Humoral immunity in phenotypes of chronic lung allograft dysfunction: A broncho-alveolar lavage fluid analysis

doi:10.1016/j.trim.2016.08.004

Transplant Immunology

Volume 38, September 2016, Pages 27-32

https://doi.org/10.1016/j.trim.2016.08.004 Get rights and content

Highlights

•
Increased levels of immunoglobulins in BAL of rCLAD patients
•
Overlap between AMR and rCLAD evidenced by increased complement proteins and DSA
•
The effect of anti B-cell therapy should be investigated specifically in rCLAD.

Abstract

Background

Recently, antibody mediated rejection (AMR) has been associated with a higher incidence of chronic lung allograft dysfunction (CLAD) and mortality after lung transplantation (LTx). We investigated markers related to AMR and matrix remodeling in CLAD, with special attention for its two phenotypes being bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD).

Methods

Immunoglobulins (IgA, IgE, IgG₁–IgG₄, total IgG and IgM) and complement (C4d and C1q) were quantified in lung lavage samples at the moment of BOS (n = 15) or RAS (n = 16) diagnosis; and were compared to stable transplant patients who served as control (n = 14). Also, airway remodeling and metalloproteinases (MMPs) were investigated via zymography and gelatin degradation. The presence of DSA was additionally assessed in blood.

Results

Total IgG, IgG₁-IgG₄ and IgM were increased in rCLAD versus control (p < 0.001) and BOS patients (p < 0.01). IgA and IgE were increased in rCLAD compared to control (respectively p < 0.05 and p < 0.01), but not to BOS. Total IgG and IgE were increased in BOS versus control (respectively p < 0.01 and p < 0.05). Complement proteins were exclusively present in rCLAD and correlated positively with immunoglobulins. Additionally, in blood, DSA were more present in rCLAD (p = 0.041). MMP-9 levels increased in RAS and BOS versus control (p < 0.001) and MMP-9 induced gelatin degradation was only increased in BOS compared to control (p < 0.01).

Conclusion

We demonstrated increased levels of immunoglobulins and complement proteins dominantly present in rCLAD. This leads to the belief that antibodies and AMR might play a more important role in rCLAD compared to BOS. Therefore, anti B-cell therapy could offer beneficial therapeutic effects in patients diagnosed with rCLAD, which needs further research.

Introduction

Despite the improved survival rates over the last decade due to better surgical techniques, perioperative management and more intense immunosuppressive prophylaxis, chronic rejection after lung transplantation (LTx) remains a major obstacle clinically referred to as chronic lung allograft dysfunction (CLAD). CLAD is defined as a chronic decline in FEV₁ (≥ 20%) compared to the best post-operative values and for which no specific other cause can be identified. Two major phenotypes within CLAD were recently proposed, being restrictive CLAD (rCLAD) or restrictive allograft syndrome (RAS) and the classical obstructive form of CLAD denominated as bronchiolitis obliterans syndrome (BOS) [1].

Chronic rejection has been regarded as a predominantly T-cell mediated process where accordingly standard triple-drug immunosuppressive therapy is given, to target T-cell activation and proliferation [2]. Recent evidence, however, indicated that B-cells might be involved in the pathogenesis of CLAD as well [3]. Activated B-lymphocytes can produce a diverse range of immunoglobulins which all possess specific biological functions and produce donor-specific antibodies (DSA) directed towards specific human leukocyte antigens (HLA) of the graft [4], [5]. Circulating DSAs promote complement activation resulting into lung injury [6].

This phenomenon is, according to the International Society for Heart and Lung Transplantation (ISHLT), denominated as antibody mediated rejection (AMR). AMR is defined by the presence of circulating DSAs, clinical allograft dysfunction and histological evidence for C4d deposition accompanied with representative lung injury [7]. Besides the importance of C4d, in cardiac transplantation, correlations have also been demonstrated between C1q positive antibodies and early AMR [8]. LTx recipients with presence of DSA have an increased risk of CLAD and suffer from a worse survival [9], [10], [11]. In a recent study of Roux et al., AMR patients seem to evolve exclusively to RAS [12]. Therefore, at the moment of CLAD diagnosis, we investigated the presence of DSAs in blood combined with complement activation and the presence of immunoglobulins in BAL. Prior work demonstrated lung morphometric differences between BOS and RAS phenotypes, despite the fact they all showed small obliterative airway lesions. In this perspective, we additionally aimed to correlate the investigated humoral markers with airway remodeling.

Therefore, we analyzed broncho alveolar lavage (BAL) fluid and blood of rejecting allografts (BOS and RAS) compared to stable transplant patients.

Section snippets

Materials and methods

Patients' characteristics included gender, age, type of LTx (single or bilateral), underlying disease and immunosuppressive therapy at moment of CLAD. CLAD was diagnosed as a persistent decline in FEV₁ of at least 20% compared to the two best post-operative values, without any other identifiable cause. A further subdivision was made between BOS and RAS: if total lung capacity (TLC) was available, RAS diagnosis was made using a decrease in TLC ≥ 10% in combination with a ≥ 20% decrease in FEV₁. If

Patient characteristics

Demographic data, summarized in Table 1, demonstrate that the control patients were older (p = 0.047), and did not show differences in gender, underlying disease, type of transplantation or immunosuppressive therapy (p > 0.05) between groups. Following diagnosis of rCLAD, median graft survival was lower compared to BOS (respectively 1.1 ± 0.3 versus 2.1 ± 0.4 years; p = 0.022). No significant difference was seen for immunosuppressive therapy. Also, the effect of azithromycin did not influence our results

Discussion

Our findings demonstrate an overlap of AMR with rCLAD evidenced by the presence of circulating DSAs and increased levels of immunoglobulins and complement proteins mainly in BAL of rCLAD patients. Additionally, the finding of increased presence and functionality of MMP-9 in lavage samples of BOS and rCLAD, points to a dysregulated or exaggerated airway wall remodeling.

AMR is characterized by the presence of donor-specific anti-HLA antibodies in the context of vascular C4d deposition in

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As shown in Fig. 1A, the IgA/IgM ratio was significantly higher in BALF than in serum, and the IgG/IgM ratios was also tended to be high in BALF, indicating that class-switched antibodies abundantly exist in the lungs. The IgG/IgA ratio in BALF ranged from 1.8 to 53 and appeared to be higher than that in healthy subjects (reported to be approximately 1) [32,33]. Next, we compared the titers of autoantibodies in paired samples.
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As previously published, a HLA screening result was considered negative if MFI <500 and positive if MFI ≥500 [22,23]. BAL was performed with 2 aliquots of sterile saline (50 mL) of which the returned fractions were pooled and processed for cell counting as previously described [18]. C4d deposition on transbronchial biopsies was assessed by immunohistochemistry at our routine clinical pathology laboratory.
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^☆: None of the authors have anything to declare

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Humoral immunity in phenotypes of chronic lung allograft dysfunction: A broncho-alveolar lavage fluid analysis☆

Highlights

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Materials and methods

Patient characteristics

Discussion

J Heart Lung Transplant.

J Heart Lung Transplant Off Publ Int Soc Heart Transplant

J Heart Lung Transplant.

J Heart Lung Transplant.

Chest

Am. J. Transplant.

Lab Investig J Tech Methods Pathol.

J. Biol. Chem.

Trends Immunol.

Am. J. Transplant.

Am. J. Transplant.

J. Heart Lung Transplant.

Mod. Pathol.

Am. J. Transplant.

Immunosuppression for lung transplantation

Proc. Am. Thorac. Soc.

An introduction to immunology and immunopathology

Allergy, Asthma Clin. Immunol.