Role of Complement Activation in Obliterative Bronchiolitis Post–Lung Transplantation

Hidemi Suzuki, Mark E. Lasbury, Lin Fan, Ragini Vittal, Elizabeth A. Mickler, Heather L. Benson, Rebecca Shilling, Qiang Wu, Daniel J. Weber, Sarah R. Wagner, Melissa Lasaro, Denise Devore, Yi Wang, George E. Sandusky, Kelsey Lipking, Pankita Pandya, John Reynolds, Robert Love, Thomas Wozniak, Hongmei Gu, Krista M. Brown and David S. Wilkes
J Immunol October 15, 2013, 191 (8) 4431-4439; DOI: https://doi.org/10.4049/jimmunol.1202242

Abstract

Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17–regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1–related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1–related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17–mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.

Introduction

Obliterative bronchiolitis (OB), characterized by fibrosis of terminal airways (1), remains the limiting factor in long-term survival of lung transplants and is synonymous with chronic rejection. Conversely, primary graft dysfunction (PGD) is an acute clinical condition characterized by hypoxemia and diffuse pulmonary infiltrates within 72 h of transplantation. PGD is the leading cause of early morbidity and mortality after transplantation and may predispose to OB (2, 3). Despite their clear importance to lung transplantation, there are no effective long-term treatments for PGD or OB, as the immune mechanisms that cause allograft dysfunction remain elusive.

Our laboratory reported that immune responses to type V collagen [col(V)] are key risk factors for PGD (4) and OB post-lung transplantation (5). We have demonstrated that passive transfer of anti-col(V) Abs to rat lung isograft recipients results in PGD-like histology and physiology (6). Injury to airway epithelial cells in this model was due to complement-dependent cytotoxicity and correlated with expression of col(V) on epithelial cells (6). Although native and transplanted lung epithelium express col(V), anti-col(V) Ab-mediated injury was limited to the transplanted lung, suggesting differential complement regulation in the transplanted lung compared with native lung.

Complement-regulatory proteins prevent the activation of the complement cascade by inhibiting the formation or activity of complement cascade components. In humans, CD55 (decay-accelerating factor [DAF]) accelerates the decay of classical and alternative pathway C3 and C5 convertases, preventing the amplification of the cascade (7). DAF is present on the membranes of virtually all blood cells (8, 9), vascular endothelium (10), and epithelial cells (11, 12). CD46 (membrane cofactor protein) is also a human CRP and acts in the cleavage of C3b and C4b, preventing the formation of the C3 and C5 convertases (13). Together, DAF and CD46 prevent the destruction of autologous cells by activating complement via inhibiting and degrading the convertases. Complement receptor 1–related protein y (Crry) protein is the rodent ortholog of CD46. Crry is of importance in rodent complement regulation as it has both CD55 and CD46 functions (14). Rodents also express DAF, but Crry is more efficient than DAF at C3 convertase inhibition, and therefore, Crry is of greater physiologic importance than CD55 in regulating local complement activation in rodents (15).

IL-17a is key to the development of PGD (5, 16, 17), and, as PGD is important for development of OB, IL-17a is also a major mediator of OB (18). PGD incidence is an independent risk factor for the development of OB (19, 20). Recent studies demonstrate complement-related functions of IL-17a. For example, CD55 expression is downregulated during clinical asthma (21). In addition, recent reports indicate a crucial role for IL-17a in asthma pathogenesis and that IL-17a is induced by C3a in a model of allergic airway inflammation (22, 23). These data are consistent with a study showing C3a induced IL-17a production in kidney transplant recipients (24). Collectively, these studies suggest interplay between IL-17a, lung expression of complement-regulatory proteins, complement activation, and inflammatory pulmonary diseases, but they have not been studied in the setting of lung transplantation. Therefore, we hypothesized that complement activation occurring in transplanted lungs is in part due to local downregulation of CRPs. In addition, we hypothesize that IL-17a is related to this process and subsequent complement activation that culminate in OB development.

Materials and Methods

Animals

C57BL/6 and C57BL/10 mice (25–30 g; Harlan, Indianapolis, IN) were used for orthotopic left lung transplantation. Animals were housed in the Laboratory Animal Resource Center at Indiana University School of Medicine, according to institutional guidelines. All studies were approved by the Indiana University School of Medicine Institutional Care and Use Committee.

Human lung tissues

Lung transplant tissues were obtained from explanted lungs harvested at that time for retransplantation due to refractory OB. Chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis was the initial indication for transplantation. Tissues were either fixed in formalin or snap frozen. The former and latter tissues were H&E stained to confirm the presence of OB. RNA and protein were extracted from frozen tissues with histologically proven OB and used for PCR and Western blotting, respectively. Normal lung tissues were obtained from lobectomy tissues collected at the time of lung cancer resection.

Murine lung transplantation and complement inhibition

The orthotopic transplantation of left lungs was performed, as previously described (18). Histological examination of lungs after 21 d, as described previously (18), identified those recipients with OB. Some mice were treated with an IL-17a:Fc fusion protein (Ad IL-17a Fc), as previously described (18). To study the effect of C5 inhibition, other lung transplant recipients were treated with anti-mouse C5 mAb (BB5.1mG1, 40 mg/kg) (Alexion, Chesire, CT) (25, 26), which blocks the generation of C5a and C5b-9, and 40 mg/kg isotype-matched irrelevant control mAb (r135.8) (Alexion) was purified from ascites. Subcutaneous injection of both reagents was performed at 2 d, 30 min before surgery and three times per week for 3 wk after transplantation (11 total injections preoperatively). All transplants were harvested on day 21 posttransplantation. Hemolytic complement activity was determined as reported (27, 28) using the same plasma samples assayed for C3a and C5a levels at day 21 posttransplantation. Preliminary studies confirmed that BB5.1 completely abrogated hemolytic complement activity.

Bronchoalveolar lavage fluid samples

Human bronchoalveolar lavage (BAL) samples were obtained as described, and clinical characterization of the human samples has been reported (29). Mouse BAL fluid samples were obtained, as previously reported (18).

Immunohistochemistry

Staining was performed on 4-μm tissue sections, as previously described (30). In brief, sections obtained from paraffin-embedded, formalin-fixed lungs underwent Ag retrieval treatment, followed by peroxide and protein blocks (1× Power Block; Biogenex, San Ramon, CA). Sections were then incubated with the following primary Abs: rabbit anti-mouse Crry (1:200), rabbit anti-mouse CD55 (1:200), rabbit anti-human CD46 (1:150), and rabbit anti-human CD55 (1:150) (all Santa Cruz Biotechnology, Santa Cruz, CA). Then sections were stained using a sensitive avidin-streptavidin-diaminobenzidine peroxidase kit (Biogenex), according to the manufacturer's instructions. Sections were counterstained with hematoxylin. Quantification of CD55 and CD46 in human tissues was conducted using the Aperio Scanscope Imaging System. In brief, the slides were imaged using the Aperio Scancscoe CS, and normal and OB bronchioles were identified. The Positive Pixel Count algorithm (Apeio Scanscope software) was used to quantify the amount of a specific stain present in a scanned slide image. A color range was specified (range of hues and saturation), as were three intensity ranges (weak, positive, and strong). For pixels that satisfied the color specification, the algorithm counted the number and intensity-sum in each intensity range, along with three additional quantities, as follows: average intensity, ratio of strong/total number, and average intensity of weak positive pixels. Default input parameters were preconfigured for brown color intensity fraction of positive to quantification in the three intensity ranges (220–175, 175–100, and 100–0). Pixels that were stained, but did not fall into the positive-color specification were considered negative-stained pixels. These pixels were counted as well, so the fraction of positive to total stained pixels was determined. The algorithm was applied to an image by using ImageScope software, which allows selection of an image region of analysis, specifying the input parameters, running the algorithm, and viewing and saving the algorithm results. Computer-assisted morphometric analysis of digital images was done using the Aperio software that came with the Aperio Digital Imaging System. Specific algorithms were used for the positive pixel analysis. Bronchioles comprised ∼15% of the entire lung section analyzed.

C3a and C5a ELISA

C3a was quantitated in human BAL fluid samples using the human C3a ELISA kit (BD Biosciences, Franklin Lakes, NJ), according to manufacturer’s instructions. Human BAL samples were obtained from lung transplant recipients and were previously described in detail (29). Mouse C3a was assessed from BAL fluids by standard ELISA. The native protein (catalogue 558618), capture Ab (I-87-1162), and biotinylated detection Ab (I87-419) were obtained from BD Pharmingen. C5a ELISA was conducted with a commercial ELISA kit per manufacturer’s protocol (R&D Systems, Minneapolis, MN).

Generation of col(V)-reactive lymphocytes

C57BL/6 mice were immunized by injecting 200 μg col(V) emulsified in 200 μl CFA into the base of the tail. To boost the initial immunization, mice received a second injection of 200 μg col(V) emulsified in 200 μl IFA, 10 d after the initial injection of CFA (16). Ten days after boosting, mice were sacrificed, draining (inguinal) lymph nodes were harvested, and individual lymph node lymphocytes were isolated, as reported (6). Lymph node cells were resuspended in 1% sterile PBS prior to coculture in MLRs.

Western blotting

Immunoblotting of soluble protein from airway epithelial cell (RLE-6TN; American Type Culture Collection, Manassas, VA) cultures incubated with IL-17a, IL-6, or IL-10 (eBioscience, San Diego, CA) for varying time periods. Cytokine concentrations used were determined by preliminary studies. Cell cultures were performed, as described (30). Primary Abs for Western blotting included CD55 (I-97; Santa Cruz Biotechnology, Santa Cruz, CA), Crry (M-180; Santa Cruz Biotechnology), and glyceraldehyde dehydrogenase (Santa Cruz Biotechnology).

Real-time PCR

Total RNA from mouse lung samples was isolated, as previously described (18). Reverse transcription and real-time PCR were performed, as described (18). Primer pairs for IL-17a and β-actin primers are published (18). Primer pairs for Crry, CD55 (human and mouse), and CD46 were purchased, as follows: mouse Crry, Mm00785297_s1 TaqMan Primer (Applied Biosystems); mouse CD55(DAF1), Mm00438377_m1 Taqman Primer (Applied Biosystems); human CD55, Hs00892618_m1 Taqman Primer (Applied Biosystems); human CD46, Hs00611256_m1 Taqman Primer (Applied Biosystems). Relative quantity of target was assessed by 2−ΔΔt method, using β-actin cycle threshold for normalization.

Mixed leukocyte reaction

Splenic CD3+ T lymphocytes were isolated from C57BL/6 or C57BL/10 mice, or from C57BL/6 mice 21 d after lung transplantation, as previously described (31). Col(V)-reactive T cells (CD3+) were also isolated from C57BL/6 mice immunized with col(V) in CFA, as reported above. Alloantigen or col(V)-reactive T cells were cocultured with irradiated T cell–depleted splenocytes isolated from normal C57BL/6 mice in MLRs, as reported in the presence or absence of exogenous C3a (10 ng/ml; R&D Systems, Minneapolis, MN). Supernatants were collected for cytokine profiles after incubating for 72 h.

Cytometric bead array

Cell-free samples of culture medium or heparinized serum were assessed for cytokines by the mouse Th1, Th2, Th17 CBA kit (BD Biosciences), as described, and analyzed by BD Cytometric Bead Array software, version 1.3, per manufacturer’s instruction.

Statistical analyses

Data are expressed as mean ± SEM. Analysis was by two-way ANOVA with paired or nonparametric t test using Prism 4 (GraphPad Software, San Diego, CA). Significance was determined by p < 0.05.

Results

Complement-regulatory protein expression

Previous results from this laboratory showing complement-mediated cytotoxicity to airway epithelium in lung isografts after passive transfer of col(v) Abs (6) led to the question as to why complement is being activated in the transplanted lung and not native lungs. To assess whether complement regulators were downregulated on epithelial cells, the targets of early and late immune-mediated injury posttransplantation, we used immunohistochemistry to detect CD55 and CD46 in lung tissues from normal humans and patients with lung transplant–associated OB. We also assessed CD55 and Crry, the murine homolog of CD46, expression in the lungs of orthotopically transplanted mice early posttransplant and in OB lesions.

In normal human tissue, CD55 and CD46 were highly expressed on the epithelial cells of the large and small airways (Fig. 1, wide arrows). These two CRPs were also expressed, to varying degrees, in the interstitial tissue of the lung (narrow arrows). However, CRP staining was seen less intensely on the vascular endothelium (data not shown). Lung tissue from patients diagnosed with clinical and histologic OB was also evaluated for CRP expression. One patient (patient 1 in Fig. 1A) showed constriction of airways without fibrotic growth into the airways or loss of epithelium. Lung tissue from this patient showed an intact epithelial layer on small airways, but CD55 and CD46 staining was nearly undetected in the epithelium or interstitium (Fig. 1A). A second patient diagnosed with OB (patient 2 in Fig. 1A) showed obliteration of the airways, with partial loss of the epithelial cell layer. This patient also showed reduced levels of CD55 and CD46 staining throughout the lesion, including the airway epithelium, indicating that OB is characterized by low levels of CRP expression on airway epithelium. In two additional patients (including those shown in Fig. 1A), quantitative immunohistochemistry was used to compare the expression of bronchiolar CD46 and CD55 in normal lungs and allografts with OB. Notably, expression of CD46 and CD55 was significantly less in bronchioles of OB lungs (Fig. 1B).

FIGURE 1.
FIGURE 1.

Epithelial cell CRP expression is diminished in human OB. (A) Complement-regulatory protein expression was assessed by immunohistochemistry on 4-μm lung sections from normal and OB specimens. In humans, sections of normal lung show discrete CD55 and CD46 staining in airway epithelial cells (wide arrows) and in the interstitium (narrow arrows). However, in lung sections from patients with OB (OB, patient 1; OB, patient 2), airway epithelial cell staining of both CD55 and CD46 is severely downregulated. Two different OB patients are represented, patient 1 showing intact airway epithelium and patient 2 showing fibrotic plugs in the airway with a loss of epithelial cell integrity. Photomicrographs are representative of two normal human samples and six human OB patients. Original magnification ×400. (B) The bar graph below the photomicrographs shows quantitation of the immunohistochemical images described above. Data shown are mean + SEM of four normal lungs and four lung transplants with histologically proven OB. CD46 normal compared with OB, p < 0.001. CD55 normal compared with OB, p < 0.036.

Orthotopic mouse lung transplants that developed OB were assessed for CD55 and Crry expression at 21 and 28 d after transplantation (Fig. 2A). Our prior studies confirm that OB occurs in ∼50% of mice at 21 or 28 d posttransplant (18). Mice diagnosed with OB 21 d postsurgery had low levels of CD55 and Crry expression on the airway epithelium particularly within OB lesions (Fig. 2A, wide arrows). CD55 staining in the interstitium was also low, although there was variable staining of Crry in the interstitium of some of these mice. In contrast, the native lung from 21-d OB mice had robust staining of both CD55 and Crry, indicating that the pathology was local and restricted to the transplanted lung. For both the transplanted and native lungs, animals with OB diagnosed at 28 d showed similar staining patterns to those of the 21-d animals (Fig. 2A). To investigate the time to loss of CRP expression, we determined CD55 and Crry transcript expression at 1 and 21 d posttransplantation. Quantitative PCR confirmed significantly lower levels of transcripts for CD55 and Crry in allograft compared with normal C57BL/10 (donor) lungs at day 1 posttransplantation, and that these changes persist at 21 d posttransplantation (Fig. 2B).

FIGURE 2.
FIGURE 2.

Airway epithelial cell CRP expression in mouse lung transplant recipients. (A) Recipient C57BL/6 mice developed OB after transplantation of left lungs from C57BL/10 donors. CD55 and Crry immunostaining in the transplanted lungs were low in airway epithelial cells at both 21 and 28 d posttransplantation. Some interstitial staining of Crry was maintained in the 21-d animals. Both CD55 and Crry are apparent in airway epithelium (wide arrows) and in the parenchymal tissue of the native lungs at both time points and are similar to staining intensities and patterns from normal humans (A) and from normal mice (data not shown). Epithelial expression of Crry and CD55 was markedly diminished in OB lesions of transplanted lungs (wide arrows). Isotype controls were carried out for either transplanted or native lungs from each time point, as staining of both lungs was performed at the same time. Images are representative of five to eight animals per condition. Original magnification ×400. (B) Quantitative PCR showing diminished transcripts for CD55 and Crry 1 and 21 d post-murine lung transplantation as compared with lungs from normal C57BL/10 mice. Data shown are mean ± SEM of three individual transplant experiments and three normal lungs.

Complement activation

Low levels of CD55, CD46, and Crry on airway epithelium during OB in humans and mice suggested that complement might be activated in the lung. ELISA showed that normal human lung BAL fluid samples contained little C3a (Fig. 3A). In contrast, patients with histologically proven OB showed a 2.5-fold higher (p < 0.05) C3a level in BAL fluid. Normal mice also had low levels of C3a in BAL fluid by ELISA (Fig. 3B). C57BL/6 mice receiving C57BL/6 isografts had similar low levels of C3a. In contrast, C3a levels were >5-fold higher in the BAL fluid from C57BL/10 lungs transplanted into C57BL/6 mice during OB compared with normal or isograft mice (Fig. 3B, p < 0.05). Therefore, low levels of CRP expression on airway epithelium correlate with increased complement activation.

FIGURE 3.
FIGURE 3.

Humans and mice with OB have increased C3a in BAL fluids. The complement split product, C3a, was assessed in BAL fluids from humans and mice. BAL fluids were obtained under standard protocols and were performed identically for normal and OB samples. (A) Normal patients (n = 10) showed significantly lower C3a levels as compared with patients with OB (n = 6). *p < 0.05 versus normal. (B) Mice with allografted or isografted lungs were lavaged. Unoperated mice of the same sex, strain, and age (normals) were also lavaged in the same manner. These normal mice (n = 5) had significantly lower levels of BAL fluids C3a as compared with mice with OB (n = 14). Isografted mice (n = 10) had C3a levels similar to those of normal mice. *p < 0.05 versus normal or isograft.

IL-17 downregulates CRP expression

CRPs are downregulated early in the course of rejection following transplantation, with reduction in Crry and CD55 transcripts noted within 1 d posttransplantation in mice (Fig. 2) at a time when IL-17a lung transcripts and protein were also increased (data not shown, manuscript in preparation). Notably, Crry and CD55 were also downregulated at day 28, the time when OB occurred; and our prior study reported that OB is IL-17 dependent (18). These data suggested that IL-17a levels and CRP expression might be linked and were further investigated by incubation of cultured rat airway epithelial cells with IL-17a or IL-6, a potent inducer of IL-17, and assessing CD55 and Crry by Western blotting. IL-17a (50 ng/ml) induced dose-dependent reductions in Crry, but not CD55, on airway epithelial cells within 6 h (Fig. 4). IL-6 did not affect expression of CD55 or Crry at any time point (data not shown).

FIGURE 4.
FIGURE 4.

IL-17a mediates downregulation of Crry in cultured rat airway epithelial cells. RLE-6TN cells were cultured to 70% confluency and then treated with 50 ng/ml IL-17a for various times. Soluble protein was separated, transferred to a solid support, and probed for Crry, CD55, and GAPDH as an internal protein-loading control. Crry isoform levels decreased significantly by 6 h. CD55 was also unaffected by IL-17a treatment. Data are representative of four experiments with the same results.

C3a stimulates alloantigen and col(V)-induced IL-17a production

The results of the airway epithelial cell experiments suggested that IL-17a controls CRP expression and, in turn, complement activation in the lung after transplantation. We asked whether a possible feed-forward loop exists, wherein complement activation, in the form of C3a (22), could affect IL-17a production. Exogenous C3a induced significantly more IL-17 production from alloantigen-primed T cells (CD3+) in coculture with allogeneic APCs (C57BL/6-derived T cell–depleted splenocytes) (Fig. 5A). In contrast, addition of C3a reduced IFN-γ production, but had no significant effect on IL-10, IL-6, or IL-2 (Fig. 5A). We have reported that immune responses to the autoantigen col(V) have key roles in the rejection response, including OB, and are IL-17 dependent. Therefore, we also determined whether C3a increased col(V)-induced IL-17 production from col(V)-reactive T cells. Indeed, exogenous col(V) significantly increased IL-17 levels, yet decreased IL-10 production from col(V)-reactive CD3+ T cells (C57BL/6) in coculture with C57BL/6 T cell–depleted splenocytes as a source of APCs (Fig. 5B).

FIGURE 5.
FIGURE 5.

C3a effects on cytokine production and lymphocyte proliferation. (A) CD3+ splenic T lymphocytes (3 × 105) derived from C57BL/6 that received lung allografts from C57BL/10 mice were incubated with T cell–depleted splenocytes from C57BL/10 mice as a source of APCs (3 × 105) in the presence and absence of C3a (10 ng/ml). (B) Pure CD3+ T cells from col(V)-immunized mice (C57BL/6, 3 × 105) were incubated with T cell–depleted splenocytes from C57BL/6 mice as a source of APCs (3 × 105) in the presence and absence of C3a (10 ng/ml). Conditioned medium was assessed for cytokines by cytometric bead array after 72-h incubation. Levels of cytokines from wells of T lymphocytes alone or APCs alone were below the level of detection. Values represent averages ± SD of three independent experiments. *p < 0.01.

IL-17a blockade effects on CRP expression, IL-17a, and complement activation

Previous results from this laboratory showed that neutralizing IL-17a using an adenoviral construct to overexpress the IL-17R (IL-17RFc) in mice undergoing orthotopic lung transplantation abrogated OB development (18). Using tissues from these animals, we asked whether neutralizing IL-17 would restore CRP expression and reduce C3a levels in the animals that did not experience OB by 21 d. In contrast to Fig. 2, showing diminished CD55 and Crry expression in the transplanted lung relative to native lung, neutralizing IL-17 resulted in comparable expression of these proteins in native and transplanted lungs (Fig. 6). Luciferase adenoviral construct control animals showed no effect of the vector on CRP staining (data not shown).

FIGURE 6.
FIGURE 6.

IL-17a blockade restores CRP expression and reduces C3a and Il-17a levels. In the mouse model of OB development after lung transplantation, some transplanted mice were treated with an Ad IL-17 Fc vector. Animals were assessed 21 d after transplantation, as this time point was common for OB development in this model (18). Lung sections were assessed for CD55 and Crry expression. Transplanted lung in Ad IL-17a Fc-treated animals showed significantly increased epithelial levels of both Crry and CD55 in airway epithelium as compared with mice with OB (Fig. 2). Transplanted lungs showed similar staining patterns and staining intensity as native lungs. A luciferase control vector indicated that target signals were not altered by the vector used to deliver the Fc fusion protein. Micrographs are representative of at least three mice. Photomicrographs are representative of four Ad IL-17a Fc-treated mice and two control vector-treated mice, counterstained with hematoxylin. Original magnification ×400.

Blockade of IL-17 activity was further investigated by comparing BAL fluid C3a levels in Ad IL-17a Fc-treated animals and allografted animals that did not have OB (Table I). BAL fluid from the transplanted lungs of the treated mice had low levels of C3a, similar to those of normal mice. Because these animals did not develop OB, the low levels of C3a might reflect robust transplant health. However, allografted mice that received the control vector and did not develop OB had high levels of BAL fluid C3a (p < 0.05). C3a in untreated animals without OB was similar to those with OB (Table I), which agrees with previous results showing that whereas only 50% of allografted mice develop OB, all showed acute rejection and increases in IL-17 (18). These data suggest that lower C3a levels were due to IL-17 blockade.

View this table:
Table I. BAL fluid levels of C3a in mice post–lung transplant

Effect of complement inhibition on OB development

Data showing decreased expression of CD55 and Crry, with associated increased complement activation during OB that is associated with increased IL-17, suggested that complement inhibition may prevent OB and diminish systemic IL-17 levels. To address this question, C5 was inhibited systemically using an anti-C5 Ab (BB5.1), as described in Materials and Methods. Acute rejection scores, the presence or absence of OB, and systemic cytokine levels were determined. In addition, serum levels of C3a and C5a were measured. Notably, C5 inhibition abrogated the development of OB 21 d posttransplantation, whereas OB occurred in 50% of control Ab-treated mice, as expected (Fig. 7A). Additionally, acute rejection scores (“A” scores) were reduced significantly in anti-C5 Ab-treated mice (Fig. 7A). Whereas mice that develop OB also had higher systemic IL-17a, IL-6, and IFN-γ levels, inhibiting C5 resulted in significantly lower levels of each cytokine (Fig. 7B). C5 inhibition resulted in significantly lower levels of C3a and C5a in plasma (Fig. 7C). Because C5 inhibition downregulated IL-17, shown to decrease Crry and CD55 in vitro (Fig. 4), we next determined whether C5 blockade recovered CRP expression in vivo. Notably, immunostaining for Crry and CD55 protein expression was recovered in mice treated with the anti-C5 inhibitor compared with that observed in normal (nontransplanted) lungs (Fig. 8). Total complement inhibition by BB5.1 Abs was confirmed by quantifying complement hemolytic activity in BB5.1 compared with control mice, as reported in Materials and Methods.

FIGURE 7.
FIGURE 7.

C5 blockade prevents OB, downregulates acute rejections, and results in lower plasma levels of C3a and C5a following murine orthotopic lung transplantation. The left lungs in C57BL/10 mice were transplanted into C57BL/6 mice. Some mice were treated with anti-C5 Ab or control Ab, as described in Materials and Methods. Twenty-one days posttransplantation, the lungs were harvested and scored for rejection pathology using standard criteria. C3a and C5a plasma levels were determined. (A) Rejection scores and histopathology. Fifty percent of control Ab-treated mice developed OB, whereas none of the anti-C5–treated mice did. Anti-C5–treated mice also revealed lower acute rejection scores (also shown in the bar graph, “A” scores, p < 0.026). Histopathology shows development of OB in control Ab-treated mice plus severe acute rejection, whereas less severe acute rejection and absence of OB were noted in anti-C5–treated mice. The lower histologic sections are trichrome stains to show connective tissue deposition. Histologic scores represent the mean ± SEM of eight mice in each group. Histology representative of eight mice in each group (original magnification ×400). (B) Plasma cytokine levels. Levels of each cytokine were measured, as reported in Materials and Methods. The groups included those that received isoytpe control Ab and are divided into those that developed OB (n = 4) and those that did not develop OB (n = 4). The other group received anti-C5 Abs (BB5.1) (n = 8). Data shown are the mean ± SEM, *p < 0.05, compared with mice that received anti-C5 treatment and did not develop OB, or that received the isotype control Ab and also did not develop OB. (C) Plasma levels of C3a and C5a were determined at 21 d posttransplantation in each group. Data represent the mean ± SEM of eight mice in each group (p < 0.0029 for C3a, and p < 0.00007 for C5a).

FIGURE 8.
FIGURE 8.

Neutralizing C5 recovers airway epithelial cell CRP expression in mouse lung transplant recipients. C57BL/6 that were recipients of orthotopic C57BL/10 lung grafts were treated with anti-C5 (BB5.1) or isotype control Abs, as reported in Materials and Methods. Twenty-one days posttransplantation, the lungs were harvested and immunostained for CD55 and Crry, as reported. Data are representative of eight mice treated with anti-C5 inhibitor (BB5.1) and five normal C57BL/10 lungs. Original magnification ×400.

Discussion

This series of experiments shows that the CRPs, CD55 and Crry (CD46), are downregulated during OB in both humans and mice. At the same time, BAL fluid C3a is increased in both species. Importantly, IL-17a mediates downregulation of Crry in cultured airway epithelial cells in vitro. Neutralizing IL-17a rescued Crry and CD55 expression in vivo and was associated with lower local levels of C3a. Finally, blocking C5 not only downregulated acute rejection and abrogated OB, but it also reduced systemic IL-17 levels and local and systemic concentrations of C3a and C5a; it also recovered Crry and CD55 expression. Data showing that C3a induces IL-17 in autoimmune and alloimmune environments and that IL-17a downregulates the expression of epithelial cell-derived Crry suggest a feed-forward loop of IL-17 induced downregulation of CRPs and complement activation.

To the best of our knowledge, this is the first study to demonstrate the integrated roles of IL-17a and complement in autoimmunity and alloimmunity of chronic lung transplant rejection. Some studies have noted the importance of IL-17a in both autoimmune- and alloimmune-mediated rejection (5, 18, 32, 33), whereas others have noted that complement may be activated during rejection pathology (34–36), but the data shown in this work suggest a mechanism by which the innate and adaptive immune systems are interconnected in the development of OB.

IL-17a–mediated downregulation of Crry (Fig. 4) suggests that pre-existing inflammatory or autoimmune conditions in the recipient, especially with exposure of col(V), can predispose to early rejection, agreeing with the results of Iwata et al. (6). Furthermore, Crry is reported to be more dominant than CD55 in local complement regulation (15). Therefore, data showing that IL-17 is linked to downregulating CD55, but not Crry, in vitro may have great physiologic significance. However, in vivo it appears that IL-17 may regulate both CD55 and Crry expression, suggesting differential effects of IL-17 in vivo compared with in vitro. Alternatively, IL-17 may induce other pathways that regulate CD55/Crry/CD46 expression.

The results of the current study suggest a feed-forward loop in which IL-17a suppresses CRP expression, leading to complement activation and production of C3a. This stimulates more IL-17a production and possible further CRP downregulation. CRP loss may be due to increased production of IL-17a. It is also notable that C5 blockade downregulated systemic production of IL-17 and IL-6, and this was associated with recovery of Crry and CD55 in the transplanted lung. An unexpected finding was how rapidly Crry and CD55 were downregulated posttransplant, which occurred within 24 h posttransplantation. This time frame corresponds to IL-17a–induced downregulation in vitro, as shown in Fig. 4. It is also notable that when we assessed the time frame of systemic IL-17a production in the C57BL/10→C57BL/6 lung transplant model, we observed IL-17a and IL-6 allograft lung transcripts were upregulated significantly at 24 h posttransplant (H. Suzuki and D. S. Wilkes, manuscript in preparation). The rapidity of this response suggests that IL-17a is induced during ischemia reperfusion injury in the transplanted lung. Indeed, Sharma et al. (37) reported robust IL-17 production from δγ-T cells within 3 h of ischemia reperfusion injury in the lung. It is interesting to speculate that IL-17a or IL-6 directly has this effect or via induction of other cytokines that act in a paracrine fashion to block transcript expression. Alternatively, each cytokine or both could induce expression of matrix metalloproteases known to cleave Crry or CD55 from the cell surface (38). However, the specific molecular mechanism of IL-17–mediated downregulation of Crry and CD55 is unknown. Furthermore, whereas the loss of Crry and CD55 is rapid in lungs transplanted into normal recipients, as shown in the mouse model, it is interesting to speculate that pretransplant conditions, such as idiopathic pulmonary fibrosis, which is associated with systemic IL-17 activity, may only accelerate CRP loss. Although these events could account for early CRP loss, data showing that Crry and CD55 are downregulated during OB suggest chronic dysregulation of CRP expression post-lung transplantation. The mechanisms for chronic loss could also be due to matrix metalloproteases that are known to be upregulated post-lung transplantation (39, 40), or perhaps mediated by chronic airway hypoxia known to occur post-lung transplantation, which can also enhance complement activation (41–43). Indeed, lung transplantation airway hypoxia has been implicated in fibrosis that could culminate in OB (42). Although intriguing, the technical limitations of bronchial artery reanastomosis in both mice and humans preclude our ability to directly answer this question. However, performing retrograde flush of the bronchial arteries at the time of donor harvest has been shown to decrease cytokines that play key roles in inflammation and immunity (44). Such an approach may prevent immune events associated with OB.

Although Abs have key roles in activating complement, recent evidence from Murakami et al. (45) suggests that Th17 development and IL-17a–mediated complement activation can be initiated in the absence of a humoral immune response. In an IL-6– and IL-17–dependent model of arthritis, microbleeding into joint, followed by transfer of Th17-polarized T cells, was sufficient to stimulate a complement-dependent fulminant autoimmune arthritis. Based on data shown in the current study, it is interesting to speculate that complement activation in the arthritis model was in part induced by IL-17– and/or IL-6–mediated downregulation of CRPs on synovial tissues. This could account for IL-17–mediated damage associated with MHC class II alleles for which tissue-specific causative Ags cannot be identified (46). Alternatively, any pre-existing condition that increases IL-17 levels, such as ischemia reperfusion injury (37) or idiopathic pulmonary fibrosis (47), could downregulate CRPs on airway epithelium of newly transplanted donor lung, as described above.

Importantly, these published reports taken together with the present data strongly suggest that any IL-17–mediated inflammatory process, in the presence of activated T cells, is sufficient to initiate complement activation and production of C3a. What is more, this initiation and amplification of the feed-forward loop may occur strictly in response to, and generate, a cellular immune response, as shown by the effects of C3a on cytokine production (Fig. 5). Humoral immunity, in the form of anti-HLA Abs or autoantibodies, may not be essential to the initiation or propagation of the loop, and this idea is currently under investigation. Alternatively, constitutive, low-level activation of the complement cascade via the alternative pathway could be amplified by IL-17a–mediated CRP downregulation and thereby initiate the feed-forward loop.

However, our results could also support a role for Abs in amplification of this loop. Complement-mediated damage, whether induced by autoantibodies or anti-HLA Abs, or infections could lead to exposure of interstitial col(V) on airway epithelial cells, thereby inducing an autoimmune response, more complement activation, and IL-17a production. Indeed, our preclinical and patient studies and those from other investigators clearly show a role for both alloimmune and autoimmune pathologies in OB (5, 32, 48). C3a induction of IL-17 in alloreactive and col(V)-reactive lymphocyte cytokine production supports the concept of a feed-forward loop of complement activation and IL-17a levels and further reductions in CRP expression that culminate in graft destruction.

In summary, the preclinical and clinical data in the current study suggest a key role for complement activation in OB pathogenesis. Whereas the results of complement inhibition in ischemia reperfusion injury post-lung transplantation have been promising, the current studies suggest a need for clinical trials in complement inhibition for the treatment of OB.

Disclosures

D.S.W. is cofounder and Chief Scientific Officer of ImmuneWorks, a biotech company developing novel therapeutics for immune-mediated lung diseases. The other authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grants HL067177, HL096845, and P01AI084853 (to D.S.W.). This work was also supported by National Heart, Lung, and Blood Institute RO1 Grants HL109288 (to R.V.) and HL109310 (to R.S.).

  • Abbreviations used in this article:

    BAL
    bronchoalveolar lavage
    col(V)
    type V collagen
    CRP
    complement-regulatory protein
    Crry
    complement receptor 1–related protein y
    DAF
    decay-accelerating factor
    OB
    obliterative bronchiolitis
    PGD
    primary graft dysfunction.

  • Received August 13, 2012.
  • Accepted August 14, 2013.

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