Antibody-mediated rejection

The introduction of both complement 4d (C4d) staining in renal allograft biopsies and sensitive methods to detect anti-human leukocyte antigen antibodies, such as single antigen bead flow assays, into tissue-typing techniques have shown the importance of antibody-mediated alloimmune response in kidney transplantation. The use of these sensitive methods, combined with the increased number of transplants in highly sensitized patients with donor-specific antibodies, or patients receiving desensitization protocols, have increased the awareness and thus the incidence of acute antibody-mediated rejection. Chronic rejection also can be mediated through alloantibodies, and the term chronic antibody-mediated rejection recently was proposed. In this review article we summarize the current knowledge of the role of alloantibodies in transplantation, the diagnosis and treatment of acute and chronic antibody-mediated rejection, and their effect on graft function and outcome.     

Relationship of B cell alloantibodies to renal allograft survival.

To define the relationship of donor-specific B lymphocyte alloantibodies to renal allograft survival, longitudinal serum samples obtained pre- and post-transplantation were examined for antibodies cytotoxic to donor B lymphocytes. Ten of 17 renal allograft recipients had antibodies to donor B lymphocytes but not T lymphocytes either pre- and/or post-transplantation. Three patients underwent successful transplants despite preformed B cell antibodies; however, seven who developed B cell antibodies only after transplantation are either undergoing chronic rejection (4) or have had severe rejection crisis (3). Seven patients with no B cell antibodies have functioning grafts. In all cases, B cell antibodies were detected before biochemical and clinical evidence of rejection. Similar findings were noted when sera of 38 renal transplant recipients were examined for B cell antibodies cytotoxic to an unrelated panel of B lymphocytes. These results demonstrate that the development of B cell alloantibodies after transplantation is often associated with rejection and that successful renal transplantation can be performed across a positive B cell crossmatch.     

Platelets Influence Vascularized Organ Transplants from Start to Finish

This review relates the basic functions of platelets to specific aspects of organ allograft rejection. Platelet activation can occur in the donor or recipient before transplantation as well as during antibody- and cell-mediated rejection. Biopsies taken during organ procurement from cadaver donors have documented that activated platelets are attached to vascular endothelial cells or leukocytes. In addition, many patients waiting for transplants have activated platelets due to the diseases that lead to organ failure or as a result of interventions used to support patients before and during transplantation. The contribution of platelets to hyperacute rejection of both allografts and xenografts is well recognized. Intravascular aggregates of platelets can also be prominent in experimental and clinical transplants that undergo acute antibody or cell-mediated rejection. In acute rejection, platelets can recruit mononuclear cells by secretion of chemokines. After contact, monocytes, macrophages and T cells interact with platelets through receptor/ligand pairs, including P-selectin/PSGL-1 and CD40/CD154. There is a potential for therapy to inhibit platelet mediated immune stimulation, but it is counterbalanced by the need to maintain coagulation in the perioperative period.     

Clinical Significance of MHC-Reactive Alloantibodies that Develop after Kidney or Kidney–pancreas Transplantation

The purpose of this study was to determine the relationships between acute rejection, anti-major histocompatibility complex (MHC) class I and/or class II-reactive alloantibody production, and chronic rejection of renal allografts following kidney or simultaneous kidney-pancreas transplantation. Sera from 277 recipients were obtained pretransplant and between 1 month and 9.5 years post-transplant (mean 2.6years). The presence of anti-MHC class I and class II alloantibodies was determined by flow cytometry using beads coated with purified MHC molecules. Eighteen percent of recipients had MHC-reactive alloantibodies detected only after transplantation by this method. The majority of these patients produced alloantibodies directed at MHC class II only (68%). The incidence of anti-MHC class II, but not anti-MHC class I, alloantibodies detected post-transplant increased as the number of previous acute rejection episodes increased (p = 0.03). Multivariate analysis demonstrated that detection of MHC class II-reactive, but not MHC class I-reactive, alloantibodies post-transplant was a significant risk factor for chronic allograft rejection, independent of acute allograft rejection. We conclude that post-transplant detectable MHC class II-reactive alloantibodies and previous acute rejection episodes are independent risk factors for chronic allograft rejection. Implementing new therapeutic strategies to curtail post-transplant alloantibody production, and avoidance of acute rejection episodes, may improve long-term graft survival by reducing the incidence of chronic allograft rejection.     

In vivo platelet activation with in vitro hyperaggregability to arachidonic acid in renal allograft recipients

Renal allograft recipients were investigated to determine the extent and possible nature of in vivo platelet activation. In 92 allografted patients stable for more than 4 months' duration, intraplatelet serotonin in circulating platelets was depleted significantly. In a further 16 patients studied serially for 12 to 16 weeks following transplantation, intraplatelet serotonin fell abruptly within 4 days from transplantation to very low levels, and remained thus for 10 weeks, rising toward normal at about 12 weeks. Although some patients showed abrupt falls in intraplatelet serotonin coincident with acute rejection episodes, there was no difference in intraplatelet serotonin in seven patients whose grafts functioned well immediately and remained stable, and seven in whom repeated rejection led to graft loss within 3 months. Thus, these tests of platelet function do not permit diagnosis of rejection or prediction of graft outcome. Plasma platelet factor 4 (PF4) concentrations, in contrast, were normal in most patients during the first 6 weeks after grafting, then rose and remained abnormal up to 13 years following the allograft in the long-term stable graft recipients. This discrepancy suggests a different mode of platelet activation in the first few weeks after grafting from subsequent months. Despite universal depletion of intraplatelet amines and alpha-granule contents only four out of 14 early allograft recipients had an abnormal bleeding time, and platelet aggregation thresholds with adenosine-5'-diphosphate and collagen were not different from controls. However, thresholds for platelet aggregation with arachidonic acid were reduced significantly (P less than 0.01) and thromboxane B2 generation was increased in vitro. There was no correlation between depletion of intraplatelet serotonin and circulating platelet-agglutinating material, but nine of 17 biopsy specimens from rejecting allografts taken during the first 3 months showed extensive glomerular localization of platelet membrane antigens and PF4.     

Cytomegalovirus-related disease and risk of acute rejection in renal transplant recipients: a cohort study with case-control analyses

The relationship between a cytomegalovirus (CMV) infection and the acute rejection of a renal transplant is not well established. The aim of the study was to document whether the clinical presentation of a CMV infection as a diffuse inflammatory disease or as a clinically asymtomatic illness is a risk factor of acute renal transplant rejection. One hundred and ninety-two consecutive renal transplant recipients were included in a historical cohort study for exposed – non exposed analyses. CMV infection after transplantation was the exposure factor. Before transplantation, 113 patients had antibodies against CMV and 79 were seronegative. The patients were divided into three groups: Group 1 consisted of 64 patients who had neither clinical signs of CMV disease nor CMV serological changes after transplantation, Group 2 consisted of 77 seropositive patients with asymptomatic viremia, and Group 3 consisted of 51 seropositive patients with clinical signs of diffuse inflammation that included fever, neutropenia, and various visceral involvements (CMV disease). Groups 2 and 3, the seropositive patients, were paired with Group 1 patients. Acute rejection was considered as CMV-induced when it occurred within one month following viremia, during the first year after transplantation. Transplant patients with CMV disease, had a significant likelihood of developing acute rejection after CMV infection or reactivation (P < 0.01). The odds ratio for developing rejection was 5.98, 95 % confidence interval: 1.21–29.40. Such a link was not documented for recipients with asymptomatic CMV infection. In conclusion, CMV disease, but not asymptomatic viremia, is a risk factor of acute renal transplant rejection. On epidemiological grounds, these results support the hypothesis that factors controlling both the viral replication and the diffuse inflammatory process are implicated in acute graft rejection. Received: 20 July 1999 Revised: 22 February 2000 Accepted: 9 June 2000     

Vascular endothelial cell apoptosis induced by anti-donor non-MHC antibodies: a possible injury pathway contributing to chronic allograft rejection.

BACKGROUND: Non-major histocompatibility complex (non-MHC) alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. METHODS: The kinetics of alloantibody production and the mechanism by which non-MHC alloantibodies cause graft injury were investigated in a Lewis-to-Fischer 344 (LEW-to-F344) rat model of cardiac transplantation. RESULTS: Flow cytometry detected that all the F344 recipients of LEW allografts produced anti-donor immunoglobulin G (IgG) antibodies reactive with LEW lymphocytes and endothelial cells. A sub-group of recipients that rejected their grafts in 30 to 60 days exhibited markedly increased levels of anti-donor IgG antibodies (n = 6, mean fluorescence intensity [MFI]:23.85 +/- 2.7) than recipients with long-surviving allografts (n = 4, MFI:11.23 +/- 0.81; p = 0.00058). Passive transfer of anti-donor sera induced chronic rejection of LEW heart allografts in an immune non-responsiveness model of F344 rats induced by intrathymic inoculation of donor-specific lymphocytes. Immunoglobulin G antibodies purified from the anti-LEW sera exhibited complement-dependent cytotoxicity against LEW vascular endothelial cells in flow-cytometric cytotoxicity assay. The targeted endothelial cells displayed early (annexin V+) and late (TUNEL+) evidence for programmed cell death. Western blot analysis of poly (ADP-ribose) polymerase (PARP) demonstrated that the 25-kD PARP-cleavage fragment was present at the lysates of the vascular endothelial cells treated with anti-donor IgG antibodies, indicating apoptosis-associated caspase activity in these cells. In situ teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining demonstrated that vascular endothelial cell apoptosis was consistently present in all LEW heart allografts with chronic rejection. CONCLUSIONS: Non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury through an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.     

Pretransplant systemic inflammation and acute rejection after renal transplantation

BACKGROUND: There are presently no established pre-transplant tests that consistently identify patients who may be at increased risk for acute rejection episodes after renal transplantation. We studied whether pretransplant serum levels of C-reactive protein (CRP), a marker for the presence of systemic inflammation, would predict the occurrence of acute rejection episodes after renal transplantation. METHODS: Pretransplant serum was tested for CRP level in 97 consecutive renal transplant recipients. Time to acute rejection after transplantation was stratified by CRP level and compared using the Kaplan-Meier method. In addition, Cox regression multivariate analysis was performed to assess whether any pretransplant covariates could independently predict the subsequent occurrence of acute rejection episodes. RESULTS: Pretransplant mean CRP levels were higher in patients who subsequently had a rejection episode versus those who had no rejection (22.2+/-2.9 vs. 11.7+/-1.8 microg/ml, respectively, P=0.003). Patients less than the median CRP value had a significantly longer time to rejection compared to those with higher CRP levels (P=0.002). Similarly, patients within the lowest CRP quartile had longer times to rejection when compared with the highest quartile (P=0.006). Cox proportional hazards regression multivariate analysis identified CRP level as the only independent pretransplant risk factor for rejection identified (P=0.044). CONCLUSIONS: Pretransplant systemic inflammation as manifested by elevated serum CRP level independently predicts the risk of acute rejection after renal transplantation and may be useful in stratifying patients at the time of transplantation according to immunological risk. Thus, assessment of pretransplant systemic inflammatory status may be helpful in prospective individualization of immunosuppression therapy after renal transplantation.     

Vascular endothelial growth factor expression in human chronic renal allograft rejection

BACKGROUND: Chronic renal allograft rejection is characterized by interstitial fibrosis and vasculopathy. Vascular endothelial growth factor (VEGF) is an endothelial mitogen with increased expression in inflammation and vasculopathy. METHODS: Renal tissue from 17 patients with chronic rejection was examined for VEGF protein and the presence of CD 68-positive macrophages, and compared to biopsies from patients with temporary allograft dysfunction, acute rejection, and native kidneys with thin membrane disease. RESULTS: In the chronic rejection group, there was markedly increased expression of VEGF protein in the interstitium (P<0.0001). In serial sections, VEGF colocalized with the expression of CD 68-positive macrophages. Significantly more macrophages were in the tubulointerstitium in tissue with chronic rejection than in those with temporary allograft dysfunction (P<0.005). Additionally, VEGF protein expression in the glomeruli and the vascular compartment of patients with chronic rejection was increased. CONCLUSION: The up-regulation of VEGF in chronic renal allograft rejection may be important in inflammation and development of fibrosis.     

Increased incidence of renal allograft thrombosis under cyclosporine immunosuppression

Six of 325 patients undergoing renal transplantation under combined cyclosporine (CsA)-prednisone immunosuppression displayed renal artery thrombosis between 4 and 12 days after transplantation. All six patients had satisfactory initial revascularization, as ascertained by radionuclide scan and renal function. In none was the thrombosis considered to be secondary to rejection, either by clinical course or upon renal biopsy. Since there was no clear etiologic factor and since none of the overlapping 297 patients treated with azathioprine-prednisone displayed this complication, these cases appear to support the hypothesis that CsA alters intravascular hemostatic homeostasis. Data in experimental models are consistent with a predisposing factor to thrombosis, namely CsA reduces the synthesis of prostacyclin stimulating factors, leading to decreased prostacyclin production by vascular endothelial cells, and to failure to inhibit platelet aggregation.     

Peritubular Capillary Damage in Acute Humoral Rejection: An Ultrastructural Study on Human Renal Allografts

The ultrastructural features of peritubular capillary (PC) damage was studied in 12 kidney allografts with acute humoral rejection (AHR). AHR manifested in diffuse linear PC staining for C4d, and histology consistent with Banff grade III in 7 recipients and Banff grade II in 5. Allografts with acute tubular necrosis served as controls. First biopsies (post-transplantation day 16.2 +/- 2.2): The intra-capillary exudate comprised monocytes (59%), polymorphonuclears (14%), lymphocytes (12%) and not otherwise specified mononuclears (15%). Three patterns of focal PC endothelial injury were observed: lysis, an increased rate of apoptosis and fragmentation. No correlation was found between the respective damage types and the inflammatory cell types or the Banff grades. Controls revealed endothelial swelling, detachment from basement membrane and fragmentation. Follow-up biopsies: Monocytes transformed into macrophages intra-luminally. The reparative changes comprised endothelial cytoplasmic protrusions, binucleated endothelial cells and capillary sprouts. Early transplant capillaropathy and transplant glomerulopathy were noted in 2 recipients. Literature data indicate that lysis is mediated by anti-HLA alloantibodies; apoptosis, demonstrated first in the present study, may be induced by non-HLA-type anti-endothelial antibodies. Fragmentation is caused by ischemia. Ongoing endothelial injury leads to transplant capillaropathy and transplant glomerulopathy, the characteristic lesions of chronic rejection.     

Immunocytological determination of lymphocytes and monocytes/macrophages in urinary sediments of renal allograft recipients.

BACKGROUND: Urinary studies using Papanicolaou staining following kidney transplantation led to the conjecture that acute allograft rejection might be accompanied by an increased lymphocyturia. However, it is difficult to distinguish lymphoid cells from other urinary cells using conventional stains. METHODS: Staining of urinary lymphocytes using FITC-labelled antibodies is complicated by a high unspecific fluorescence that limits the evaluation. Therefore, we developed a method to stain urinary lymphocytes using enzyme-linked antibodies. The cells were cytocentrifuged onto microscope slides and were fixed. RESULTS: By means of a combined evaluation of Papanicolaou and immunocytochemical staining, CD3-positive pan T cells, CD4-positive T-helper cells, CD8-positive cytotoxic/suppressor cells, and CD14-positive monocytes/macrophages of urinary sediments were determined in 41 kidney graft recipients following renal transplantation. During periods of normal graft function, neither positive lymphocytes nor positive monocytes/macrophages were found in the urinary sediments. However, in the course of acute allograft rejection a significant increase in positive lymphocytes and positive monocytes/macrophages could be observed. Interestingly, in cases of acute allograft rejection the distribution of urinary lymphocytes and monocytes was comparable to the distribution of infiltrating immunocompetent cells in renal allograft biopsies. CONCLUSION: The present study demonstrates that immunocytochemical staining via enzyme-conjugated antibodies is a reliable method to visualize T lymphocytes and monocytes/macrophages in the urinary sediment, and that this technique may be of special diagnostic value in the diagnosis of acute allograft rejection.     

Platelet-mediated activation of endothelial cells: implications for the pathogenesis of transplant rejection

BACKGROUND: Platelets exert their normal functions at sites of endothelial disruption by plugging discontinuities in blood vessels and secreting products that promote thrombosis, inflammation, and the healing of wounds. Whether platelets might induce these changes in xenograft blood vessels, leading to development of acute vascular rejection, has been uncertain. METHODS: To examine the role of human platelets in modulation of xenograft endothelium, pig endothelial cells were treated with human platelets. RESULTS: Treatment of quiescent porcine endothelial cells with human platelets modulated the endothelial cells. Whereas resting human platelets caused little change in normal porcine endothelial cells, platelets activated with small amounts of thrombin induced striking changes in the endothelial cells, including the induction of tissue factor activity, the expression of E-selectin, and the secretion of endothelin-1. These changes were induced, at least in part, by interleukin-1 (IL-1) associated with the platelet surface and were modified by the secretion of transforming growth factor-beta (TGF-beta). CONCLUSION: These findings may explain how the activation of platelets at an early point in the rejection of vascularized organ xenografts or in chronic diseases might contribute to thrombotic, ischemic, and inflammatory changes characteristic of an organ xenograft undergoing rejection.     

Release of platelet-activating factor from rabbit heart perfused in vitro by sera with transplantation alloantibodies

During "hyperacute rejection" of rabbit heart perfused with transplantation alloantibodies, platelet activating factor (PAF) was released into the coronary effluent, which appeared to have physicochemical and functional properties similar to the 1-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (synthetic PAF) and to PAF obtained from IgE-sensitized rabbit basophils. The release of PAF was associated with an early tachycardia, followed by increasing bradycardia and conduction arrhythmias, as well as decrease of coronary flow and of amplitude of electrogram. The heart stopped beating within 30 min. The release of PAF as well as the "rejection" required the presence of fresh rabbit serum as a source of complement. The PAF receptor antagonist SRI 63-072 in a dose of 0.6 mg, reversed by 70% the reduction of coronary flow within 2-4 min after its addition to the perfusate; ED50 was 0.4 mg. Bradycardia and arrhythmia were reduced; however, the normal electrical activity was only occasionally restored. The cessation of heart action was delayed up to 50 min after the beginning of perfusion with transplantation alloantibodies and complement, but it was not prevented. These results suggest that PAF is released during "rejection" of the heart perfused in vitro with serum containing transplantation alloantibodies in the absence of inflammatory cells and that this mediator is at least in part responsible for the deterioration of cardiac function.     

De novo anti-HLA antibodies in renal allograft recipients: a cross-section study

Background

The occurrence of anti-HLA antibodies plays a well established role in solid organ rejection. The development of x-MAP multiple bead technology (Luminex) has enabled more accurate detection and definition of these alloantibodies.

Methods

In 267 kidney transplant patients with stable allograft function for ≥3 years, we analyzed the presence of anti-HLA antibodies by Luminex technology. These patients had no alloantibodies before transplantation, and the immunosuppression treatment was: tacrolimus, cyclosporine, mycophenolate mofetil, prednisone, everolimus, and/or sirolimus.

Results

Fifteen of the 267 patients showed anti-HLA class I antibodies and 12 showed anti-HLA class II antibodies, Seven patients had donor-specific antibodies (DSA): 1 anti-HLA class I, 5 anti-HLA class II, and 1 with both classes. No differences were found between DSA and the use or not of any specific therapy. However, in the retrospective review, we found a higher incidence of acute rejection episodes in the immediate posttransplant period among patients who developed class II DSA than those without DSA.

Conclusions

The prevalence of patients with normal renal function who develop DSA beyond 3 years after transplantation was relatively low. Steroid or withdrawal replacement of calcineurin inhibitors with inhibitors of mammalian target of rapamycin seem to not be risk factors to increase the development of DSA. The finding that patients who developed DSA showed a higher rate of previous acute rejection episodes suggested that they should be monitored more frequently for HLA antibodies.     

Platelets an Inflammatory Force in Transplantation

Platelet interactions with dendritic cells, T cells and B cells have been best studied in vasculitis and atherosclerosis, but similar mechanisms may contribute to acute and chronic vascular lesions in transplants. In acute inflammation, platelets adhere to vessels and release mediators that increase endothelial cell activation and leukocyte recruitment. Adherent platelets can also augment antibody and cellular immune responses. Activated platelets recruit T cells and initiate a feedback loop. In this loop, platelets secrete chemokines to recruit T cells, and then activated T cells stimulate platelets through CD40-CD154 interactions to secrete more chemokines thereby recruiting more T cells. The interaction of platelets and T cells is enhanced by P-selectin/PSGL-1 stimulation. Both helper and cytotoxic T cells are stimulated by platelets. Antibody production that is stimulated through increased helper T-cell function can activate complement. This sets up another activation loop because platelets express receptors for antibodies and complement. In addition to inflammation, platelets stimulate repair by releasing growth factors and chemokines to recruit circulating vascular progenitor cells. These repair mechanisms could promote the replacement of donor parenchmal cells with recipient cells and contribute to vascuplopathy. This review discusses the interplay of platelets and the immune system in relation to transplantation.     

Acute transplant glomerulopathy with monocyte rich infiltrate

Acute transplant glomerulopathy refers to alloimmune mediated endothelial injury and glomerular inflammation that typically occurs early post-kidney transplantation. We report a case of a 48-year old woman with end stage renal disease from lupus nephritis who developed an unexplained rise in serum creatinine 2 months after renal transplant. As immunosuppression, she received alemtuzumab induction followed by a tacrolimus, mycophenolate mofetil and prednisone maintenance regimen. Her biopsy revealed severe glomerular endothelial injury associated with monocyte/macrophage-rich infiltrate in addition to mild acute tubulointerstitial cellular rejection. We briefly discuss acute transplant glomerulitis, its pathology and association with chronic/overt transplant glomerulopathy, C4d negative antibody-mediated rejection and the significance of monocytes in rejection. We also postulate that alemtuzumab induction may have contributed to the unusual pattern of monocyte-rich transplant glomerulitis.     

Uterus transplantation: Histological findings in explants at elective hysterectomy

Uterus transplantation has enabled women with absolute uterine factor infertility to carry a pregnancy. The first human uterus transplantation trial was initiated in 2013 in Gothenburg, Sweden. It was completed with 7 transplantations with long-term allograft survival and 9 children born from 6 women. In the present study we describe the histopathology of these 7 allografts, which were removed at 22-83 months after transplantation, and compare findings to control cases. Morphological findings in a subset of explants included linear subepithelial inflammation and perivascular stromal inflammation in the cervix, small inflammatory foci in the myometrium, and intimal inflammation in larger arteries. The average number of T cells, B cells, and macrophages was higher in transplants compared to normal controls, but variability was high among transplants. Chronic-active vascular rejection was seen in 2 of 7 transplants, both showed also inflammation in the cervix. Further, the inflammation seen in the cervix reflected the inflammation in the myometrium, suggesting that cervical biopsies are suitable to monitor rejection. However, the degree of inflammation and signs of rejection in explants did not reflect on the possibility to become pregnant in this limited series.