Factor B of the alternative complement pathway regulates development of airway hyperresponsiveness and inflammation

Taube et al. 10.1073/pnas.0602357103.

Supporting Information

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Supporting Figure 5
Supporting Table 1
Supporting Figure 6
Supporting Figure 7
Supporting Table 2
Supporting Figure 8
Supporting Figure 9
Supporting Text

Fig. 5. C3 deposition in large airways. (A) Challenged-only WT mice. (B) Challenged-only fB-/- mice. (C) Sensitized and challenged WT mice. (D) Sensitized and challenged fB-/- mice. Arrow indicates C3 deposition along the epithelium of a large airway. (Original magnification: ´ 200.)

Fig. 6. Tissue inflammation and goblet cell metaplasia in fB-/- mice. Tissue inflammation was evaluated 48 h after the last challenge by using hematoxylin and eosin staining (H&E) and periodic acid/Schiff (PAS) staining for goblet cells in challenged-only WT mice (fB+/+), sensitized and challenged WT mice (fB+/+), challenged-only factor B-deficient mice (fB-/-), and sensitized and challenged factor B-deficient mice (fB-/-). (Bars: 50 m m.)

Fig. 7. Cytokine levels in fB-/- mice. Levels of IL-4, IL-5, IL-13, IL-12, and IFN-g were determined in BAL fluid 48 h after the last challenge in challenged-only fB+/+ mice (white bar, n = 12), sensitized and challenged fB+/+ mice (black bar, n = 12), challenged-only fB-/- mice (light gray, n = 12), and sensitized and challenged-only fB-/- mice (dark gray bar, n = 12) mice. Mean ± SEM are given. *P < 0.05 compared with all other groups. #P < 0.05 compared with fB+/+ challenged-only mice and fB-/- challenged-only mice.

Fig. 8. Effect of sensitization and challenge with ragweed in factor B-deficient mice. Mice were sensitized and challenged with ragweed as described in Materials and Methods. (A) Forty-eight hours after the last challenge, changes in airway resistance in response to MCh were assessed. Ragweed-sensitized and -challenged fB+/+ mice (solid squares, n = 8), challenged-only fB+/+ mice (open squares, n = 8), fB-/- mice (open circles, n = 8), and ragweed-sensitized and -challenged fB-/- mice (solid circles, n = 8). (B) Total cell number (TCC), macrophage (Mac), lymphocyte (Lym), neutrophil (Neu), and eosinophil (Eos) numbers were evaluated in BAL fluid. Sensitized and challenged fB+/+ mice (black bars, n = 8), challenged-only fB+/+ mice (white bars, n = 8), challenged-only fB-/- mice (light gray bars, n = 8), and sensitized and challenged fB-/- mice (dark gray bars, n = 8). Mean ± SEM are given. *P < 0.05 compared with all other groups; #P < 0.05 compared with challenged-only fB+/+ mice and challenged-only fB-/- mice.

Fig. 9. Administration of a factor B-neutralizing antibody to sensitized and challenged WT and C4-/- mice decreases AHR and airway inflammation. Tissue inflammation, goblet cell hyperplasia, and peribronchial eosinophil inflammation were evaluated 48 h after the last challenge by using hematoxylin and eosin staining (H&E), periodic acid/Schiff staining (PAS), and staining with an antibody to major basic protein (a -MBP). Challenged-only mice (C), sensitized and challenged mice treated with systemic control Ab (S/C control Ab), treatment of sensitized and challenged mice with systemic (S/C anti-fB i.p.), or inhaled (S/C anti-fB inhal) antibody. (Bar: 50 mm.)

Supporting Text

Results

Complement Activation Occurs After Sensitization and Challenge. Evidence for activation of complement in WT but not fB-/- mice was obtained by staining for C3 deposition in the lungs. When evaluated by immunofluorescence, challenged-only mice (without prior sensitization) showed virtually no C3 deposition (Fig. 5 A and B). WT mice that were subjected to both sensitization and challenge had a marked increase in the intensity of staining for C3, particularly in the large airways (Fig. 5C). Little C3 deposition was evident in the large airways of fB-/- mice that had undergone sensitization and challenge (Fig. 5D). These findings further indicate that complement activation occurs in sensitized and challenged mice and that activation is much greater in mice with an intact alternative pathway. Lung sections were also stained for IgG, but no IgG was detectable in any of the lung sections studied from sensitized and challenged WT and fB-/- mice (images not shown). The absence of IgG is consistent with non-Ig mediated activation of the alternative pathway after sensitization and challenge and with previous studies demonstrating that B cells are not critical to the development of AHR by using this approach (1).

Activation of the Alternative Pathway Is Critical for the Development Airway Inflammation. In parallel and preceding the increases in BAL fluid, allergen sensitization and airway challenge led to an increase in peribronchial inflammation and marked eosinophil infiltration compared with mice that were only challenged (Fig. 6). However, sensitized and challenged fB-/- mice showed markedly reduced peribronchial inflammation (Fig. 6) compared with sensitized and challenged control mice. To quantitate eosinophil accumulation in the lung tissue, sections were stained with an antibody to major basic protein. In challenged-only mice, only few eosinophils were detected in the peribronchial tissue (Table 1). Sensitization and subsequent allergen challenge of fB+/+ mice resulted in significantly increased peribronchial eosinophil numbers. In contrast, sensitized and challenged fB-/- mice showed significantly fewer peribronchial eosinophils. Sensitized and challenged C4-/- mice showed similar increases in peribronchial eosinophil numbers compared with the sensitized and challenged respective WT mice (Table 1).

Another hallmark of allergic airway disease is goblet cell metaplasia of airway epithelial cells. Lungs were stained with periodic acid-Schiff (PAS) to identify mucus-containing cells in the airway epithelium. In sensitized and challenged mice a large number of cells staining positive for mucus were found (Fig. 6 and Table 1) in contrast to challenged-only mice, where no PAS-positive cells were detectable. Sensitized and challenged fB-/- showed significantly fewer mucus-containing cells in the airway epithelium compared with the sensitized and challenged WT mice. In sensitized and challenged C4-/- mice, the number of goblet cells was similar to the sensitized and challenged WT mice (Fig. 6 and Table 1).

Complement Activation Through the Alternative Pathway Regulates Cytokine Levels in BAL Fluid. T helper 2 cytokine production by T cells plays a key role in the induction of allergic airway inflammation and AHR. To evaluate the cytokine response after allergen challenge, we quantitated IL-4, IL-5, IL-12, IL-13, and IFN-g in the BAL fluid 48 h after the last OVA challenge. Sensitization and challenge of WT mice resulted in significant increases in IL-4, IL-5, and IL-13 and lower IL-12 and IFN-g levels compared with challenged-only mice (Fig. 7). Sensitized and challenged fB-/- mice showed reduced levels of IL-4, IL-5, and IL-13 in the BAL fluid. However, levels of IL-12 and IFN-g were significantly higher in sensitized and challenged fB-/- mice compared with sensitized and challenged WT mice and similar to challenged-only mice.

Failure of Development of AHR and Airway Inflammation in Factor B-Deficient Mice Is Not Specific to OVA. To determine whether the absence of AHR after allergen sensitization and challenge was caused by a specific unresponsiveness to OVA, fB-/- and WT mice were sensitized and challenged with ragweed. Ragweed-sensitized and -challenged fB-/- mice showed a decrease in responsiveness to MCh, whereas fB+/+ mice developed a strong response to MCh (Fig. 8A). Similarly, airway inflammation and eosinophil numbers in BAL fluid were reduced in ragweed sensitized and challenged fB-/- mice compared with the fB+/+ mice (Fig. 8B).

Treatment with a Factor B-Neutralizing Antibody Inhibits the Development of AHR in Sensitized and Challenged C57BL/6 and C4-/- Mice. Lung tissue inflammation was reduced in systemic or nebulized anti-fB mAb-treated mice compared with the control mice (Fig. 9). This reduction in inflammation was also reflected in a reduction of peribronchial eosinophil numbers (Table 1). Numbers of goblet cells were also significantly lower in sensitized and challenged mice treated with nebulized and systemic anti-fB mAb compared with control mice (Fig. 9 and Table 1).

Methods

Purification of Factor B. The affinity column was created by binding goat anti-human properdin factor B (DiaSorin, Stillwater, MN) to CNBr-activated Separose (Amersham Pharmacia) according to the manufacturer’s instructions. C57BL/6 mice were bled by cardiac puncture, and the blood was collected into syringes containing 50 m l of 500 mM EDTA to prevent alternative pathway activation. The blood was centrifuged at 700 ´ g for 15 min, and the plasma was collected. The plasma was then diluted 1:1 with buffer (50 mM e -amino caproic acid/10 mM EDTA /2 mM, benzamidinein PBS, pH 7.4) and passed through a 0.22-m m filter (GE Water Technologies). The plasma was added to the affinity column, and the column was washed with 10 column volumes of buffer. The factor B was eluted with 5 M LiCl₂ and dialyzed overnight against PBS. The purity of the factor B was then checked by electrophoresis on a 10% Tris-glycine gel and stained with Comassie.

Generation of Anti-fB Antibody. Briefly, fB-/- mice were immunized with a recombinant fusion protein created from the second and third short consensus repeat (SCR) domains from the factor B gene and an Ig heavy chain. The SCR domains were chosen because they are part of the deleted segment of the factor B gene in the fB-/- mice. fB-/- mice were then immunized with this protein and then boosted four times at 3-week intervals. One day after the last injection, spleen cells were fused with myeloma cells at the University of Colorado Monoclonal Antibody Center. Anti-fB mAb-secreting clones were then identified and characterized as described (2). One of the hybridomas, 1379, was used for these experiments. 1379 was purified from tissue culture supernatant with a Protein-G Sepharose column (Pharmacia). LPS was removed from the purified mAb by using polymyxin (Sigma-Aldrich). The Limulus Amebocyte lysate assay (BioWhittaker) was used according to the manufacturer’s instructions to verify that the mAb had LPS levels < 1 unit/mg of mAb. The purity of the mAb was then checked by electrophoresis on a 10% Tris-glycine gel and stained with Comassie.

Immunofluorescence Studies. For immunofluorescence, lungs were inflated through the trachea with a 1:1 mixture of OCT compound (Sakura Finetek, Torrance, CA) and PBS and were then snap-frozen in liquid nitrogen. Four-micrometer sections were cut with a cryostat and stored at -80°C. The slides were later fixed with acetone and blocked for 20 min with 10% whole goat serum (Cappel) in PBS. The slides were then incubated overnight at 4°C with FITC-conjugated goat anti-mouse C3 (Cappel) diluted 1:150 in PBS or with FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) diluted 1:150 in PBS. Because staining for IgG was negative, this antibody served as a control to verify specificity of staining with the anti-C3 antibody. The slides were counterstained with hematoxylin (Vector Laboratories). Imaging was performed (in a blinded manner) on a Nikon T2000 inverted fluorescent microscope with SLIDEBOOK software (Intelligent Imaging Innovations, Denver).

1. Hamelmann, E., Takeda, K., Schwarze, J., Vella, A. T., Irvin, C. G. & Gelfand, E. W. (1999) Am. J. Respir. Cell Mol. Biol. 21, 480-489.

2. Thurman, J. M., Kraus, D. M., Girardi, G., Hourcade, D., Kang, H. J., Royer, P. A., Mitchell, L. M., Giclas, P. C., Salmon, J., Gilkeson, G., et al. (2005) Mol. Immunol. 42, 87-97.