Objectives: Endothelium is the major tissue for hyperacute and acute rejection. Binding of antibody to endothelium activates several immunologic mechanisms. Antiendothelial cell antibodies are a group of nonhuman leukocyte antigen antibodies that may play a role in the induction of an immunologic reaction that triggers inflammation. The aim of this study was to investigate whether there was an association between antiendothelial cell antibody positivity and panel reactive antibody positivity in renal transplant patients.

Materials and Methods: In this study, we investigated the association between anti­endothelial cell antibodies and panel reactive antibody Class I class II crossmatch positivity in patients, and compared these results with results from 100 healthy volunteers. All serum samples were analyzed by bead-based technology for calculated panel reactive antibody positivity; in addition, slides were used, each containing human umbilical vein endothelial cells and capillary-rich tissue for antiendothelial cell antibody positivity.

Results: Antiendothelial cell antibodies was positive in 48 of 89 patients (panel reactive antibody Class I class II negative), 22 of 35 patients (class I-positive), 25 of 39 patients (class II-positive), 26 of 40 (class I- class II positive), and 37 of 57 serologic and flow cytometry crossmatch-positive patients (P ≤ .016), and ultimately, in 122 of 205 patients and 25 of 100 volunteers (P ≤ .001). Antiendothelial cell antibody positivity was more frequent in panel reactive antibody-positive than negative patients and the control group.

Conclusions: Binding of antiendothelial cell antibodies to endothelial cells may activate complement by the classical pathway and cause up-regulation of adhesion molecules. This study questioned the antigenic specificity of anti­endothelial cell antibodies. Our study results showed that antiendothelial cell antibodies may play an important role for graft destruction, independent of panel reactive antibody and crossmatch positivity.

Key words : End-stage renal disease, Human leukocyte antigen, Non-HLA antibody

Introduction

Antibodies against the endothelium can damage the graft cells via a complement-dependent mechanism and/or stimulation of a proinflammatory and proliferation signaling mechanism. Endothelium expresses both human leukocyte antigen (HLA) and non-HLA antigens. Antiendothelial cell antibodies (AECAs) are a family of non-HLA antibodies that target the endothelial cells via antigenic determinants on the cell membrane. These antibodies are mainly immunoglobulin G (IgG) antibodies binding through the Fab domain. The AECAs effects are well-known in autoimmune diseases and some inflammatory processes. They might contribute to the pathogenesis of systemic vasculitis-associated diseases by acti­vation of endothelial cells, direct cytotoxic effects due to complement-dependent cytotoxicity, indirect cytotoxic effects secondary to antibody-dependent cytotoxicity, induction of coagulation, induction of apoptosis through the binding of phospholipids or heat shock protein, or induction of endothelial cell activation.¹ The targeted antigens of AECA are a large group including matrix proteins and extracellular molecules.^2,3 The AECAs induce the expression of adhesion molecules, E-selectin, intracellular adhesion molecule 1, vascular cell adhesion molecule 1, some interleukins including interleukin 1, interleukin 6, and interleukin 8, and monocyte chemoattractant protein^5,14. Panel reactive antibody (PRA) is defined as the percentage of HLA antigens of a panel reacting with a patient’s serum. The presence of anti-HLA antibodies are at high risk for acute and chronic antibody mediated rejection.⁶ Studies have shown that a substantial effect of antibody ligation of class I molecules on the surface of endothelial cells is the induction of proliferation.⁷ Anti-HLA class II antibodies play an important role for chronic rejection by stimulating endothelial cell proliferation. The aim of this study was to retrospectively investigate whether there was an association between AECA positivity and PRA positivity in renal transplant patients.

Materials and Methods

Subjects and assays
In this retrospective study, we investigated the association between AECA and PRA class I and class II positivity in patients. For this purpose, we compared the results from 205 patients and 100 healthy volunteers (previous AECA study results). All samples collected from patients were stored at -20°C until testing. Samples were analyzed by bead-based technology, with screening and identification for class I and II and single antigen for donor-specific antibodies, for calculated PRA positivity. For AECA positivity, each sample was diluted 1:10 in phosphate-buffered saline (PBS) and polysorbate (Tween, Sigma-Aldrich, St. Louis, MO, USA). We used slides containing biochips coated with frozen sections of human umbilical vein endothelial cells (HUVEC) and capillary-rich tissue such as skeletal muscle (Euroimmun, FB 1960-1005-2, Lübeck, Germany). The test specificity for HUVEC was 100% and monkey skeletal muscle was 100%; the sensitivity for HUVEC was 100% and monkey skeletal muscle was 97%. Frozen sections of primate skeletal muscle and cultivated HUVEC covering the reaction areas of the biochip slides were incubated with a dilute serum sample. A reaction was considered positive when specific antibodies (IgG) attached to the antigens. The bound antibodies were stained with fluorescein-labeled antihuman antibodies and visualized by fluorescence microscopy (Figure 1). Results were determined qualitatively and quantitatively.

Statistical analyses
Statistical analysis was performed using a statistical package (SPSS, Version 17.0, SPSS Inc., Armonk, NY, USA). Data were summarized according to the frequency distribution. The categorical variables between groups were analyzed with chi-square test or Fisher exact test. Values of P ≤ .05 were considered statistically significant.

Results

The results showed that the PRA-positive (PRA ≥ 60%) patient group has increased AECA levels. The AECA test was positive in 48 of 89 patients (PRA class I class II negative; P = 1.00), 22 of 35 patients (PRA class I-positive; P = .047), 25 of 39 patients (PRA class II-positive; P = .005), 26 of 40 patients (PRA class I class II positive, P = .0001), and 37 of 57 of the serologic and flow cytometry (FCM) crossmatch-positive patients (P = .016). The AECA test was positive in 122 of 205 patients and 25 of 100 volunteers (P < .001) (Table 1). The AECA positivity was more frequent in PRA-positive patients than the PRA-negative patient and control groups and independent from PRA mean fluorescence intensity (MFI) values. The PRA positivity was more frequent in female than male patients (Figure 2). No significant correlation was found between MFI value or donor-specific antibodies and occurrence of AECA.

Discussion

In our study, we showed that PRA-positive renal transplant patients had increased levels of AECA. The AECA recognized constitutively expressed or cytokine induced antigens such as β2-glycoprotein I, platelet factor 4, DNA, and membrane or cyto­skeleton proteins such as tubulin, vimentin, laminin, vinculin, and annexin V.⁸ Multiple pathogenic roles of AECA have been elicited in diseases that involve the vascular system such as vasculitis and atherosclerosis.⁹ Previous studies showed that the development of AECAs was significantly associated with cytomegalovirus infection and especially associated with the level of cytomegalovirus antigenemia positivity.^10,11 The AECAs may trigger an inflammatory process and induce endothelial cell apoptosis and necrosis.¹² The AECAs are detected in a wide range of clinical pathologies that involve the vascular system, including autoimmune disease, infectious disease, and vasculopathies, but they also can be observed in healthy individuals.^13,14 Alloimmune-mediated injury of the endothelium leads to the aberrant expression of self proteins and subsequent generation of autoantibodies. It is accepted that blood group antigens and HLA class I and II are not targets for AECA-associated autoimmune disease.¹⁵ The AECAs may be joined to destructive potential of PRA on graft endothelial cells.

In conclusion, alloimmune-mediated injury of the endothelium leads to the aberrant expression of self proteins and subsequent generation of auto-antibodies. Occurrence of AECAs is not significantly associated with acute vascular rejection alone, but may be related to PRA positivity and/or may aggravate the inflammatory process together.

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