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Background Antibodies against HLA antigens cause refractory allograft rejection with vasculopathy in some, but not all, patients.
Methods We studied 33 kidney-transplant recipients who had refractory vascular rejection. Thirteen had donor-specific anti-HLA antibodies, whereas 20 did not. Malignant hypertension was present in 16 of the patients without anti-HLA antibodies, 4 of whom had seizures. The remaining 17 patients had no malignant hypertension. We hypothesized that activating antibodies targeting the angiotensin II type 1 (AT1) receptor might be involved.
Results Activating IgG antibodies targeting the AT1 receptor were detected in serum from all 16 patients with malignant hypertension and without anti-HLA antibodies, but in no other patients. These receptor-activating antibodies are subclass IgG1 and IgG3 antibodies that bind to two different epitopes on the second extracellular loop of the AT1 receptor. Tissue factor expression was increased in renal-biopsy specimens from patients with these antibodies. In vitro stimulation of vascular cells with an AT1-receptor–activating antibody induced phosphorylation of ERK 1/2 kinase and increased the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor-
Conclusions A non-HLA, AT1-receptor–mediated pathway may contribute to refractory vascular rejection, and affected patients might benefit from removal of AT1-receptor antibodies or from pharmacologic blockade of AT1 receptors.
B. The AT1 antagonist losartan blocked agonistic AT1-receptor antibody–mediated effects, and passive antibody transfer induced vasculopathy and hypertension in a rat kidney-transplantation model.
We studied kidney-transplant recipients who had severe allograft dysfunction but did not have anti-HLA antibodies; in these patients, rejection was invariably accompanied by accelerated hypertension, and even convulsions, in a manner reminiscent of preeclampsia. In fact, the first patient we examined in this group (the index patient) had had preeclampsia 16 years earlier. Immunologic similarities between graft rejection and fetal survival in pregnancies complicated by preeclampsia are well recognized.6 We previously reported that agonistic antibodies that target the angiotensin II type 1 (AT1) receptor may develop in women with preeclampsia after the 20th week of gestation,7 and we observed agonistic AT1-receptor activity in nine kidney-transplant recipients during an episode of rejection.8 We then reasoned that similar mechanisms might be present in refractory allograft rejection and initiated a comprehensive investigation of this issue.
Methods
Study Patients and Biopsies
We studied all patients with steroid-refractory acute renal-allograft rejection seen among those who received kidney transplants at the Charité University Hospital Campus Mitte in Berlin from January 1, 2000, through July 31, 2004. Serum samples were obtained prospectively from all patients during rejection episodes and were screened for donor-specific anti-HLA antibodies and also analyzed for agonistic antibodies targeting the AT1 receptor. Serum samples from patients with steroid-refractory acute vascular rejection from three collaborating centers (the Charité University Hospital Campus Virchow Clinic, and the University Hospital Erlangen–Nürnberg, both in Germany, and the Ospedale Civile in Bergamo, Italy) were also examined. Written informed consent to use serum samples for research purposes was obtained from each patient while he or she was awaiting transplantation. The institutional review board of Charité University Hospital Berlin approved the protocols.
Allograft-biopsy specimens were processed by standard techniques and graded according to the Banff 97 classification, with updated scoring of the degree of humoral rejection.9,10 Biopsy specimens were stained with polyclonal antibodies for C4d (Biomedica)11,12 and tissue factor (provided by T. Luther, Dresden).
Initial immunosuppressive therapy consisted of a calcineurin inhibitor, mycophenolate mofetil, methylprednisolone, and antibody against interleukin-2 receptor for induction. Patients who were positive for donor-specific anti-HLA antibodies or C4d or who had vascular rejection manifested as endarteritis or necrotizing vasculitis with fibrinoid necrosis were treated with plasmapheresis together with intravenous immune globulin. Patients initially treated with cyclosporine were switched to high-dose tacrolimus when refractory rejection was detected.
Bioassays and Analysis of Epitopes
Patients' serum samples were examined for the presence of AT1-receptor–activating IgG in a functional bioassay that involves spontaneously beating cultured neonatal-rat cardiomyocytes that express several G-protein–coupled receptors, including the AT1 receptor. Changes in the activation state of the cells is measured directly by counting the beating rate by means of computerized image analysis. Isolation, cultivation, and detection procedures have been described in detail previously.13 The dose–response relationship between activating IgG or angiotensin II concentration and the spontaneous beating rate is linear.
To study the specificity of the AT1 receptor–mediated response, IgG-stimulated cells were treated with an AT1-receptor blocker or angiotensin II type 2 (AT2)–receptor blocker. Short, overlapping synthetic peptides corresponding to the sequence of the second extracellular loop of the AT1 receptor were used for identification of epitopes. To identify the IgG subclass, IgG from 10 patients from whom sufficient serum was available were treated with murine monoclonal antihuman IgG1, IgG2, IgG3, and IgG4 antibodies.
Surface-Plasmon-Resonance Analysis
Binding of AT1-receptor antibody was verified and quantified by surface-plasmon-resonance analysis (BIAcore). The IgG fraction from affected patients or control human IgG was allowed to pass over biotinylated peptides loaded on a streptavidin-immobilized chip in a biosensor (BIAcore) at different flow rates. After the association phase and the dissociation phase, the relative increase in response of AT1-receptor peptide and control peptide (
2-adrenergic receptor) flow channels was determined. The kinetic variables were calculated with BIA evaluation software (Biosensor, version 3.1).
Assays
Human coronary-artery endothelial cells and vascular smooth-muscle cells were stimulated with AT1-receptor antibodies in the presence or absence of AT1-receptor blocker or AT2-receptor blocker. Protein extraction, sodium dodecyl sulfate–poly-acrylamide-gel electrophoresis, and membrane treatment were performed as described by Dechend et al.14 Primary antibodies against polyclonal extracellular signal–regulated kinase (ERK) 1/2 and phosphorylated ERK 1/2 were used. Triplicate experiments with six patients' IgG preparations were performed. Electromobility-shift assays in vascular smooth-muscle cells stimulated with angiotensin II, AT1-receptor antibodies, or control IgG were performed as described previously.14
Animal Model of Kidney Transplantation
The low-responder Fischer 344-to-Lewis life supporting rat kidney-transplantation model was used to test the physiological effects of various antibodies. AT1-receptor antibodies (a pool derived from two donors), control IgG, or vehicle was continuously infused into recipient rats by means of intraperitoneal, seven-day-release, osmotic minipumps (Alzet, Charles River Laboratories). After implantation of the minipumps, one kidney was transplanted into each rat (cold-ischemia time was two hours), and kidneys were harvested after seven days (n=4 per treatment group).15 For continuous blood-pressure monitoring, telemetric transmitters were placed into the abdominal aortas of six additional rodent recipients, before transplantation. The histologic features of the rat allografts were analyzed after harvesting with standard techniques. Direct immunofluorescence with fluorescein isothiocyanate–labeled antihuman IgG (Dako) was used to determine intragraft binding of AT1-receptor antibodies; cryosections were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (Vector).
Statistical Analysis
Continuous data are presented as medians (with ranges). We performed comparisons between groups with use of Fisher's exact test for categorical values and the Mann–Whitney U test for continuous variables.
Results
Thirty-three patients with steroid-refractory rejection were identified (23 at Charité University Hospital Campus Mitte and 10 at collaborating centers). Thirteen had detectable donor-specific anti-HLA antibodies. Sixteen patients who did not have donor-specific anti-HLA antibodies had malignant hypertension, followed by convulsions in four. Four patients had neither donor-specific anti-HLA antibodies nor malignant hypertension. Figure 1 shows representative biopsy specimens from patients with malignant hypertension and refractory rejection who had no donor-specific anti-HLA antibodies; these showed either endarteritis (Figure 1A) or fibrinoid necrosis (Figure 1B). Patients whose biopsy specimens had evidence of fibrinoid necrosis underwent magnetic resonance angiography studies that showed multiple perfusion defects consistent with cortical infarctions (Figure 1C). Subsequently, patients with anti-HLA antibodies had substantially better graft survival than did those who had malignant hypertension but did not have anti-HLA antibodies (Figure 1D).
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Specific angiotensin II–receptor antagonists were used to test whether the agonistic response to patients' IgG was specific for the AT1 receptor. Losartan, but not the AT2-receptor antagonist PD 123319, abolished the functional bioassay response (Figure 2B). Angiotensin II and IgG from patients achieved results in the bioassay. Thus, IgG fractions of our patients' serum contained AT1-receptor antibodies.
The demographic data from the 16 patients who were positive for AT1-receptor antibodies were compared with those for the 13 patients with donor-specific anti-HLA-antibodies. As shown in Table 1, patients with anti-HLA antibodies were more likely to have staining for C4d in their renal-biopsy specimens and had less rapid allograft loss. Otherwise, there were no significant differences in clinical or demographic characteristics between the two groups.
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In comparison, rejections in association with AT1-receptor antibodies had a similar incidence (10 episodes, accounting for 43.5 percent of steroid-refractory rejection episodes and 8.4 percent of all rejection episodes). The remaining four steroid-refractory rejection episodes at Charité Campus Mitte (accounting for 17.4 percent of steroid-refractory episodes and 3.4 percent of all rejection episodes) occurred in patients who had neither AT1-receptor antibodies nor donor-specific anti-HLA antibodies. The crude prevalence of the rejection episodes associated with AT1-receptor antibodies (10 among 278 kidney transplantations performed) during four years was 3.6 percent.
IgG fractions possessing agonistic activity were used to examine possible epitopes on the second extracellular loop of the AT1 receptor, to which the antibodies would bind. Two amino acid sequences, AFHYESQ and ENTNIT, inhibited the agonistic activity of IgG obtained from the patients (Figure 2C), indicating that these sites contain the IgG-binding epitopes for the antibody. Neutralization experiments in which monoclonal antihuman IgG1, IgG2, IgG3, and IgG4 antibodies were added to AT1-receptor antibodies demonstrated that antibodies of the IgG1 and IgG3 subclass are responsible for the agonist-like effect of these antibodies (Figure 2D).
The specificity of the bioassay findings was assessed with surface-plasmon-resonance analysis, which confirmed binding of AT1-receptor antibodies to the peptide of the second extracellular loop of the AT1 receptor. The BIA evaluation program used to analyze the results calculated the dissociation rate constant at 1.05±0.29x10–3 per second and the pseudomolecular association rate constant at 5.02±1.40x10–3 per second, revealing a high binding affinity of AT1-receptor antibodies to the AT1 receptor.
Thus, IgG fractions from patients contained AT1-receptor antibodies of the IgG1 and IgG3 subclasses that bound to the second extracellular loop of the AT1 receptor. Further studies were performed, on the basis of our hypothesis that these antibodies played a role in the pathogenesis of refractory vascular rejection. Vascular lesions in grafts that are rejected because of the presence of anti-HLA antibodies are generally attributed to complement activation. However, patients with AT1-receptor antibodies did not have complement-fixing cytotoxic anti-HLA antibodies in their serum, and their renal-biopsy specimens generally did not show staining for C4d. AT1-receptor antibodies were examined to see whether they could initiate signaling mediated by ERK 1/2 in vitro by means of endothelial cells and vascular smooth-muscle cells (Figure 3A and Figure 3B). AT1-receptor antibody induced phosphorylation of ERK 1/2 (Figure 3A and Figure 3B), which peaked at 10 minutes without a change in total ERK 1/2 in the cells.
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B (NF-B), a transcription factor pivotal in initiating immune responses and inflammatory disease,16 when studied in nuclear extracts from vascular smooth-muscle cells, was activated by AT1-receptor antibodies, but not by IgG from control patients (Figure 3D).
Tissue factor, which initiates the extrinsic coagulation pathway, is a target gene for AP-1 and NF-B. Biopsy specimens obtained from patients during episodes of rejection mediated by AT1-receptor antibodies revealed intense staining of tubular cells, inflammatory infiltrate, and peritubular capillaries (Figure 4A). In contrast, only weakly positive staining was seen in biopsy specimens obtained after patients had been treated with losartan and plasmapheresis (Figure 4B). Treatment consisting of plasmapheresis, intravenous immune globulin, and 100 mg of losartan daily in seven patients with AT1-receptor antibodies resulted in significantly improved allograft survival, as compared with that in patients receiving standard antirejection treatment (Figure 4C). Serum from four patients with the longest rejection-free follow-up (who are still receiving losartan) became negative for AT1-receptor antibodies (Figure 4D).
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Patients with refractory vascular allograft rejection and accelerated hypertension but without anti-HLA antibodies were found to have pathogenic antibodies directed at two epitopes of the second extracellular loop of the AT1 receptor in their serum. AT1-receptor antibodies induced phosphorylation of ERK 1/2, AP-1 activation, and NF-B activation in vascular cells. An AT1-receptor antagonist blocked the effects mediated by AT1-receptor antibodies. Passive infusion of AT1-receptor antibodies in a rat renal-transplantation model induced vascular changes and increased blood pressure. These data suggest that AT1-receptor antibodies may be directly involved in the pathogenesis of vascular rejection.
Earlier we had observed AT1-receptor activity during steroid-refractory, C4d-negative renal-allograft rejection in nine patients with hypertension, but not in eight patients with steroid-sensitive cellular rejection. In the present study, we increased the number of patients studied and clearly defined the pretransplantation activity of IgG1- and IgG3-subclass antibodies that bound to AT1-receptor epitopes and induced signaling cascades. The clinical presentation of transplant recipients with AT1-receptor antibodies appears similar to that of transplant recipients with anti-HLA antibodies, but with significantly worse allograft survival.
Similar agonistic antibodies against the AT1 receptor have been reported in patients with preeclampsia.7 Recently, other investigators confirmed the existence of AT1-receptor antibodies in patients with preeclampsia17 and also demonstrated that the antibodies induced mobilization of intracellular calcium and activation of a transcription factor, nuclear factor of activated T cells, in AT1-receptor–transfected Chinese-hamster-ovary cells.17 The index patient in the present report had had preeclampsia many years earlier. Nevertheless, the antibodies in this patient and the other transplant recipients bound to an epitope that did not entirely coincide with the antibodies described earlier in the patients with preeclampsia.7
Antibodies to G-protein receptors have been identified in some patients with idiopathic dilated cardiomyopathy18 or malignant hypertension.19 In Graves' disease, autoantibodies activate the thyrotropin G-protein receptor, increasing target-organ activity.20 Anti–endothelial-cell antibodies are non-HLA antibodies that may be related to the pathogenesis of allograft rejection.21,22 However, research on anti–endothelial-cell antibodies is hampered by the heterogeneity of endothelial cells in various vascular beds, a fact that makes identifying a standard detection method difficult.23 Moreover, initially detected target antigens of anti–endothelial-cell antibodies were of unclear relevance.23
We suggest that AT1-receptor antibodies have similarities to anti–endothelial-cell antibodies since endothelial cells have one AT1 receptor,24 and AT1-receptor antibodies induced phosphorylation of ERK 1/2 in endothelial cells. We further suggest that binding of AT1-receptor antibodies to the AT1 receptor is a critical step for activating the downstream signaling cascade, mimicking the action of angiotensin II and inducing damage to the allograft. Emerging information has established that angiotensin II acts as an inflammatory cytokine participating in various vascular disorders.25
AT1-receptor antibodies may induce inflammatory responses and contribute to allograft rejection by means of activation of NF-B target genes. Blockade of NF-B activity with decoy oligodeoxynucleotides reduced tubulointerstitial infiltration in rat renal allografts.26 Expression of tissue factor as regulated by NF-B and AP-1 may increase procoagulatory activity of the injured vessels.
We previously reported NF-B and AP-1 subunit expression in biopsy specimens from several kidney-transplant recipients.8 We now report that biopsy specimens of patients with rejection associated with AT1-receptor antibodies had evidence of increased tissue factor expression and secondary thrombotic occlusions and that tissue factor was reduced after losartan treatment. It has previously been recognized that the coagulation system has a role in the pathogenesis of rejection.27 An anticardiolipin antibody–mediated increase in tissue factor expression contributes to thrombosis in patients with the antiphospholipid syndrome.28 In rat cardiac allografts, tissue factor plays a critical role in clotting abnormalities and transplant arteriosclerosis.29 Lesions in allografts in rats infused with AT1-receptor antibodies resemble those observed in kidney-transplant recipients.
It is unknown whether AT1-receptor antibodies function only through proinflammatory and procoagulatory activity or also by means of specific immune responses. Experimental studies have implicated the renin–angiotensin system in the regulation of the specific immune response and immune-mediated renal injury.30,31 Since AT1 receptors are present on human mononuclear cells,32,33 an effect of AT1-receptor antibodies on T lymphocytes, monocytes, and dendritic cells appears to be likely. Furthermore, rejection in association with circulating anti-HLA antibodies is commonly characterized by a high incidence of severe vascular lesions with fibrinoid necrosis, whereas endarteritis and mononuclear cell tubulitis predominate in T-cell–mediated rejection.34
We first observed endarteritis and later fibrinoid necrosis, tubulitis, and interstitial-cell infiltration in patients' biopsy specimens. These characteristics may define a distinct type of kidney-transplant rejection mediated by AT1-receptor antibodies. The incidence of rejection among patients positive for AT1-receptor antibodies in this study is similar to that of rejection due to donor-specific anti-HLA antibodies. However, it remains unclear why AT1-receptor antibodies develop in certain patients and why these antibodies target the allograft.
Molecular mimicry may be important; cross-reactivity with microbial antigens has an important role in other pathologic processes associated with antibodies directed against G-protein receptors — for example, myasthenia gravis or the dilated cardiomyopathy seen in Chagas' disease.35,36 We have not tested the possibility that the AT1 receptors in patients in whom AT1-receptor antibodies develop might harbor specific polymorphisms.37 We speculate that post-transplantation reperfusion injury may alter the intragraft expression of AT1 receptor, change its density, or cause conformational changes. A permissive environmental phenomenon might enhance local intragraft immunoreactivity owing to an activated innate immune response.
Transplantation nephrologists have been reluctant to use angiotensin II blockers because of possible decreases in organ perfusion and filtration. However, this concern may be outweighed by the potential advantages of these drugs.38 The seven patients discussed here who were treated with losartan, plasmapheresis, and intravenous immunoglobulin had amelioration of the antibody-mediated rejection process and remain free of rejection while receiving losartan. However, their treatment was neither randomly assigned nor blinded, and the numbers of patients are too small to permit us to draw firm conclusions. We speculate that detection of AT1-receptor antibodies in patients on a waiting list for a transplant might identify those at risk for refractory rejection.
Dr. Fritsche reports having served as a consultant to Novartis, Hexal, Shire, and GDL, being a stockholder in Novartis, receiving grant support from Novartis and Wyeth, and receiving lecture fees from Hoffmann–La Roche. Dr. Kintscher reports having served on the advisory boards of Novartis and Takeda, receiving grant support from Boehringer Ingelheim, and receiving lecture fees from Takeda. Dr. Unger reports serving as a consultant to Novartis, Abbott, Takeda, and Merck and having received grant support from Takeda, Novartis, Bayer, and Boehringer Ingelheim. Dr. Neumayer reports having received consulting fees from Novartis and Shire and lecture fees from Novartis and Wyeth. Dr. Luft reports having served as a consultant and receiving grant support from Novartis, Boehringer Ingelheim, and CVRx.
We are indebted to Professor U. Frei of Humboldt University in Berlin, Professor G. Remuzzi and Dr. N. Perico of the Mario Negri Institute in Bergamo, Italy, and Professor K. Amann and Dr. C. Hugo of the University of Nürnberg–Erlangen for providing samples.
Source Information
From the Departments of Nephrology (D.D., L.F., M.N.-K., D.E., K.B., H.-H.N.) and Pathology (B.R.), and the Center for Cardiovascular Research (D.D., M.N.-K., U.K., T.U.), Charité University Hospital; the Franz Volhard Clinic and HELIOS Klinikum (J.H.B., R.D., I.M., F.C.L.); the Max Delbrück Center for Molecular Medicine (D.N.M., R.P., G.W.); and the HLA Laboratory (C.S.) — all in Berlin; and the Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France (J.H.).
Address reprint requests to Dr. Dragun at the Department of Nephrology and Transplantation, Charité University Hospital, Campus Mitte, Schumannstr. 20–21, 10117 Berlin, Germany, or at duska.dragun{at}charite.de.
References
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B decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts. FASEB J 2000;14:815-822. -subunit that has biologic activity and reacts immunochemically with herpes simplex virus. J Clin Invest 1989;84:1174-1180. [Web of Science][Medline]
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Related Letters:
Angiotensin II Type 1–Receptor Activating Antibodies in Renal-Allograft Rejection
Hilbrands L., Hoitsma A., Wetzels J., Ansari M. J., Tinckam K., Chandraker A., Dragun D., Fritsche L., Luft F. C.
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N Engl J Med 2005;
352:2027-2028, May 12, 2005.
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