R&D Systems) as per the manufacturer’s instructions, and the assays were sensitive to 5 pg/ml for IL-5 and 1.5 pg/ml for IL-13.
Total IgE and Ag-specific Ig measurements
Serum IgE titers were determined by two-site sandwich ELISA using an ELISA kit (BD PharMingen, San Diego, CA) as per manufacturer’s specifications, and the assay was sensitive to 2 ng/ml. For the Ag-specific Ab analyses, wells of microtiter plates were precoated with 40 µl of OVA in PBS and blocked with 5% BSA in PBS with 0.05% Tween 20. Serum diluted 1/500 was then added and incubated for 1 h at 37°C. Plates were washed, and biotinylated isotype-specific Abs (anti-IgE, IgG1, and IgG2a; Jackson ImmunoResearch Laboratories) were added for an additional hour of incubation. Plates were washed again and streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories) was added for 30 min at room temperature. After a final wash, plates were developed with 0.5 M nitrophenyl phosphate in 0.1 M 2-amino-2-methyl-1-propanol buffer (Sigma-Aldrich). Data are expressed as OD405.
Statistical analysis
Statistical analysis was performed using the Prism software package (GraphPad, San Diego, CA). Statistical significance of all results were assessed using the two-tailed, unpaired Student’s t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mice deficient in the C3aR lack a functional receptor that can
bind the complement anaphylatoxin C3a (41). In our
laboratory, mouse models of pulmonary allergy have demonstrated that
the expression of this receptor is increased on airway smooth muscle
after Ag challenge (39) and that AHR, lung inflammation,
and immune responses are attenuated in mice deficient in C3, the parent
protein of C3a (15). Collectively, these studies suggest
that C3a and its receptor, C3aR, are important molecular mediators of
pulmonary allergy and, therefore, potential therapeutic targets in the
treatment of asthma. To further establish the role that C3aR plays in
allergic lung disease, we subjected C3aR-deficient mice to an in vivo
mouse model of pulmonary allergy. C3aR-/- and
C3aR+/+ mice were challenged with a mixed Ag
preparation comprised of A. fumigatus cell culture filtrate
prepared free of living organisms (42) and OVA. OVA was
specifically added to the preparation to detect Ag-specific Ig levels
after AHR measurements and evaluate immune responses during the course
of disease. Twenty-four hours after the last challenge, AHR to
increasing doses of ACh was evaluated by airway plethysmography. Within
this model, mice challenged with the Ag preparation are expected to
have greater sensitivity to ACh, and, as a result, the provocative
concentration of ACh required to induce a 200% increase in airway
resistance above baseline will be lower for these mice compared with
PBS-challenged controls (9). As shown in Fig. 1
, Ag-challenged wild-type mice developed
an asthma-like response characterized by an increase in AHR as revealed
by enhanced sensitivity when exposed to ACh. In contrast, AHR was
abrogated in Ag-challenged C3aR-/- mice as
shown by the attenuation in ACh sensitivity relative to challenged
wild-type littermates. Furthermore, the AHR in the
C3aR-/- mice approached that observed in
PBS-challenged C3aR+/+ and
C3aR-/- controls, demonstrating an important
role for this complement anaphylatoxin receptor in allergic airway
responsiveness.
|
An additional facet of asthma is the pulmonary inflammation
characterized by the presence of white blood cells, specifically
eosinophils, recruited into the airways (5). Eosinophils
are chemotactic to the C3a anaphylatoxin (26), and mice
deficient in the C3aR could potentially have reduced eosinophil
recruitment into the lung after exposure to Ag. To determine whether
the C3aR contributes to eosinophil recruitment, lungs from challenged
mice were lavaged after the AHR measurements, and the BAL fluid was
examined for influx of white blood cells (Fig. 2). Quantitation of white blood cells
from the BAL of PBS-challenged C3aR+/+ and
C3aR-/- mice revealed a population of cells
comprised predominantly of macrophages with few neutrophils,
eosinophils, and lymphocytes. Challenging wild-type mice with the mixed
Ag preparation resulted in an increase in all cell types relative to
PBS controls, and the BAL cell population consisted mainly of
eosinophils and neutrophils. However, challenged C3aR-deficient mice
revealed a marked reduction in all inflammatory cells, including an
88% reduction in eosinophils as well as a 79% decrease in airway
neutrophils and a 54% decrease in airway lymphocytes. The reduced
presence of inflammatory cells in the BAL fluid was further
corroborated by H&E stained lung sections from challenged C3aR knockout
mice (Fig. 3), which revealed diminished
infiltration of eosinophils into the peribronchial region between the
pulmonary blood vessels and the airways relative to their wild-type
counterparts (Fig. 3). Collectively, these results demonstrate that
mice deficient in the C3aR not only have deficits in lung eosinophil
recruitment after challenge but also have defects in neutrophil and
lymphocyte recruitment as well.
|
|
Mucus hypersecretion is another characteristic feature
contributing to the airway obstruction in asthma. Overproduction of
mucus in the airways is the result of increased proliferation of goblet
cells and their increased production of mucin subsequent to challenge
with allergen (1). Measurement of BAL mucin levels in
Ag-challenged C3aR-/- mice revealed a
significant 52% reduction in airway mucin levels compared with the
Ag-challenged controls (Fig. 4) and
indicate that the C3aR participates in airway mucin production.
Furthermore, this data is consistent with the attenuated AHR and airway
eosinophil recruitment observed in challenged C3aR-deficient mice and
strongly suggests that the C3aR contributes to much of the pulmonary
inflammation in this model.
|
production
in Ag-challenged lungs
Previous reports have described elevated levels of Th2 cytokines
in asthma (1) and the importance of the Th2 response in
mouse models of pulmonary allergy (9, 11). Considering
these observations and that challenged C3aR-/-
mice had a 54% decrease in levels of airway lymphocytes (Fig. 2
), we
performed additional studies to understand the role of the C3aR with
respect to T cell effector function. To assess T cell activation in
C3aR-/- mice, IL-4 and IFN- production in
the lung was quantitated by ELISPOT and evaluated as a marker
of Th2 vs Th1 responses after challenge (Fig. 5, top panel). Consistent with
this model, wild-type mice challenged with the mixed Ag preparation
exhibited a pronounced Th2 response in the lung as demonstrated by the
elevated levels of cells expressing IL-4 and lack of cells expressing
IFN-. In contrast to the elevated numbers of IL-4-producing cells in
the wild-type animals, cells expressing IL-4 were reduced 59% in the
lungs of Ag-challenged C3aR-/- mice. Lungs from
Ag-challenged C3aR-deficient and wild-type mice did not have measurable
IFN--producing cells above the background limits for the assay, and
analysis of BAL fluid by ELISA also revealed no detectable quantities
of IFN- protein in either challenged wild-type or C3aR-deficient
animals (data not shown).
|
, bottom panel). Challenging
C3aR-/- mice with the mixed Ag preparation did
not have a pronounced effect on IL-5 expression in the airways, and the
IL-5 levels detected in the BAL fluid of these mice was below the
sensitivity limits of the assay. Challenging
C3aR-/- mice did increase BAL IL-13 levels
above PBS controls. However, IL-13 levels in the knockout mice were
still reduced 77% compared with Ag-challenged wild-type animals. These
results and the reduced numbers of IL-4-producing cells demonstrate
that the C3aR is involved in IL-4, IL-5, and IL-13 regulation in the
lung and indicate that the C3a anaphylatoxin and its receptor are
potentially linked to adaptive immunity by promoting Th2 responses in
this model. Effect of C3aR deficiency on Ag-specific Ig levels
The preceding data suggest that the dramatic reduction in the
airway responses and pulmonary inflammation observed in Ag-challenged
C3aR-/- mice is due to a defect in Th2
function. To gain further insight into the mechanisms linking
complement to adaptive immunity, we determined total and Ag-specific Ab
responses in wild-type and C3aR-/- mice.
Increased production of IgE Abs during the course of asthma is another
characteristic of the disease (46), and examination of
total serum IgE in Ag-challenged C3aR+/+ and
C3aR-/- mice revealed elevated levels of IgE
compared with their PBS controls. However, total IgE levels in
challenged C3aR-deficient mice were significantly reduced by 40%
compared with the similarly challenged wild-type mice (Fig. 6, top panel). Ag-specific
IgE, IgG1, and IgG2a isotypes were also examined in
C3aR-/- mice (Fig. 6, bottom panel),
and although C3aR knockout and wild-type mice had comparable levels of
IgG2a, Ag-specific IgE and IgG1 levels were decreased 70% and 64%,
respectively, in the Ag-challenged C3aR-/-
animals. These results are consistent with the premise that IgE and
IgG1 production, which is controlled by T cell production of IL-4 and
IL-13 (47), should be reduced as a result of decreased
IL-4 and IL-13 production in the C3aR-deficient mice. Moreover,
expression of IgG2a, which is regulated by Th1 cells and IFN-
(48), would be similar between C3aR-deficient and
wild-type mice because no IFN- production was detected in these
animals. Collectively, these results strongly suggest that the
decreased IgE levels result from the reduced IL-4 and IL-13 production
in the C3aR-deficient mice and reveal a defect in Th2 effector function
in the absence of the C3aR.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The AHR findings from this study are consistent with previous reports examining the role of C3 or C3a in rodent models of pulmonary allergy. ACh is a cholinergic agonist that can induce bronchoconstriction in mice (49), and the increased sensitivity of airway smooth muscle to ACh is thought to originate from the release of spasmogenic substances during the effector phase of asthma which cause bronchoconstriction and airway inflammation (reviewed in Ref. 50). When comparing the airway responses of C3aR-deficient mice with C3-deficient mice in the A. fumigatus/OVA model (15) or with C3aR-deficient rodents in the OVA model (18, 40), Ag-challenged animals deficient in C3 or C3aR had problems mounting immediate bronchospastic responses or late-phase AHR relative to their wild-type controls. Although the level of reduction in the airway response varies between the different reports, these studies collectively highlight the ability of C3a to regulate airway tone in these rodent models. Given these findings, one could postulate that production of complement components in the airways (51) could provide a local source of C3. After challenge with an allergen, proteolytic cleavage of C3 from classical, lectin, or alternative pathway activation or from proteases present in the A. fumigatus extract (20) or released from activated mast cells (52) could generate C3a in the airways leading to subsequent bronchial smooth muscle contraction. Considering that elevated levels of the C3a peptide have been detected in the BAL of human asthma patients (16, 17, 18, 19) and that the C3aR has been detected on airway smooth muscle in human lung (39), C3a and its receptor are implicated as regulators of AHR in asthma. However, it currently is unclear whether C3a modulates AHR directly by binding C3aR on airway smooth muscle cells or indirectly by stimulating release of mediators of bronchoconstriction from other cells bearing C3aR.
In addition to the airway responses, both C3- and C3aR-deficient strains had similar deficits in IL-4 expression and IgE serum levels in the A. fumigatus/OVA model (15), and, although not previously reported, Ag-challenged C3-deficient mice also have decreases in IL-5 and IL-13 expression comparable to the C3aR-deficient mice (data not shown). Curiously though, white blood cell recruitment into the airways was dramatically different between the two knockouts. Specifically, C3-deficient mice had a 42% reduction in lung eosinophil levels whereas C3aR-deficient mice had an 88% reduction when compared with their wild-type controls. One possible explanation for this difference comes from studies examining the role of Mac-1 (CD11b/CD18) in pulmonary allergic disease (53). Widely known as an adhesion molecule involved in leukocyte diapedesis, Mac-1 also binds C3bi (54), another complement activation fragment derived from C3. When challenged with Ag, mice deficient in Mac-1 exhibit increased eosinophil recruitment into the airways and lung parenchyma relative to wild-type controls. These findings are in contrast to the observations described in this study where challenged C3aR-deficient mice have dramatically reduced levels of airway eosinophils relative to wild-type controls. Knowing that C3-deficient mice lack both the C3a and C3bi peptides, one could postulate that the reduction in airway eosinophils that results from the lack of C3a in challenged C3-deficient mice is compromised by the concomitant absence of C3bi.
Likewise, inconsistencies in the lung eosinophil levels and the immune responses exist when comparing data from this study with reports examining pulmonary allergy in C3aR-deficient mice (18) and guinea pigs (40). Although deficits in lung eosinophil recruitment were observed in this study, no significant differences in eosinophil recruitment were detected between OVA-challenged wild-type and C3aR knockout mice or OVA-challenged wild-type and C3aR-deficient guinea pigs. Moreover, total serum IgE levels and BAL levels of IL-4, IL-5, and IL-13 were reported to be similar between challenged wild-type and C3aR knockout mice (18). These responses could possibly be explained by differences in the animals used for the study. The knockout mice used in this study were backcrossed eight generations onto the C57BL/6 background whereas the previously published reports used C3aR-deficient guinea pigs (40) or C3aR knockout mice that were backcrossed three generations on the BALB/c background (18). Alternatively, differences in Ag might also explain these findings. The studies by Humbles et al. (18) and Bautsch et al. (40) used OVA to induce pulmonary allergy whereas this study used extracts from A. fumigatus combined with OVA. In contrast to OVA alone, A. fumigatus is an allergen relevant to human allergic lung disease and has been shown to activate complement and release complement anaphylatoxins in vitro (20).
Studies have also examined whether the complement anaphylatoxin C5a and its receptor, C5aR, are primary mediators in pulmonary allergy models. Recent studies have demonstrated that inhibition of C5a using a C5aR antagonist will reduce late airway responses in OVA-challenged rats (55) and have implicated the gene encoding the fifth component of the complement system (C5), which is the parent protein for C5a, as a susceptibility locus for allergen-induced AHR (56). C5a has also been shown to regulate the production of IL-12 from macrophages (57), a cytokine which suppresses Th2 responses, and the expression of IL-4 and IL-13 from basophils (58), which may contribute to Th2 development in the initial stages of asthma. Because IL-4, IL-13, IgE, and mucus production were not completely inhibited in C3aR-deficient mice, it is possible that the C3aR and C5aR may have overlapping functions in regulating the underlying inflammatory and immune responses in this model. Additional studies directly comparing mice deficient in the C3aR and C5aR will be required to resolve these issues.
Perhaps the most significant findings reported in this study are the combined defects in AHR, eosinophil recruitment, IgE responses, and mucus hypersecretion after Ag challenge. Previous studies have established that IL-4, IL-5, and IL-13 regulate these functions in similar pulmonary allergy models (9, 10) and suggest that the reduced presence of IL-4, IL-5, and IL-13 in the lungs of C3aR-deficient mice are the central cause for these observed defects. Although the C3aR has been detected on human T lymphocytes in vitro (37), expression of the C3aR has not been demonstrated on mouse T lymphocytes nor does the receptor have a known role in regulating T cell responses in vivo. One explanation for these findings is that T lymphocytes lack the C3aR which is critical for the direct development of Th2 responses in this model. Alternatively, one could hypothesize that the C3aR has a vital regulatory role on many cell types that contribute to the pulmonary inflammation and immune responses. The C3aR has been detected on both human and mouse bronchial epithelial and smooth muscle cells (39) and on human B lymphocytes (38), eosinophils (35), and mast cells (36), cell types which are involved in the development of asthma. Although this report demonstrates that the C3aR is important for both Th2 development and experimental allergic lung disease, further investigation will be required to distinguish the distinct roles played by the C3aR regarding the function of T lymphocytes and other supporting cell types in asthma.
In conclusion, the study presented in this report substantiates previous reports describing C3aR-mediated regulation of AHR and documents a novel regulatory role for the C3aR in the development of Th2 responses in a mouse model of pulmonary allergy. These results lay the foundation for additional studies directed at assessing the role of the C3aR on CD4+ T lymphocytes in vitro as well as delineating in vivo the mechanisms by which C3a contributes to airway responses, mucus hypersecretion, lung eosinophil recruitment, and IgE production. Given the important contribution of Th2 cytokine production in asthma, further study of C3a-mediated regulation of T lymphocytes may shed light in the causative events leading to AHR and airway inflammation in this disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Biological Allergy Research, ALK-Abelló, Hoersholm, Denmark.
3 Address correspondence and reprint requests to Dr. Rick A. Wetsel, Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas, 2121 West Holcombe Boulevard, Suite 907, Houston, TX 77030. E-mail address: Rick.A.Wetsel{at}uth.tmc.edu
4 Abbreviations used in this paper: AHR, airway hyperresponsiveness; i.n., intranasal; ACh, acetylcholine; BAL, bronchoalveolar lavage.
Received for publication July 22, 2002. Accepted for publication September 9, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and 
and prostaglandin E2. Proc. Natl. Acad. Sci. USA 85:6880., IL-6, and the polyclonal immune response. J. Immunol. 159:4279.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  N. Mizutani, T. Nabe, and S. Yoshino Complement C3a Regulates Late Asthmatic Response and Airway Hyperresponsiveness in Mice J. Immunol., September 15, 2009; 183(6): 4039 - 4046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhang, I. P. Lewkowich, G. Kohl, J. R. Clark, M. Wills-Karp, and J. Kohl A Protective Role for C5a in the Development of Allergic Asthma Associated with Altered Levels of B7-H1 and B7-DC on Plasmacytoid Dendritic Cells J. Immunol., April 15, 2009; 182(8): 5123 - 5130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. G. Therien, V. Bernier, S. Weicker, P. Tawa, J.-P. Falgueyret, M.-C. Mathieu, J. Honsberger, V. Pomerleau, A. Robichaud, R. Stocco, et al. Adenovirus IL-13-Induced Airway Disease in Mice: A Corticosteroid-Resistant Model of Severe Asthma Am. J. Respir. Cell Mol. Biol., July 1, 2008; 39(1): 26 - 35. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Basham, M. Sathe, J. Grein, T. McClanahan, A. D'Andrea, E. Lees, and A. Rascle In vivo identification of novel STAT5 target genes Nucleic Acids Res., June 1, 2008; 36(11): 3802 - 3818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Wills-Karp Complement Activation Pathways: A Bridge between Innate and Adaptive Immune Responses in Asthma Proceedings of the ATS, July 1, 2007; 4(3): 247 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Dillard, R. A. Wetsel, and S. M. Drouin Complement C3a Regulates Muc5ac Expression by Airway Clara Cells Independently of Th2 Responses Am. J. Respir. Crit. Care Med., June 15, 2007; 175(12): 1250 - 1258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Melendi, S. J. Hoffman, R. A. Karron, P. M. Irusta, F. R. Laham, A. Humbles, B. Schofield, C.-H. Pan, R. Rabold, B. Thumar, et al. C5 Modulates Airway Hyperreactivity and Pulmonary Eosinophilia during Enhanced Respiratory Syncytial Virus Disease by Decreasing C3a Receptor Expression J. Virol., January 15, 2007; 81(2): 991 - 999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Mueller-Ortiz, T. J. Hollmann, D. L. Haviland, and R. A. Wetsel Ablation of the complement C3a anaphylatoxin receptor causes enhanced killing of Pseudomonas aeruginosa in a mouse model of pneumonia Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L157 - L165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Taube, J. M. Thurman, K. Takeda, A. Joetham, N. Miyahara, M. C. Carroll, A. Dakhama, P. C. Giclas, V. M. Holers, and E. W. Gelfand Factor B of the alternative complement pathway regulates development of airway hyperresponsiveness and inflammation PNAS, May 23, 2006; 103(21): 8084 - 8089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Drouin, M. Sinha, G. Sfyroera, J. D. Lambris, and R. A. Wetsel A Protective Role for the Fifth Complement Component (C5) in Allergic Airway Disease Am. J. Respir. Crit. Care Med., April 15, 2006; 173(8): 852 - 857. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhou, H. Patel, K. Li, Q. Peng, M.-B. Villiers, and S. H. Sacks Macrophages from C3-deficient mice have impaired potency to stimulate alloreactive T cells Blood, March 15, 2006; 107(6): 2461 - 2469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Schaefer, S. Konrad, J. Thalmann, C. Rheinheimer, K. Johswich, B. Sohns, and A. Klos The Transcription Factors AP-1 and Ets Are Regulators of C3a Receptor Expression J. Biol. Chem., December 23, 2005; 280(51): 42113 - 42123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. P. Gerard, B. Lu, P. Liu, S. Craig, Y. Fujiwara, S. Okinaga, and C. Gerard An Anti-inflammatory Function for the Complement Anaphylatoxin C5a-binding Protein, C5L2 J. Biol. Chem., December 2, 2005; 280(48): 39677 - 39680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Honczarenko, M. Z. Ratajczak, A. Nicholson-Weller, and L. E. Silberstein Complement C3a Enhances CXCL12 (SDF-1)-Mediated Chemotaxis of Bone Marrow Hematopoietic Cells Independently of C3a Receptor J. Immunol., September 15, 2005; 175(6): 3698 - 3706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. B. Martin and B. K. Martin Characterization of the Murine C3a Receptor Enhancer-Promoter: Expression Control by an Activator Protein 1 Sequence and an Ets-Like Site J. Immunol., September 1, 2005; 175(5): 3123 - 3132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Therien, R. Baelder, and J. Kohl Agonist Activity of the Small Molecule C3aR Ligand SB 290157 J. Immunol., June 15, 2005; 174(12): 7479 - 7480. [Full Text] [PDF]
|
|
|
|
|
|
R. Baelder, B. Fuchs, W. Bautsch, J. Zwirner, J. Kohl, H. G Hoymann, T. Glaab, V. Erpenbeck, N. Krug, and A. Braun Pharmacological Targeting of Anaphylatoxin Receptors during the Effector Phase of Allergic Asthma Suppresses Airway Hyperresponsiveness and Airway Inflammation J. Immunol., January 15, 2005; 174(2): 783 - 789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Boos, I. L. Campbell, R. Ames, R. A. Wetsel, and S. R. Barnum Deletion of the Complement Anaphylatoxin C3a Receptor Attenuates, Whereas Ectopic Expression of C3a in the Brain Exacerbates, Experimental Autoimmune Encephalomyelitis J. Immunol., October 1, 2004; 173(7): 4708 - 4714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ahamed, R. T. Venkatesha, E. B. Thangam, and H. Ali C3a Enhances Nerve Growth Factor-Induced NFAT Activation and Chemokine Production in a Human Mast Cell Line, HMC-1 J. Immunol., June 1, 2004; 172(11): 6961 - 6968. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
C. Taube, Y.-H. Rha, K. Takeda, J.-W. Park, A. Joetham, A. Balhorn, A. Dakhama, P. C. Giclas, V. M. Holers, and E. W. Gelfand Inhibition of Complement Activation Decreases Airway Inflammation and Hyperresponsiveness Am. J. Respir. Crit. Care Med., December 1, 2003; 168(11): 1333 - 1341. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
