Published ahead of print on September 18, 2003, doi:10.1164/rccm.200306-739OC
© 2003 American Thoracic Society Inhibition of Complement Activation Decreases Airway Inflammation and Hyperresponsiveness ,Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center; and Department of Medicine, Division of Rheumatology, University of Colorado Health Sciences Center, Denver, Colorado Correspondence and requests for reprints should be addressed to Erwin W. Gelfand, M.D., National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: gelfande{at}njc.org
Studies in murine models have suggested the involvement of the complement anaphylatoxins (C3a and C5a) in the development of allergic asthma. We investigated the effects of inhibiting complement activation after sensitization but before allergen challenge on the development of allergic airway inflammation and airway hyperresponsiveness. To prevent complement activation, we used a recombinant soluble form of the mouse membrane complement inhibitor complement receptor-related gene y (Crry) fused to the IgG1 hinge, CH2 and CH3 domains (Crry-Ig), which has decay-accelerating activity for both the classic and alternative pathways of complement as well as cofactor activity for factor I-mediated cleavage of C3b and C4b. C57BL/6 mice were sensitized (Days 1 and 14) and challenged (Days 24–26) with ovalbumin. Crry-Ig was administered after allergen sensitization either as an intraperitoneal injection or by nebulization before allergen challenge. Crry-Ig significantly prevented the development of airway hyperresponsiveness, decreased airway and lung eosinophilia as well as the numbers of lung lymphocytes, decreased levels of interleukin (IL)-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and decreased serum ovalbumin-specific IgE and IgG1. These results suggest that prevention of complement activation may have a therapeutic role in the treatment of allergic airway inflammation and asthma in sensitized individuals.
Key Words: mouse model • lung function • lymphocytes • cytokines Allergic asthma is a complex syndrome that has been characterized by airway obstruction, airway inflammation, and airway hyperresponsiveness (AHR) (1). It is well established that acquired immunity plays an important role in the development of these responses, and CD4+ lymphocytes in particular have been shown to be pivotal for orchestrating the development of airway inflammation and AHR (2). Additionally, components of the innate immune system may also play a role in the development of allergic airway inflammation and AHR, with the complement pathway being the focus of recent research. Biologically active fragments derived from the C3, C4, and C5 family of proteins mediate many of the functions of the complement pathway (3). The anaphylatoxins C3a and C5a are liberated as activation by-products and are potent proinflammatory mediators that bind to specific cell surface receptors and cause leukocyte activation, smooth muscle contraction, and increased vascular permeability (4). In patients with allergic asthma, increased levels of C3a (4) and C5a (5) in bronchoalveolar lavage (BAL) fluid have been described after allergen challenge, suggesting activation of the complement pathway through an allergen-induced mechanism. Also, receptors for C3a and C5a are expressed on bronchial epithelial cells and bronchial smooth muscle cells in humans and mice (6), and after allergen challenge of sensitized mice, increased expression of C3a receptors on bronchial smooth muscle has been reported (6). Interestingly, different components of the complement system appear to exhibit different functions in the development of AHR. C5-deficient mice show an increased susceptibility to the development of airway inflammation and AHR (7), whereas C3 or C3a receptor-deficient animals appear protected (4, 8–10). Based on these studies and the apparently different roles for C3a and C5a in the development phase of AHR before sensitization, it is unclear whether targeting the activation of complement after sensitization but during the allergen challenge phase might abolish or aggravate the response. A number of complement inhibitors are currently being developed and tested as therapeutic agents for human inflammatory, ischemic, and autoimmune diseases (11). In humans, soluble recombinant complement receptor type 1 (sCR1/CD35) has been developed as an inhibitor that can be used in vivo and has demonstrated therapeutic benefit in well-established models of acute ischemia and inflammation (12). The murine homologues of human sCR1 are mouse CR1 and complement receptor-related gene y (Crry). Crry is a widely expressed, membrane-bound intrinsic complement regulatory protein (13, 14), and in direct comparisons between the two inhibitors, Crry was found to be a more potent inhibitor of complement activation (15). A recombinant soluble form of the mouse membrane complement inhibitor Crry fused to mouse IgG1 hinge, CH2 and CH3 domains (Crry-Ig), has been created and shown to inhibit antibody-induced glomerulonephritis effectively (16), to block antibody-induced fetal loss (17), and to attenuate intestinal damage after the onset of mesenteric ischemia/reperfusion injury in mice (18). In this study, we investigated the effects of systemic and local treatment of allergen-sensitized mice with Crry-Ig in a model of allergic airway inflammation and AHR. We demonstrated that treatment with Crry-Ig is effective in blocking the development of airway inflammation as well as AHR even when administered after sensitization but before airway challenge. Some of the results of these studies have been previously reported in an abstract (19).
Female C57BL/6 mice, 8 to 12 weeks of age, were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained on ovalbumin (OVA)-free diets. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center. Mice were sensitized by intraperitoneal injection of 20 µg of OVA (Grade V; Sigma Chemical Co., St. Louis, MO) suspended in 2.25 mg aluminum hydroxide (Alum Imuject; Pierce, Rockford, IL) on Days 1 and 14 and then challenged via the airways, using nebulized OVA (1% in phosphate-buffered saline [PBS]), with an ultrasonic nebulizer (DeVilbiss Health Care, Somerset, PA) for 20 minutes daily on Days 24, 25, and 26. Two hours before each OVA challenge and 36 hours before measurement of airway responsiveness, mouse Crry-Ig (16) was administered to mice either by intraperitoneal injection (3 mg per treatment per mouse) or by nebulization. For nebulization, four mice were placed in a plexiglass box, and 15 mg of Crry-Ig (in 3 ml of PBS) were nebulized using an ultrasonic nebulizer (DeVilbiss Health Care). As a control, rat IgG (Sigma) at the same dose and volume was injected intraperitoneally or nebulized at the same time points. Both treatments with control antibody had no effect on the development of AHR, airway inflammation, and cytokine levels. Therefore, for reasons of brevity, only data of intraperitoneally injected control antibodies are presented. On Day 28, AHR was assessed, and animals were sacrificed the same day for the collection of BAL fluid, blood, and lung tissue. In a different protocol, Crry-Ig treatment was administered only during the sensitization phase. Crry-Ig (3 mg per mouse per injection) was administered intraperitoneally on Day 1 before the first intraperitoneal sensitization and then every other day until Day 19. Mice were challenged as described previously here on Days 24–26, and AHR was assessed. Airway resistance and dynamic compliance were determined as a change in airway function after increasing concentrations of aerosolized methacholine in anesthetized, tracheostomized mice (20). After each aerosolized methacholine challenge, the data were continuously collected for 1 to 5 minutes, and maximum values of airway resistance and minimum values of dynamic compliance were taken to express changes in these functional parameters (20).
After assessment of airway function, lungs were lavaged via the tracheal tube with Hank's balanced salt solution (1 x 1 ml, 37°C). A number of BAL cells were counted by using a cell counter (Coulter Counter; Coulter Co., Hialeah, FL). Differential cell counts were made from cytocentrifuged preparations, and percentage and absolute numbers of each cell type were calculated. Cytokine levels were assessed by ELISA in BAL fluid (21). Interferon- Tissue sections were stained with hematoxylin and eosin, periodic acid-Schiff, and immunohistochemically for cells containing eosinophilic major basic protein, using a rabbit anti-mouse major basic protein antibody (provided by J. J. Lee, Mayo Clinic, Scottsdale, AZ) (22). Slides were examined in a blinded fashion, and numbers of eosinophils in the peribronchial tissue and goblet cells were analyzed separately using the IPLab2 software (Signal Analytics, Vienna, VA). Lung cells were isolated by enzymatic digestion (23). Peribronchial lymph node (PBLN) and spleen cells were purified as previously described (24, 25). Mononuclear cells (MNCs) were purified by density-gradient centrifugation (ICN Biomedicals, Aurora, OH) and were counted (Coulter Counter). The ratio of CD3-, CD4-, and CD8-positive cells was assessed by flow cytometry analysis using fluorescein isothiocyanate–conjugated monoclonal rat anti-mouse CD3 and phycoerythrin-conjugated rat anti-mouse CD4 or CD8 (all PharMigen). Absolute numbers of CD4-positive (CD3+/CD4+) and CD8-positive (CD3+/CD8+) T cells were calculated by multiplying the number of total MNCs in the lung and the percentage of either CD3/CD4 or CD3/CD8 cells. To measure in vitro cytokine production lung, PBLN and spleen cells were plated at 4 x 105 per ml in 96-well round bottom tissue culture plates and incubated with medium alone or OVA (10 µg/ml) for 5 days in a humidified atmosphere of 5% CO2 at 37°C. Cell free supernates were harvested and stored at -80°C pending cytokine ELISA assays. Serum levels of total IgE and OVA-specific IgE and IgG1 were measured by ELISA as previously described (21). The OVA-specific antibody titers of samples were related to internal pooled standards, which were arbitrarily assigned to be 500 ELISA units. The total IgE level was calculated by comparison with a known mouse IgE standard (55 3481; PharMingen). C3 was measured by radial immunodiffusion using agarose containing a high titered anti-human C3 antibody that cross-reacts with mouse C3 (intact and converted). A pool of normal mouse (C57BL/6 background) sera was used to generate a standard curve, and the results were reported as the percentage of the normal mouse serum pool. Analysis of variance was used to determine the level of difference between all groups. Single pairs of groups were compared by Student's t test. Comparisons for all pairs were performed by the Tukey-Kramer honestly significant difference test for airway responsiveness and histology data; p values for significance were set at 0.05. Values for all measurements are expressed as the mean ± SEM.
Treatment with Systemic Crry-Ig Increases Serum Levels of C3 To assess whether treatment with Crry-Ig affects levels of C3 in vivo, we measured C3 levels in serum at 48 hours after the last allergen challenge. Serum levels of C3 were similar in challenged only, sensitized and challenged, and sensitized and challenged mice treated with a control antibody or nebulized Crry-Ig (Figure 1) . In contrast, mice treated with systemic Crry-Ig showed significantly (p < 0.05) increased levels of serum C3 (Figure 1). Thus, any potential effects of Crry-Ig in this model are not due to decreasing serum C3 levels.
Treatment with Crry-Ig Inhibits the Development of AHR To determine the effect of complement inhibition on the development of AHR, OVA-sensitized and nonsensitized mice were challenged with an aerosol of 1% OVA on 3 consecutive days. The response to methacholine in nonsensitized mice challenged with OVA alone showed small, dose-dependent increases in airway resistance and reductions in dynamic compliance (Figure 2) . After OVA sensitization and challenge, increases in airway resistance values and decreases in dynamic compliance values were significantly higher throughout the methacholine dose–response curve (Figure 2). Administration of Crry-Ig, either intraperitoneally or by nebulization, significantly prevented the increases in airway resistance and reductions in dynamic compliance throughout the methacholine dose–response curve (Figure 2). In contrast, treatment of sensitized and challenged mice with rat IgG as a control had no significant effect on the development of AHR (Figure 2).
Treatment with Crry-Ig Decreases Airway Eosinophilia Eosinophilic airway inflammation is an important outcome after allergen challenge of sensitized mice. To determine the effects of Crry-Ig on eosinophilic inflammation after allergen sensitization and challenge, the cellular content of BAL fluid was assessed. Sensitized and challenged mice demonstrated a significant (p < 0.001) increase in total cell count and eosinophil number in BAL fluid when compared with the nonsensitized, challenged mice, which had almost no eosinophils in the BAL fluid (Figure 3) . Sensitized mice treated with either systemic or nebulized Crry-Ig during challenge showed significantly (p < 0.05) lower total cell counts and numbers of eosinophils when compared with the sensitized and challenged animals (Figure 3). Treatment with the control IgG had no effect on eosinophil accumulation in the BAL.
Crry-Ig Treatment Decreases Eosinophil and Lymphocyte Accumulation in Lung Tissue Lung tissue was obtained and processed 48 hours after the last allergen challenge. Sensitized and challenged animals (Figure 4C) demonstrated an increase in peribronchial inflammation compared with the nonsensitized, challenged alone animals (Figure 4A). Mice treated with either systemic (Figure 4E) or nebulized (Figure 4G) Crry-Ig showed markedly reduced peribronchial inflammation. To quantitate eosinophil infiltration in the lung, tissue was stained with anti-major basic protein (Figure 5) . In mice challenged alone, few eosinophils were detected in the peribronchial tissues (Figures 5A and 6) . In contrast, sensitization and subsequent airway challenge resulted in a significant increase in peribronchial eosinophil numbers (Figures 5B and 6). Treatment with Crry-Ig, either systemically (Figures 5C and 6) or by nebulization (Figures 5D and 6), significantly decreased eosinophil accumulation in the peribronchial areas. Treatment with the control antibody did not significantly alter the level of eosinophil infiltration (Figure 6).
To assess the number of CD4- and CD8-positive lymphocytes in the lung, MNCs were isolated from the lung as described in METHODS. Sensitized and challenged animals showed a significant (p < 0.01) increase in the number of MNCs in the lung (mean ± SEM: 2.42 x 106 ± 0.1 8 x 106 MNCs per lung, n = 8) compared with animals challenged alone (1.29 x 106 ± 0.15 x 106 MNCs per lung, n = 8). This increase in MNC was also reflected in T-lymphocyte numbers in the lung. Sensitized and challenged mice treated with systemic Crry-Ig (1.44 x 106 ± 0.12 x 106 MNCs per lung, n = 8) showed no significant increase in total MNC number in the lung. Sensitization and challenge led to a significant increase in CD3 positive/CD4 positive (CD3+/CD4+) as well as CD3+/CD8+ cells compared with challenged only control subjects (Figure 7) . In contrast, sensitized and challenged mice treated with systemic Crry-Ig showed no significant increase in CD3+/CD4+ and CD3+/CD8+ compared with the challenged only control subjects (Figure 7).
Crry-Ig Prevents Goblet Cell Hyperplasia and Mucin Hyperproduction in Airway Epithelium Lung sections were stained with periodic acid-Schiff to identify mucus-containing cells in the airway epithelium. A large number of cells staining positive for mucus were found in sensitized and challenged mice (Figures 4D and 6) compared with mice challenged alone (Figures 4B and 6). Treatment with either systemic (Figures 4F and 6) or nebulized (Figures 4H and 6) Crry-Ig significantly reduced goblet cell hyperplasia. No differences in goblet cell mucus production were found after treatment with control antibody (Figure 6).
Treatment with Crry-Ig Alters Cytokine Production in BAL Fluid
Crry-Ig Treatment Decreases Production of IL-5 and IL-13 by Lung Cells in Response to OVA In Vitro To monitor local cytokine production, MNCs, isolated from lungs, PBLN, and spleen were cultured together with OVA. Concentrations of IL-5, IL-13, and IFN-  in culture supernates were measured by ELISA. Sensitization and subsequent airway challenge with OVA resulted in increased production of IL-5 and IL-13 by lung, PBLN and spleen cells in OVA-stimulated cultures (Figure 9)
. Lung cells from mice treated with systemic Crry-Ig showed significantly lower IL-5 and IL-13 production in OVA-stimulated cultures compared with untreated mice (Figure 9). However, this effect was not detected in cultures of cells from PBLNs and spleen. In cultures of PBLN and spleen cells, no differences in cytokine levels were found between untreated sensitized and challenged animals and animals treated with systemic Crry-Ig (Figure 9).
Crry-Ig Inhibits Production of OVA-specific IgE in Serum Serum levels of total and OVA-specific IgE were measured 2 days after the last airway challenge. Sensitization and challenge with OVA resulted in significantly increased serum levels of total IgE, OVA-specific IgE, and IgG1 compared with challenged only mice (Table 1) . Treatment with systemic or nebulized Crry-Ig reduced serum levels of total IgE, OVA-IgE, and OVA-IgG1 (Table 1). Treatment with control antibody had no effect on serum levels (Table 1).
Crry-Ig Does Not Affect the Development of AHR or Airway Inflammation When Administered during the Sensitization Phase To assess further whether inhibition of complement activation during sensitization of the animals also affects the development of allergic airway disease, mice were treated with Crry-Ig only during the sensitization period as described in METHODS. Mice treated with Crry-Ig during sensitization still developed AHR, similar to sensitized and challenged control mice, and was significantly higher compared with sensitized and challenged mice treated with Crry-Ig during the challenge phase (Figure 10) . In parallel, treatment with Crry-Ig during sensitization did not prevent the accumulation of inflammatory cells including eosinophils in the BAL fluid (Figure 10).
Studies in complement/complement receptor gene-altered animals have suggested a role for the complement system in the development of AHR after allergen sensitization and challenge. In this study, we show that inhibition of complement activation during allergen challenge but after sensitization of normal mice decreases airway inflammation and prevents the development of AHR. Furthermore, we demonstrate that inhibition of complement activation affects not only the influx of inflammatory cells into the lung and AHR but also T-cell function, as shown by decreases in Th2 cytokine levels in BAL fluid. After allergen challenge of patients with asthma, elevated concentrations of C3a and C5a have been detected in BAL fluid (4, 5) compared with nonatopic control patients, suggesting the triggering of complement activation during the challenge phase. Allergen–IgG immune complexes could trigger activation of the classic pathway (26), and certain antigens may directly activate C3 via the alternative pathway. In addition, neutral tryptase released from mast cells or pulmonary macrophages may directly (proteolytically) cleave either C3 or C5 (27, 28). In these experiments, we chose to inhibit the activation of complement in normal hosts and inhibit complement activation before allergen challenge, but after sensitization was fully completed. This is a different approach from experiments with genetically altered mice, where complement activation or receptor binding is inhibited throughout the sensitization and challenge phases. For our purposes, we used Crry-Ig, a recombinant soluble form of the mouse membrane complement inhibitor Crry fused to the mouse IgG1 hinge, CH2 and CH3 domains (16); it demonstrates the same abilities but has a significantly longer half-life than Crry alone. Crry is a membrane-bound intrinsic complement regulatory protein, which prevents C3 and C4 activation on self-membranes in mice by inhibiting C3 convertase (15). Furthermore, Crry exhibits combined decay-accelerating and factor I cofactor activity for C3b and C4b, blocking activation of C3 by both the classic and alternative pathways. Crry-Ig can therefore be considered a direct inhibitor of C3 activation. As a consequence, C3-dependent C5 activation is also inhibited. However, the possibility of direct C5 activation, as suggested in models of sepsis (29), cannot completely be excluded. Crry-Ig has been shown to effectively inhibit complement-induced injury in different animal models of complement-induced diseases (16–18). In this study, inhibition of complement activation not only prevented the development of AHR but also prevented the development of airway and lung tissue eosinophilia, goblet cell hyperplasia, as well as the bias toward Th2 cytokine production in BAL fluid and the elevation of serum allergen-specific IgE/IgG1 levels. All of these effects were independent of the route of Crry-Ig administration. Interestingly, only systemic (intraperitoneal injection) administration of Crry-Ig significantly increased serum concentrations of C3, suggesting a decrease in the turnover of C3 after sensitization and challenge and therefore an increase in systemic C3 levels. This finding is similar to results in mice overexpressing a soluble form of Crry systemically under the control of the metallothionein promoter, where higher serum levels of C3 could be found compared with nontransgenic mice, probably because of decreased spontaneous turnover by the alternative pathway (30). However, both systemic as well as nebulized Crry-Ig were similarly effective in reducing AHR, lung eosinophil inflammation, Th2 cytokine levels in BAL fluid, and goblet cell hyperplasia, suggesting that local complement activation in the lung (4, 5) can be an important trigger for development of AHR and airway inflammation. The development of AHR as well as early and late airway responses has been linked to C3 or expression of the C3a receptor. After allergen challenge, sensitized guinea pigs with a deficiency of the C3a receptor developed a significantly lower degree of bronchoconstriction compared with wild-type animals (9). Similarly, the presence of a functional C3a receptor as well as C3 has been shown to be necessary for the development of airway dysfunction (4, 8, 31). Findings from different groups suggested that the C3a receptor is important to the development of AHR but that the full development of airway inflammation and Th2 cytokine production may be regulated by C3 independently of the expression of the C3a receptor (4, 9, 31). However, more recent work using aspergillus to induce allergic airway disease showed a decrease in airway inflammation as well as levels of Th2 cytokines after allergen challenge of C3a receptor-deficient animals (8). In this context, some of the inconsistencies may be explained by the overlapping and potentially distinct roles played by both parent molecule C3 and the C3a peptide, which regulates a number of inflammatory functions through interaction with its receptor. C3 deficiency, unlike C3a receptor deficiency, may also lead to a failure to generate C3bi or C5a, peptides also involved in the control (promotion/inhibition) of inflammatory responses (8). Importantly, the absence of C3 or C3a receptors during the sensitization phase (as in knockout mice) may have profound effects on the subsequent response to allergen challenge, rendering it difficult if not impossible to determine what are the actual targets. In this study, inhibition of complement activation only during the challenge phase decreased both the development of AHR as well as airway inflammation, suggesting that complement activation at this stage of the response in normal sensitized mice is involved in both responses. Furthermore, the data indicate that in sensitized mice, prevention of local complement activation in the lung during allergen challenge is sufficient to inhibit the allergen-induced responses. In contrast, administration of Crry-Ig during the sensitization phase was not effective in preventing the development of AHR or airway inflammation. Complement activation during the initial immunization stage has been linked to the induction of an adaptive (T–cell mediated) immune response (32), but T-cell functions are not primarily affected in C3- and C4-deficient mice (33). It is therefore not surprising that inhibition of complement activation during the sensitization phase did not affect the development of AHR and airway inflammation in this T-cell–dependent model (2).
In addition to the role of C3 in the development of AHR, the C5 locus has been identified as a susceptibility gene in the development of AHR (7). A deletion in the coding sequence of C5 resulted in C5 deficiency, which did not prevent the development of AHR but led to AHR development. The data suggested that in C5-deficient mice, the development of Th1 responses was impaired, favoring expression of Th2 responses. The authors found that the C5a receptor regulated the production of IL-12 in human monocytes in vitro, and macrophages from C5-deficient mice had diminished IL-12 productive capacity in vivo (7). However, C5a-mediated inhibition of IL-12 has only been demonstrated in vitro, and two recent reports describe downregulation of IL-12 production in macrophages in the presence of C5a in vitro (34, 35). In this study, mice treated with Crry-Ig had similar levels of IL-10, IL-12, and IFN- Our findings of decreased AHR and airway inflammation after inhibition of complement activation are in agreement with recent findings by Abe and colleagues (36). These authors demonstrated that in sensitized rats, treatment with an sCR1 as well as C5a receptor antagonist inhibited early and late airway response after allergen exposure. sCR1 is considered the human homologue for Crry-Ig, and both exhibit identical inhibitory activities on C3 convertases. The suppression of the late airway response by sCR1 could be reversed by intratracheal instillation of rat C5a desArg. In contrast, in this model, C3a desArg had no influence on the development of late airway response. Their findings and these ones suggest that complement inhibition after allergen exposure offers an effective approach to prevent either early and late airway responses (36) as well as the development of AHR. Treatment with Crry-Ig decreased the development of AHR as well as airway inflammation and resulted in decreased levels of IL-4, IL-5, and IL-13 in BAL fluid. Some of these cytokines have been directly linked to the development of AHR and airway inflammation and goblet cell hyperplasia (22, 37–41). Interestingly, treatment with Crry-Ig decreased the number of CD4- as well as CD8-positive lymphocytes in the lung after allergen exposure, and MNCs isolated from the lung showed decreased production of Th2 cytokines after antigen stimulation in vitro. However, this effect of Crry-Ig treatment was only apparent in the lung, and no difference was found in production of these cytokines after OVA stimulation by MNCs obtained from spleen and PBLNs. The proliferative responses of PBLN and spleen cells to the antigen were similarly not affected by Crry-Ig treatment (data not shown). Overall, these results suggest that inhibition of complement activation after allergen challenge of a sensitized host does affect the recruitment of lymphocytes to the lung, which respond to antigen by secretion of Th2 cytokines. Indeed, during virus infection, C3-deficient mice demonstrated impaired recruitment of virus-specific CD4-positive as well as CD8-positive effector T cells to the lung (42). Human T lymphocytes do express a C3a receptor (43). However, in this study, inhibition of complement activation did not appear to affect T-cell function, cytokine production, or proliferative responses of MNCs in compartments other than lung, suggesting that there was no direct effect of the Crry-Ig treatment on lymphocyte function per se, but rather an interference with recruitment of antigen-reactive cells to the lung. In other models, C3 has been directly linked to the production of Th2 cytokines (44) and has also been associated with responses to thymus-dependent antigens (33). In addition, C3a and C5a may have additional activities on other cell types, including eosinophils and airway smooth muscle cells, which are involved in AHR, and these effects cannot be excluded at this time (6, 45–48). In this study, an inhibitor of complement activation inhibiting both the classic and alternative pathways prevented allergen challenge-induced responses in previously sensitized hosts when administered systemically or by inhalation. Inhibition of complement activation in this way appears to preferentially target the development of Th2 responses in the lung, lung eosinophilia, IgE/IgG1 antibody production, and goblet cell hyperplasia. To a large extent, this is likely through the reduction in accumulation of antigen-specific T cells to the target organ and the consequent reduction in the production of IL-4, IL-5, and IL-13. To this end, local inhibition of complement activation in the lung even in sensitized hosts may be an effective therapeutic approach for the treatment of allergic asthma.
The authors thank L.N. Cunningham and D. Nabighian (National Jewish Medical and Research Center, Denver, CO) for their assistance and Dr. J. J. Lee (Mayo Clinic, Scottsdale, AZ) for providing the anti-major basic protein antibody.
Supported by National Institutes of Health grants HL-36577 and HL-61005, Environmental Protection Agency grant R825702 (to E.W.G.), and National Institutes of Health grant AI-31105 (to V.M.H.) and DFG (Ta 275/2–1) (to C.T.). Conflict of Interest Statement: C.T. has no declared conflict of interest; Y-H.R. has no declared conflict of interest; K.T. has no declared conflict of interest; J-W.P. has no declared conflict of interest; A.J. has no declared conflict of interest; A.B. has no declared conflict of interest; A.D. has no declared conflict of interest; P.C.G. has no declared conflict of interest; V.M.H. has no declared conflict of interest; E.W.G. has no declared conflict of interest.
* Both authors contributed equally to the work.
Received in original form June 4, 2003; accepted in final form September 8, 2003
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