Complement in asthma: sensitivity to activation and generation of C3a and C5a via the different complement pathways

Complement in asthma: sensitivity to activation and generation of C3a and C5a via the different complement pathways

ORIGINAL ARTICLES Complement in asthma: sensitivity to activation and generation of C3a and C5a via the different complement pathways SVEN K. WUST, MA...

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ORIGINAL ARTICLES Complement in asthma: sensitivity to activation and generation of C3a and C5a via the different complement pathways SVEN K. WUST, MALCOLM N. BLUMENTHAL, EDWARD O. CORAZALLA, BARBARA A. BENSON, and AGUSTIN P. DALMASSO MINNEAPOLIS, MINN

Studies in rodent models suggested that complement may play a critical role in susceptibility to airway hyperresponsiveness (AHR) and as a mediator of bronchial obstruction and inflammation in asthma. Complement may participate in susceptibility to asthma because of an intrinsic abnormality in complement activation and generation of C3a, C5a, or other products that affect cellular responses, resulting in TH2 predominance and asthma susceptibility. Alternatively, an intrinsic abnormality in the cellular response to complement activation products could determine susceptibility to asthma. In this study, the authors investigated whether complement in patients with atopic asthma versus nonatopic controls possesses an increased propensity to become activated. Despite reports that total complement plasma levels in unchallenged asthmatics are normal, an abnormal sensitivity of complement to activation may exist if an isoform or a polymorphic variant of a complement protein was present and resulted in gain or loss of function without associated changes in total complement levels. Therefore, complement activation was induced in vitro in plasma of asthmatics and controls using activators of the classical, alternative, and lectin pathways and measured C3a, other C3 fragments, and C5a. For each pathway, similar amounts of generated fragments, as well as C3a/C3 and C5a/C5 ratios, were found in asthmatics and controls. Also, similar basal plasma levels of C3a and C5a were found in both groups; however, mannan-binding lectin (MBL) levels were moderately elevated in asthmatics. In conclusion, the results suggest that, in asthmatic patients, complement activation does not exhibit an abnormal sensitivity to activation by any of the known activation pathways. (Translational Research 2006;148:157–163) Abbreviations: AHR ⫽ airway hyperresponsiveness; BAL ⫽ brochoalveolar lavage; BSA ⫽ bovine serum albumin; C3aR ⫽ C3a receptor; C5aR ⫽ C5a receptor; CR2 ⫽ complement receptor 2; EDTA ⫽ ethylene diamine tetraacetic acid; EGTA ⫽ ethylene glycol bis(2-aminoethyl ether) tetraacetic acid; ELISA ⫽ enzyme-linked immunosorbent assay; FEV1 ⫽ forced expiratory volume in the first second of a forced vital capacity maneuver; GVB ⫽ gelatinVeronal buffer; IL ⫽ interleukin; LPS ⫽ lipopolysaccharide; MBL ⫽ mannan-binding lectin; PBS ⫽ phosphate buffered solution; PD20FEV1 ⫽ provocative dose of methacholine causing a 20% drop in FEV1; SD ⫽ standard deviation; SEM ⫽ standard error of the mean From the Department of Medicine, Department of Surgery, and Department of Laboratory Medicine, School of Medicine, University of Minnesota, Minneapolis, Minn. Supported by grants from the University of Minnesota Academic Health Center Faculty Research Development Program, the National Center for Research Resources of the National Institutes of Health (M01-RR00400), and the Singulair Investigator Initiated Studies Program from Merck & Co., Inc. S.K.W. was supported by the Alfred Michael Fellowship from the Lillehei Heart Institute, University of Minnesota.

Submitted for publication November 7, 2005; revision submitted March 9, 2006; accepted for publication May 11, 2006. Reprint requests: Agustin P. Dalmasso, MD, Department of Surgery, MMC 220, University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455. e-mail: [email protected]. 1931-5244/$ – see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.trsl.2006.05.004

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Complement has been considered to be an important factor in the pathophysiology of asthma. Biologically active fragments derived from complement proteins can result from activation of complement through the classical, alternative, and lectin pathways, and by the direct action of certain proteolytic enzymes on C3 or C5.1–3 In asthma, infections and allergens that affect the respiratory tract may activate complement locally.4,5 The complement fragments C3a and C5a may participate as mediators of asthma because of their ability to recruit and activate leukocytes, increase vascular permeability, stimulate contraction of smooth muscle, and trigger degranulation of mast cells. In serum of patients with asthma, reductions in complement levels and increased levels of complement activation fragments after allergen-induced bronchospasm have been found in some studies6,7 but not in others.8,9 C5a was detected in BAL fluid from atopic asthmatics,10 and increased levels of C3a and C5a were found in the BAL fluid of asthmatics after allergen-induced bronchospasm, suggesting that complement activation products may play a role as effectors of asthma.11,12 Moreover, studies in mouse models of AHR and inflammation have also suggested that both C5a and C5b-9 are important in asthma pathogenesis.13,14 In addition to a possible role as a mediator of asthma pathophysiology, complement has been proposed as a susceptibility factor in the induction of AHR and asthma,15,16 conditions that are thought to result from complex interactions between host genetic factors and environmental factors. Among multiple genes that have been linked to asthma susceptibility are the C5 (9q34) and C5aR (19q.13.3) gene-containing segments; however, the associations were found to be weak.17 Importantly, studies using rodent models of antigen-induced AHR suggest that complement participates in the development of AHR and inflammation.11,17–20 Mice lacking C3 showed reduced AHR and lung eosinophilia as well as a reduced expression of the TH2 phenotype when challenged with allergen19 and reduced AHR 2 days after exposure to airborne particulate matter.21 Mice20 and guinea pigs18 lacking the C3aR exhibited protection from the development of AHR. On the other hand, after antigen sensitization, C5-deficient mice were found to be more responsive to methacholine challenge compared with mice with normal complement.17 In vitro experiments had shown that C5a may suppress IL-12 production by monocytes,22 and because IL-12 is important for promotion of TH1 differentiation,23 these studies suggest that anomalies in C5a formation may influence IL-12 production, TH2 differentiation, and susceptibility to asthma. Moreover, C3b can inhibit IL-12 production.24,25 AHR to nonspecific stimuli is one hallmark of aller-

gic asthma in humans. Therefore, it is possible that in humans, as in rodent models, complement participates in the development of susceptibility to asthma. The authors hypothesized that this role of complement may be mediated by an abnormality in one or both of the following processes. First, complement in asthmatics may exhibit an intrinsic abnormality in one of its activation mechanisms that would result in overproduction of activation fragments, which then may induce susceptibility to asthma. If present, an abnormal sensitivity of complement to activation would suggest that one complement protein, from initiation of activation through C5 activation, may be present in abnormal concentration or as an isoform or polymorphic variant with a gain of function, or a genetic variant of an inhibitor with a loss of function. Either occurrence may result in larger amounts of biologically active fragments such as C3a and C5a, which, in turn, may act on cells of the innate immune system that may then favor asthma development. A second mechanism through which complement may participate in the development of susceptibility to asthma is that, despite a normal level of complement fragments generated during complement activation, cells of the innate immune system in asthmatics are abnormally responsive to the regulatory effects of complement followed by the development of susceptibility to asthma. In the studies reported here, the first hypothesis was investigated, namely whether complement in asthmatic subjects is abnormally sensitive to activation by the classical, alternative, or lectin pathway, resulting in excessive formation of biologically active fragments. The basal level of C3a, C5a, and MBL was also quantified in subjects with allergic asthma in comparison with nonatopic normal controls. METHODS Study subjects, pulmonary function tests, and blood collection. All study participants were recruited and tested for

atopic asthma at the University of Minnesota General Clinical Research Center. The study was approved by the Human Subjects Institutional Review Board at the University of Minnesota, and written informed consent was obtained from all participants. The research complies with the principles of the Declaration of Helsinki. All individuals were administered a detailed health questionnaire, underwent a limited physical examination, had allergen skin testing using a standard protocol, and received spirometry along with broncho-reversibility or methacholine challenge studies.26 In all subjects with a baseline forced expiratory volume in one second (FEV1) ⱖ70% of predicted and a post-bronchodilator change in FEV1 that was ⬍12% of baseline, a methacholine bronchial challenge was performed with increasing concentrations of methacholine until a ⱖ20% decline in FEV1 occurred (ie, PD20FEV1), up to 25 mg/mL. For subjects whose FEV1 was ⬍70% of predicted, bronchodilator reversibil-

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ity testing was performed using albuterol. A change in FEV1 of 12% of the baseline or greater was considered positive. Atopy was defined as immediate reactivity to allergen skin testing to 1 or more of 14 common environmental allergens.27 Skin test results were considered “positive” if the mean wheal diameter was 3 mm greater than that of the negative control. To qualify as an atopic asthmatic, subjects were required to have 1 or more positive skin tests, 2 of 3 symptoms commonly associated with asthma (cough, wheeze, and shortness of breath), a physician’s diagnosis of asthma, and airway reversibility with albuterol or a positive methacholine challenge. Participants with nonreactive skin tests and a negative methacholine bronchial challenge were enrolled as nonatopic controls. Individuals were excluded if they had any of the following: (1) birth weight ⬍ 2 kg, (2) congenital or acquired heart or lung disease, (3) a possible conflicting diagnosis such as emphysema, (4) any active or recent respiratory tract infection, (5) autoimmune diseases such as lupus or other complement-activating disease process, (6) a 5-pack/year or greater history of cigarette or equivalent tobacco use, and (7) taking any medications that may have influenced the screening process or laboratory assays. Subjects in the atopic asthmatic group were assigned a severity score (severe persistent, moderate persistent, or mild) according to the current asthma guidelines of the Heart, Lung, and Blood Institute of the National Institutes of Health, USPHS.28 For subjects satisfying all inclusion and exclusion criteria for atopic asthma or control, venous blood was drawn into a 10-mL EDTA tube and a 15-mL serum tube. The EDTA tube was placed immediately on ice and centrifuged at 4°C to obtain plasma within 1 h of collection. The serum tube was allowed to clot at room temperature and then centrifuged at 4°C. Aliquots of plasma and serum were frozen in liquid nitrogen and stored at ⫺70°C until testing. Complement activation. Assessment of plasma complement activation through the classical, alternative, and lectin pathways was performed by ELISA as described before by Roos et al,29 with minor modifications. Briefly, 96-well flatbottomed microtiter plates (Nunc, Rochester, NY) were coated with an activating substance specific for the pathway of interest in 0.1 M NaHCO3/Na2CO3, pH 9.6, (100 ␮L/well) overnight at room temperature and then blocked with 1% BSA in PBS/0.05% Tween-20 for 1 h at 37°C. The activating substances were human IgM at 2 ␮g/mL for the classic pathway, lipopolysaccharide from Salmonella typhosa at 5 ␮g/mL for the alternative pathway, and mannan at 100 ␮g/mL for the lectin pathway. Plasma samples were diluted in a GVB pH 7.5, which consisted of 5 mM Veronal, 145 mM NaCl, 0.1% gelatin, 0.05% Tween-20, and the appropriate divalent cations or EGTA for the various pathways, or 10 mM EDTA as a nonactivated control. The divalent cations or EGTA in the GVB were as follows: 0.15 mM CaCl2 and 1 mM MgCl2 for the classic pathway, 5 mM MgCl2 and 10 mM EGTA for the alternative pathway, and 2 mM CaCl2 and 1 mM MgCl2 for the lectin pathway. For the classical and alternative pathway activations, different plasma concentrations were added to the respective microtiter plates, which were then incubated at 37°C for 1 h. For the lectin pathway activation, the plasma was diluted 1:10 with GVB containing

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20 ␮g/mL of anti-C1q mAb (Quidel, San Diego, Calif) and incubated for 15 min at 4°C. Different plasma concentrations were then added to the mannan-coated microtiter plates, which were first incubated for 1 h at 4°C and then at 37°C for 1 h. After the incubation, the supernatants were harvested, mixed with an equal volume of GVB containing 25 mM EDTA and 50-␮g/mL Futhan (BD Bioscience, San Diego, Calif) mixed 1:1 with the sample diluent for the C5a ELISA kit (BD Bioscience), and stored at ⫺80°C for subsequent C3a and C5a measurements. After removal of the supernatants, the microtiter plates were washed with PBS/0.05% Tween-20 and used for assessing binding of C3 fragments. Complement assays and measurement of MBL. Basal levels of total serum complement were measured using a microtiter hemolytic assay, as previously described.30 Serum levels of C3 and C4 were determined by nephelometry. C5 levels were quantified using a radial immunodiffusion kit (The Binding Site, San Diego, Calif.); normal values given by the manufacturer are 132.4 ⫾ 20 ␮g/mL. Immunoassays of basal plasma C3a (Quidel) and C5a (BD Bioscience) were performed according to the manufacturer’s instructions. MBL serum concentrations were measured using an immunoassay kit specific for oligomeric MBL according to the manufacturer’s instructions (ScimedX Corp., Denville, NJ). C3a and C5a in the supernatants from the complement activation experiments were measured by immunoassay according to the manufacturer’s instructions (BD Bioscience). C3 deposition was assessed using an anti-C3c mAb (Quidel) that reacts with C3b and iC3b, followed by donkey anti-mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, Penn). A 100-␮L antibody solution was added to the washed microtiter plates and incubated for 1 h at room temperature. The plates were then washed 4 times with PBS/ 0.05% Tween-20. Overall, 100 ␮L of TMB solution (Pierce Chemical, Rockford, Ill) was added for 30 min, and then sulfuric acid was added to stop the reaction. The absorbance at 450 nm (A450) was read using a Vmax Kinetic plate reader (Molecular Devices, Sunnyvale, Calif). Statistical analysis. Results are expressed as means ⫾ standard error. Statistical analysis was performed using Student t-tests assuming equal variance of the means. A P value of less than 0.05 was considered significant. RESULTS Pulmonary function assessment. The pulmonary test results obtained in the study subjects are shown in Table I. Twelve atopic asthmatics (6 women) with a mean age of 36 ⫾ 13 years and 12 nonatopic control subjects (7 women) with a mean age of 33 ⫾ 11 years were included in the study. The asthmatic group consisted of 2 patients with severe persistent asthma, 7 with moderate persistent asthma, and 3 with mild asthma. In the asthmatic group, the mean FEV1 of 73.2% ⫾ 15% with a change in FEV1 post-bronchodilator treatment of 16.8% ⫾ 6% was in marked contrast to the nonatopic control group with a mean FEV1 of 97.2% ⫾ 9.1% (⬎ 80% in all subjects). Given the high

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Table I. Pulmonary function tests of study subjects (mean ⫾ SD) Study Groups*

n

% FEV1†

% Change post-BD‡

PD20FEV1§

Atopic asthma subjects Severe asthma subgroup Moderate asthma subgroup Mild asthma subgroup Nonatopic controls

12 2 7 3 12

73.2 ⫾ 15.1 56.0 ⫾ 2.8 68.1 ⫾ 4.9 93.0 ⫾ 14.2 97.2 ⫾ 9.1

16.8 ⫾ 6.1 20.5 ⫾ 7.8 17.3 ⫾ 4.6 13.3 ⫾ 9.0 ND

16 (n ⫽ 1) ⬎25

*Asthma subgroups according to NIH guidelines for severity, using % FEV1 ⬍ 60% as severe, 60% to 80% as moderate, and ⬎ 80% as mild. † FEV1, forced expiratory volume in first second of a forced vital capacity maneuver. ‡ % Change post-bronchodilator. § PD20FEV1, provocative dose of methacholine (mg/mL) causing a 20% drop in FEV1.

degree of airway obstruction as well as the significant improvement after bronchodilator treatment, only 1 subject in the asthmatic group met the criteria for the methacholine bronchial challenge. Overall, 11 of 12 subjects showed reversibility to the beta2 agonist albuterol of 12% or greater, and 1 subject had only a reversibility of 4%, which was not considered significant and therefore required a methacholine challenge. The PD20FEV1 for this subject was 16 mg/ mL, which was considered a significant reaction for bronchial hyperreactivity. All control subjects underwent a methacholine bronchial challenge that caused no significant PD20FEV1 at 25 mg/mL. Basal levels of complement, C3a and C5a, and MBL. Total complement, C3, C4, and C5 levels were

quantified in serum of all study subjects and found to be similar in asthmatics and controls (Table II). Plasma levels of C3a and C5a were also measured before plasma was activated; these levels were found to be low and similar in both groups (Table II). The basal MBL serum level was elevated in the atopic asthmatic subjects with a mean of 2549 ⫾ 231 ng/mL and a range of 19 to 6508 ng/mL, compared with the levels in nonatopic control subjects with a mean of 1590 ⫾ 148 ng/mL and a range of 7.8 to 5238 ng/mL (Table II). However, this elevation was not statistically significant (P ⫽ 0.18). Classical, alternative, and lectin complement pathway activation. The concentrations of C3a and C5a gener-

ated in the microtiter plate supernatants after activation of plasma with plastic-bound IgM, LPS, or mannan to activate the classical, alternative, and lectin pathways, respectively, were not statistically different between patients with atopic asthma and nonatopic controls for all activation pathways (Figs 1–3). Results of C5a generation after classical pathway activation are given for the 2% plasma concentration only because C5a in more diluted samples was below the detection threshold of the assay (Fig 1). The degree of C3b/iC3b deposition after activation of the classical, alternative, and lectin

Table II. Levels of complement and mannanbinding lectin in study subjects* Normal (n ⴝ 12)

Total complement (units) C3 (␮g/mL) C4 (␮g/mL) C5 (␮g/mL) C3a (ng/mL) C5a (ng/mL) MBL (ng/mL)

Asthmatic (n ⴝ 12)

P

137.9 ⫾ 5.1

142.9 ⫾ 3.8

0.47

1120 ⫾ 92 215 ⫾ 15 132.9 ⫾ 15.0 71.1 ⫾ 7.0 4.5 ⫾ 0.6 1590 ⫾ 148

1117 ⫾ 78 198 ⫾ 11 141.2 ⫾ 9.4 71.8 ⫾ 4.8 4.5 ⫾ 0.5 2549 ⫾ 231

0.89 0.39 0.73 0.93 0.94 0.18

Abbreviation: MBL, mannan-binding lectin. *Mean ⫾ SEM serum levels of CH50, C3, C4, C5, and MBL, and plasma levels of C3a and C5a.

pathways was also similar between patients with atopic asthma and nonatopic controls for all pathways (Figs 1–3). The C3a/C3 and C5a/C5 ratios in activated samples were determined because the serum concentration of C3 and C5, respectively, would influence the degree of complement fragment generated. The respective ratios were found to be almost identical in asthmatics and controls for both the classical pathway and the alternative pathway, as well as the C3a/C3 ratio for the lectin pathway (Figs 1–3). For the lectin pathway, the C5a/C5 ratio was slightly lower in asthmatics than controls, but this difference was not significant (P ⬎ 0.1 for all plasma concentrations tested) (Fig 3). Finally, the relationship to complement activation in the asthmatic subjects using the subgroups of mild, moderate, or severe asthma was evaluated and the degree of complement activation was found not to change in comparison with the results obtained with all asthmatics as a single group (results not shown). DISCUSSION

In this study, whether an increased generation of C3a, other C3 fragments, and C5a existed in plasma of individuals with atopic asthma, when plasma is incu-

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Fig 1. Deposition of C3 fragments and generation of C3a and C5a after activation of the classical complement pathway in plasma from patients with atopic asthma (n ⫽ 12) and nonatopic normal controls (n ⫽ 12). C3a/C3 and C5a/C5 ratios were calculated using C3 and C5 plasma levels as shown in Table II. The classical pathway was activated using human IgM bound to microtiter plates. Binding of C3 fragments was assessed by ELISA, and generation of C3a and C5a was measured in the supernatants by enzyme immunoassay, as described in the Materials and Methods section. Generation of C5a was measured after complement activation in 2.0% plasma. Patients versus controls, P ⬎ 0.2 for all plasma concentrations tested.

bated in vitro with activators of the classical, alternative, and lectin pathways, in comparison with nonatopic controls was investigated. To carry out these studies, it was assumed that, if an enhanced sensitivity of complement to activation is important in the induction of susceptibility to asthma, this enhanced sensitivity persists after the individual presents with the clinical manifestations of asthma. Moreover, the study design required subjects that were free from an acute exacerbation of asthma and had no evidence of a concomitant process that may activate complement such as an infection or autoimmune disease. The interest in performing this study developed from recent findings in rodent models of allergic asthma suggesting that C3a and C5a may play a fundamental role as susceptibility factors in the development of AHR and asthma.11,16 –20 In asthma patients, an abnormal sensitivity of comple-

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Fig 2. Deposition of C3 fragments and generation of C3a and C5a after activation of the alternative complement pathway in plasma from patients with atopic asthma (n ⫽ 12) and nonatopic normal controls (n ⫽ 12). C3a/C3 and C5a/C5 ratios were calculated using C3 and C5 plasma levels as shown in Table II. The alternative pathway was activated using S. typhosa lipopolysaccharide bound to microtiter plates. Patients versus controls, P ⬎ 0.2 for all plasma concentrations tested.

ment to activation would indicate that at least 1 complement protein, from initiation of activation through C5 activation, may be present in abnormal concentration or that an isoform or a polymorphic variant of that protein may result in gain or loss of function. However, no significant difference was found in the production of C3a, C3b/iC3b, and C5a, nor in the C3a/C3 or C5a/C5 ratios between atopic asthmatic and nonatopic control subjects when their plasma complement was activated in vitro by any of the pathways. These results suggest that, if complement participates in development of susceptibility to asthma in humans, this role is not because of an intrinsic abnormality in complement activation that would cause excessive generation of biologically active fragments. The results do not exclude the possibility that C3a and C5a production may be abnormally elevated had other complement activators been chosen for the studies; however, the activators used are considered to be prototypic activators for initiating the

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Fig 3. Deposition of C3 fragments and generation of C3a and C5a after activation of the lectin complement pathway in plasma from patients with atopic asthma (n ⫽ 12) and nonatopic normal controls (n ⫽ 12). C3a/C3 and C5a/C5 ratios were calculated using C3 and C5 plasma levels as shown in Table II. The lectin pathway was activated using mannan bound to microtiter plates. Patients versus controls, P ⬎ 0.1 for all plasma concentrations tested.

respective pathways. Therefore, the results favor the alternative hypothesis for the contribution of complement to asthma susceptibility. Cells of the innate immune system, such as monocytes, basophils, macrophages, and mast cells, which are known to participate in immune regulation and are under significant control by complement, may display an abnormality that results in a dysregulated response to normally activated complement. A defective C3aR or C5aR, or a defective signaling pathway in these cells, may be 1 mechanism that underscores the development of increased susceptibility to AHR and asthma. For example, monocytes and macrophages secrete IL-12, which promotes the differentiation of naive T lymphocytes to TH1 lymphocytes, but C5a inhibits the production of IL-12 by monocytes.22 Thus, C5a may favor the predominance of a TH2 phenotype, which is thought to contribute in a fundamental manner to the pathogenesis of asthma.31,32 Moreover, C5a can stimulate basophils to produce IL-4 and IL-13,33 which promote the differentiation of naive T lymphocytes to a TH2 phenotype34 –36 and

induce B cells to undergo isotype switching to IgE production.37 Through these mechanisms, complement might induce susceptibility to asthma in humans by interacting with cells of the innate immune system that modulate adaptive immunity. A role of complement in asthma may also involve CR2 localized on B cells, which can bind C3d and is part of the B cell receptor complex.38 Ligation of this receptor lowers the threshold for B cell activation and enhances the immune response to T cell-dependent antigens.38,39 In the authors’ studies, they also found that the C3a and C5a plasma levels were similar in asthmatics and nonatopic controls and that serum MBL levels were elevated in the asthmatic subjects, without reaching statistical significance. The moderately elevated MBL levels in asthma subjects may likely be a result of chronic airway inflammation causing increased synthesis of acute-phase proteins, of which MBL is a member.40 The MBL level was considered deficient (⬍50 ng/mL) in 4 subjects, 2 in each group. As a result of the relative small number of subjects in the study, MBL assessment in more individuals would be of interest, especially as low levels of MBL have been associated with several pulmonary conditions, including increased susceptibility to respiratory infections in childhood41 and increased severity of pulmonary disease in cystic fibrosis.42 In conclusion, this study suggests that the plasma complement of patients with allergic asthma does not exhibit an abnormal sensitivity to activation. Instead, these findings suggest that, if complement is a factor in the development of susceptibility to AHR and asthma, this role could be a result of an abnormal response of cells of the innate immune system to activated complement. Special thanks to Jeramy Anderson for excellent technical assistance.

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