Tineke Kardol-Hoefnagel

A Promoter Polymorphism in the CD59 Complement Regulatory Protein Gene in Donor Lungs Correlates With a Higher Risk for Chronic Rejection After Lung Transplantation

Complement activation leads primarily to membrane attack complex formation and subsequent target cell lysis. Protection against self‐damage is regulated by complement regulatory proteins, including CD46, CD55, and CD59. Within their promoter regions, single‐nucleotide polymorphisms (SNPs) are present that could influence transcription. We analyzed these SNPs and investigated their influence on protein expression levels. A single SNP configuration in the promoter region of CD59 was found correlating with lower CD59 expression on lung endothelial cells (p = 0.016) and monocytes (p = 0.013). Lung endothelial cells with this SNP configuration secreted more profibrotic cytokine IL‐6 (p = 0.047) and fibroblast growth factor β (p = 0.036) on exposure to sublytic complement activation than cells with the opposing configuration, whereas monocytes were more susceptible to antibody‐mediated complement lysis (p < 0.0001). Analysis of 137 lung transplant donors indicated that this CD59 SNP configuration correlates with impaired long‐term survival (p = 0.094) and a significantly higher incidence of bronchiolitis obliterans syndrome (p = 0.046) in the recipient. These findings support a role for complement in the pathogenesis of this posttransplant complication and are the first to show a deleterious association of a donor CD59 promoter polymorphism in lung transplantation.

Soluble CD59 is a Novel Biomarker for the Prediction of Obstructive Chronic Lung Allograft Dysfunction after Lung Transplantation

CD59 is a complement regulatory protein that inhibits membrane attack complex formation. A soluble form of CD59 (sCD59) is present in various body fluids and is associated with cellular damage after acute myocardial infarction. Lung transplantation (LTx) is the final treatment for end-stage lung diseases, however overall survival is hampered by chronic lung allograft dysfunction development, which presents itself obstructively as the bronchiolitis obliterans syndrome (BOS). We hypothesized that, due to cellular damage and activation during chronic inflammation, sCD59 serum levels can be used as biomarker preceding BOS development. We analyzed sCD59 serum concentrations in 90 LTx patients, of whom 20 developed BOS. We observed that BOS patients exhibited higher sCD59 serum concentrations at the time of diagnosis compared to clinically matched non-BOS patients (p = 0.018). Furthermore, sCD59 titers were elevated at 6 months post-LTx (p = 0.0020), when patients had no BOS-related symptoms. Survival-analysis showed that LTx patients with sCD59 titers ≥400 pg/ml 6 months post-LTx have a significant (p < 0.0001) lower chance of BOS-free survival than patients with titers ≤400 pg/ml, 32% vs. 80% respectively, which was confirmed by multivariate analysis (hazard ratio 6.2, p < 0.0001). We propose that circulating sCD59 levels constitute a novel biomarker to identify patients at risk for BOS following LTx.

Profiling of peripheral blood mononuclear cells does not accurately predict the bronchiolitis obliterans syndrome after lung transplantation

After lung transplantation (LTx), circulating mononuclear cell composition and their subsets may be predictive for the bronchiolitis obliterans syndrome (BOS). We investigated the cellular composition in patients developing BOS, or not, by analyzing peripheral blood taken at multiple time points after transplantation. PBMCs of 11 BOS and 39 non-BOS patients were analyzed by FACS for monocytes, dendritic cells, NK-, NKT-, B- and T cells as well as B- and T cell subsets. Analysis of blood samples taken monthly during the first year post-LTx showed that circulating NK, NKT and dendritic cell percentages were not indicative of BOS development, whereas increases in T cells, monocytes and lowered fractions of B cells were related to BOS development. B- and T cell subset analysis at month 5 post-LTx indicated that IgM+IgD- memory B cells and central memory CD8+ T cells were decreased, whereas NKT cells were increased in BOS patients compared to non-BOS patients. Prior to BOS diagnosis, the composition of specific mononuclear cells on a group level differs from patients remaining BOS free. However, given the overlap in percentages of cellular frequencies between the patient groups investigated, this analysis does not allow prediction or risk stratification for development of BOS in individual patients.

Single Nucleotide Polymorphisms of the High Affinity IgG Receptor FcγRI Reduce Immune Complex Binding and Downstream Effector Functions

Binding of IgG Abs to FcγRs on immune cells induces FcγR cross-linking that leads to cellular effector functions, such as phagocytosis, Ab-dependent cellular cytotoxicity, and cytokine release. However, polymorphisms in low affinity FcγRs have been associated with altered avidity toward IgG, thereby substantially impacting clinical outcomes of multimodular therapy when targeting cancer or autoimmune diseases with mAbs as well as the frequency and severity of autoimmune diseases. In this context, we investigated the consequences of three nonsynonymous single nucleotide polymorphisms (SNPs) for the high affinity receptor for IgG, FcγRI. Only SNP V39I, located in the extracellular domain of FcγRI, reduces immune-complex binding of FcγRI whereas monomeric IgG binding is unaffected. This leads to reduced FcγRI effector functions, including Fc receptor γ-chain signaling and intracellular calcium mobilization. SNPs I301M and I338T, located in the transmembrane or intracellular domain, respectively, have no influence on monomeric IgG or immune complex binding, but FcRγ signaling is decreased for both SNPs, especially for I338T. We also found that the frequency of these SNPs in a cohort of healthy Dutch individuals is very low within the population. To our knowledge, this study addresses for the first time the biological consequences of SNPs in the high affinity FcγR, and reveals reduction in several FcγRI functions, which have the potential to alter efficacy of therapeutic Abs.

Association Between Promoter Polymorphisms in CD46 and CD59 in Kidney Donors and Transplant Outcome

Complement regulating proteins, including CD46, CD55, and CD59, protect cells against self-damage. Because of their expression on the donor endothelium, they are hypothesized to be involved in accommodation. Polymorphisms in their promoter regions may affect their expression. The aim of this study was to investigate if donor polymorphisms in complement regulating proteins influence kidney transplant outcomes. We included 306 kidney transplantations between 2005 and 2010. Five polymorphisms in the promoters of CD46, CD55, and CD59 were genotyped. A CD59 promoter polymorphism (rs147788946) in donors was associated with a lower 1-year rejection-free survival [adjusted hazard ratio (aHR) 2.18, 95% CI 1.12–4.24] and a trend toward impaired 5-year graft survival (p = 0.08). Patients receiving a kidney with at least one G allele for the CD46 promoter polymorphism rs2796267 (A/G) showed a lower rejection-free survival, though this became borderline significant after adjustment for potential confounders (aHR 1.87, 95% CI 0.96–3.65). A second CD46 promoter polymorphism (rs2796268, A/G), was also associated with a lower freedom from acute rejection in the presence of at least one G allele (aHR 1.95, 95% CI 1.03–3.68). Finally, the combined presence of both favorable genotypes of rs2796267 and rs147788946 had an additional protective effect both on acute rejection (p = 0.006) and graft survival (p = 0.03). These findings could help to identify patients who could benefit from intensified immunosuppressive therapy or novel complement inhibitory therapeutics.

Reduced expression of membrane complement regulatory protein CD59 on leukocytes following lung transplantation

Cellular protection against undesired effects of complement activation is provided by expression of membrane-bound complement regulatory proteins including CD59. This protein prevents membrane attack complex formation and is considered to be involved in graft accommodation. Also, CD59 downregulates CD4+ and CD8+ T-cell activation and proliferation. It is unknown whether CD59 expression is affected by transplantation. The aim of this study was to evaluate the quantitative CD59 antigen expression on distinct leukocyte subsets following lung transplantation (n = 26) and to investigate whether this differs from pretransplantation (n = 9). The results show that CD59 expression on leukocytes is significantly lower posttransplantation compared with healthy controls (p = 0.002) and pretransplantation (p < 0.0001). Moreover, the CD59 expression diminishes posttransplantation on all distinct lymphocyte subsets (p < 0.02). This effect appeared to be specific for CD59 since the expression of other surface markers remained stable or inclined following transplantation. The highest antigen expression posttransplantation was observed on CD4+ T cells and monocytes (p ≤ 0.002). These findings show that CD59 expression on leukocytes diminishes posttransplantation, which could result in decreased resistance against complement and enhanced T-cell activation. If such reduction in CD59 expression also occurs on endothelial cells from the transplanted organ, this could lead to a change into a prothrombotic and proinflammatory phenotype.

Serum intestinal fatty acid–binding protein in the noninvasive diagnosis of celiac disease

Current diagnostic guidelines for celiac disease (CD) in pediatric patients require a duodenal biopsy if the IgA anti‐tissue transglutaminase (tTG) is below 10x the upper limit of normal (ULN). Additional markers may enable a noninvasive diagnosis in this group. Serum intestinal‐fatty acid‐binding protein (I‐FABP), a marker for intestinal epithelial damage, could be useful in this respect. A total of 95 children with a clinical suspicion of CD and tTG 1‐10x ULN were investigated. All had a duodenal biopsy and analysis of serum I‐FABP. A control group of 161 children with familial short stature and normal tTG was included. I‐FABP levels in the 71 patients with tTG 1‐10x ULN and biopsy‐proven CD (median 725 pg/mL) were not significantly different (p = 0.13) from the levels in the 24 patients with a tTG 1‐10x ULN but a normal biopsy (median 497 pg/mL). However, when combining tTG and I‐FABP levels, 11/24 patients could have been diagnosed noninvasively if tTG is ≥ 50 U/mL and I‐FABP ≥880 pg/mL or in 12/19 patients if tTG is ≥ 60 U/mL and I‐FABP ≥ 620 pg/mL. Therefore, addition of I‐FABP to the diagnostic procedure of CD may provide a noninvasive diagnosis in patients with a tTG ≥ 50 U/mL.

Association between a Single Donor TARC/CCL17 Promotor Polymorphism and Obstructive Chronic Lung Allograft Dysfunction after Lung Transplantation

Lung transplantation (LTx) outcome is hampered by development of chronic rejection, often manifested as the bronchiolitis obliterans syndrome (BOS). Low serum levels of thymus and activation-regulated chemokine (TARC/CCL17), a chemoattractant, measured during the first month post-LTx are predictive for BOS development. Since TARC/CCL17 promotor polymorphisms correlate with serum TARC/CCL17 levels, we investigated seven single-nucleotide polymorphisms (SNPs) within this region and their potential association with LTx outcome. We analyzed donor and patient SNP configurations and haplotypes and observed a trend between a donor SNP (rs223899) configuration and patient TARC/CCL17 serum levels post-LTx (p = 0.066). Interestingly, this SNP configuration in patients did not show any correlation with pre-LTx TARC/CCL17 serum levels (p = 0.776). Survival analysis showed that receiving a graft from a donor heterozygous for rs223899 has a disadvantageous impact on transplantation outcome. When stratified per donor SNP genotype, patients receiving a transplant from a heterozygous donor showed a lower BOS-free survival (p = 0.023) and survival rate (p = 0.0079). Since rs223899 is located within a NFκB binding site, heterozygosity at this position could result in a reduced TARC/CCL17 expression. Our data indicate that a single TARC/CCL17 promotor SNP in the donor correlates with lower serum TARC/CCL17 levels measured 1 month after LTx and affects clinical outcome after LTx.

Antibodies against Apoptotic Cells Present in End-stage Lung Disease Patients Do Not Correlate with Clinical Outcome after Lung Transplantation

Antibodies against HLA and non-HLA are associated with transplantation outcome. Recently, pretransplant serum IgG antibody levels against apoptotic cells were found to correlate with kidney allograft loss. We investigated the presence of these antibodies in lung transplantation (LTx) patients and evaluated the correlation of pre-LTx serum levels of IgG antibodies against apoptotic cells with LTx outcome. These cells included donor lung endothelial cells (ECs) obtained from lung perfusion fluid collected during LTx procedure. Cells were isolated, expanded in vitro, and analyzed as targets for antiapoptotic cell reactivity. Cultured cells exhibited EC morphology and were CD31+, CD13+, and vWF+. End-stage lung disease patients showed elevated serum IgG levels against apoptotic lung EC (p = 0.0018) compared to healthy controls. Interestingly, the levels of circulating antibodies directed against either apoptotic Jurkat cells or apoptotic lung ECs did not correlate, suggesting a target cell specificity. We observed no correlation between chronic or acute rejection and pre-LTx serum levels of antiapoptotic antibodies. Also, these levels did not differ between matched patients developing chronic rejection or not during follow-up or at the time of diagnosis, as they remained as high as prior to transplantation. Thus, circulating levels of antiapoptotic cell antibodies are elevated in end-stage lung disease patients, but our data do not correlate with outcome after LTx.