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Agonistic antibodies directed at cell surface receptors and cardiovascular disease
Journal of the American Society of Hypertension 2(1) (2008) 8 –14
Review Article
Agonistic antibodies directed at cell surface receptors and cardiovascular disease Friedrich C. Luft, MD*, Ralf Dechend, MD, Duska Dragun, MD, Dominik N. Müller, PhD, and Gerd Wallukat, PhD Experimental and Clinical Research Center, Medical Faculty of the Charité, Max-Delbrück Center for Molecular Medicine, HELIOSKlinikum, Berlin, Germany Manuscript received June 20, 2007 and accepted August 20, 2007
Abstract Antibodies directed at receptors can block or stimulate them. Hallmark example of the latter action is Graves’ disease where antibodies directed at the thyroid-stimulating hormone receptor exert an agonistic action. Recently, compelling evidence has been presented regarding agonistic antibodies directed against the alpha-adrenergic receptor, the beta-adrenergic receptors, the angiotensin II AT1 receptor, and the platelet-derived growth factor-alpha receptor. The antibodies could play a pathogenic role in various cardiovascular diseases, including hypertension, cardiomyopathy, pre-eclampsia, acute humoral rejection, and connective tissue disease. The mechanisms that result in the production of these antibodies are unclear, automated assays to determine their presence are beset with technical difficulties, and the therapeutic implications are uncertain. Nevertheless, the signaling phenomena resulting from these antibodies are well established and mechanistic studies are being intensively pursued. The discovery of agonistic antibodies may provide additional therapeutic avenues. © 2008 American Society of Hypertension. All rights reserved. Keywords : Adrenergic receptors; AT1 receptor; antibodies; angiotensin.
Introduction Agonistic antibodies that stimulate G protein-coupled receptors are accepted as causing diseases of the thyroid gland. Agonistic antithyrotropin receptor antibodies (TSAb) that mimic the action of thyrotropin (TSH) characterize Graves’ disease. Nonetheless, the immunological mechanisms of TSAb production remain obscure. Recently, KimSaijo et al produced a transgenic mouse that expresses a patient-derived TSAb.1 Their model may prove useful to explain Graves’ disease. Autoantibodies directed against specific epitopes in the insulin receptor are rarely the cause of either recurrent hypoglycemia or a severe form of insulin resistance (type B insulin resistance).2 In cancer chemotherapy, antibodies against the death receptors DR4 and DR5 of The Deutsche Forschungsgemeinschaft has supported the work of Drs. Luft and Müller. Further support for this work has been provided by the Bundesministerium für Bildung und Forschung. Conflict of interest: none. *Corresponding author: Friedrich C. Luft, MD, Experimental and Clinical Research Center, Lindenbergerweg 80, 13125 Berlin, Germany. Tel: 49 30 9406 4249; fax: 49 30 9406 4220. E-mail: [email protected]
tumor necrosis factor-alpha-related apoptosis-inducing ligand are interesting therapeutic targets, because agonistic antibodies against DR5 and DR2 induce apoptosis in cancer cells.3 Furthermore, agonistic anti-CD40 antibodies profoundly suppress the immune response to infection with lymphocytic choriomeningitis virus.4 Thus agonistic antibodies directed against cell-surface receptors appear to have an in vivo functional capacity. The purpose of this article is to critically review the notion that such antibodies can cause or amplify cardiovascular disease. In terms of targets, we will focus on the ␣-adrenergic receptor, the -adrenergic receptor, the angiotensin (Ang) II AT1 receptor, and the platelet-derived growth factor (PDGF) receptor. The focus of our report is directed at the question: “What is the evidence that these antibodies actually cause or augment any cardiovascular disease?”
Alpha 1-adrenergic Receptor Antibodies in Patients with Hypertension Fu et al first described and characterized agonistic antibodies directed against an extracellular immunogenic epitope on the human ␣1-adrenergic receptor in the heart.5
1933-1711/08/$ – see front matter © 2008 American Society of Hypertension. All rights reserved. doi:10.1016/j.jash.2007.08.005
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The group subsequently studied patients with malignant hypertension and found these antibodies in 7 of 11 patients.6 The antibodies were also found in normotensive subjects, drawing question as to their relevance. Luther et al investigated the incidence of auto-antibodies against the ␣1adrenoceptor in patients with primary hypertension.7 They examined the immunoglobulin fractions of sera from 54 patients with primary hypertension and 26 normotensive control subjects. They relied on the same bioassay used by Fu et al5,6 to assess the spontaneously beating rate of neonatal rat cardiomyocytes subjected to sera or immune globulin fractions from affected patients. Sera from 24 patients (44%) and three subjects (12%) were positive for agonistic antibodies directed at the ␣1-adrenergic receptor. The authors then performed an epitope analysis of 16 autoantibody-positive immunoglobulin fractions and found that in two thirds of cases, the antibodies were directed against the first extracellular loop of the ␣1-adrenoceptor. In one-third of cases, the antibody was directed against the second extraceullar loop. The autoantibodies exerted a positive chronotropic effect on isolated neonatal rat cardiomyocytes, an effect that was blocked pharmacologically by ␣1-adrenergic antagonists. The authors concluded that because the functional characteristics of the autoantibodies showed no desensitization phenomena, they could play a role in elevating peripheral vascular resistance and promoting cardiac hypertrophy in patients with primary hypertension. Unfortunately, there are no animal models to help clarify these mechanisms. Thus, the role of agonistic ␣1-adrenergic receptor antibodies in human disease remains unproved.
Beta-adrenergic Receptor Antibodies in Patients with Heart Disease Wallukat and Wollenberger identified antibodies directed at -adrenergic receptors in the serum of asthmatic patients and patients with dilative cardiomyopathy.8 In the asthmatic patients, the antibodies were inhibitory and decreased the spontaneous beating rate of neonatal rat cardiomyocytes. In the cardiomyopathy patients, the antibodies exerted a stimulating effect and increased the spontaneous beating rate of neonatal rat cardiomyocytes. The authors attributed this chronotropic effect to a putative agonistic autoantibody directed at -adrenergic receptors. Thus, the asthmatic patients appeared to produce inhibitory antibodies to the 2adrenergic receptor, while the cardiomyopathy patients produced an agonistic antibody to the 1-adrenergic receptor. The group subsequently demonstrated that the latter antibody epitope was confined to the second extracellular loop of the 1-adrenergic receptor, although an epitope on the first extracellular loop was also found.9 Details on the precise amino acid sequence addressed by the antibodies were worked out in a subsequent study.10 Trypanosomiasis is perhaps the most common infectious cause of cardiomyopathy worldwide. A particularly novel observation was
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that Trypanosoma cruzi is able to induce a functional autoimmune response against the cardiovascular human 1adrenergic receptor through a molecular mimicry mechanism.11 Jahns et al provided direct evidence that an autoimmune attack directed against the cardiac 1-adrenergic receptor plays a causal role in dilative cardiomyopathy.12 They immunized inbred rats against the second extracellular 1adrenergic receptor loop that has 100% sequence identity between human and rat every month over the course of a year. The rats developed receptor-stimulating anti-1-adrenergic receptor autoantibodies and after 9 months, progressive severe left ventricular dilatation and dysfunction were apparent. Next, they transferred sera from anti-1adrenergic receptor antibody-positive and antibody-negative animals every month to healthy rats of the same strain. All anti-1-adrenergic receptor antibody-positive-transferred rats also developed a similar cardiomyopathy. These experiments in essence satisfied Koch’s postulate. The disease was produced. A suitable mechanism was demonstrated. This mechanism was then transferred to healthy animals that subsequently contracted the disease. Störk et al followed 65 patients with dilative cardiomyopathy and 40 patients with ischemic cardiomyopathy for 10.7 ⫾ 2.5 years.13 One-quarter of the former (17/65) and 13% of the latter (5/40) harbored agonistic antibodies. In univariate and multivariable Cox regression analysis, the presence of stimulating anti-1-adrenergic receptor was associated with increased all-cause and cardiovascular mortality risk in dilative cardiomyopathy, but not in ischemic cardiomyopathy. Similar data were reported by Staudt et al.14 They reported that in the majority of dilative cardiomyopathy patients, disturbances of humoral immunity with production of cardiodepressant antibodies played a functional role. They then presented evidence that cardiodepressant antibodies predict hemodynamic benefits following immunoabsorption. Immunoglobulin absorption has been suggested as a potential therapy for cardiomyopathy associated with agonistic anti-1-adrenergic receptor antibody-induced cardiomyopathy. Non-randomized, non-blinded observational studies are encouraging.15,16 A randomized study is in progress and preliminary data are available. Staudt et al randomized 22 patients with ejection fractions less than 35% to two treatments.17 Eleven patients received a course of four immunoadsorptions, while another 11 patients received only 1 immunoabsorption session. All sessions consisted of five consecutive days of treatment. After 3 and 6 months, there were no significant differences between the two groups with respect to ejection fraction and all measured hemodynamic parameters. Because no patient was randomized to “no immunoabsorption,” conclusions from this study are limited. Another question is whether or not pharmacological beta blockade is an adequate treatment for patients with agonistic antibodies directed against the 1-adrenergic re-
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ceptor. In vitro studies showed that bisoprolol influenced 1-adrenergic receptor expression, which was otherwise down-regulated by anti-1-adrenergic receptor autoantibodies, suggesting that beta blockade does indeed influence the signaling induced by the autoantibodies.16 However, a careful clinical evaluation has not been performed. Thus, beta blockers are surely indicated for any patient with congestive heart failure. But whether or not beta blockade is specifically helpful in patients with agonistic autoantibodies or whether or not such a therapy supplants therapies aimed at removal of agonistic autoantibodies is currently unknown.
Agonistic Anti-AT1 Receptor Antibodies Fu et al produced antibodies against a synthetic peptide corresponding to amino acids 165 to 191 of the second extraceullar loop of the Ang II, AT1 receptor.18 The authors found that the antibodies did not interfere with ligand binding properties of the receptor, but did exert an agonist effect. They next immunized rats with the AT1 receptor peptides for 3 months.19 The rats developed circulating antibodies directed at the AT1 receptor that exerted a chronotropic effect in the neonatal rat cardiomyocyte bioassay. However, the rats did not develop hypertension by tail-cuff measurement and had no evidence for target organ damage. The studies may have been incomplete. Radiotelemetric blood pressure measurements would be considered standard today. Furthermore, Jahns et al required almost a year before their immunization experiments caused a demonstrable effect.12 We managed a 36-year-old woman with malignant hypertension who related having had pre-eclampsia 17 years earlier. The Wallukat laboratory (Max Delbrück Center, Berlin-Buch, Germany) tested her serum for agonistic AT1 receptor antibodies with a positive result. We next began a study of preeclamptic (de novo hypertension and proteinuria after 20 weeks pregnancy) patients. We found that women with preeclampsia regularly develop agonistic AT1 receptor antibodies. In a single patient, we serendipitously had serum samples available for antibody testing prior to the development of overt disease. The antibodies developed with the syndrome but nonetheless regressed about 4 to 6 weeks after pregnancy in all the patients tested. The report was greeted with some skepticism; however, we were able to show that the IgG3 fraction from the patients functioned as well in a Western blot as any available commercial antibody.20 In the meantime, Fu et al published a report about the presence of the antibodies in patients with malignant hypertension.21 We next showed that the antibodies from the patients exert a signaling effect. We studied the effects of AT1 receptor agonistic antibodies on reactive oxygen species (ROS), nicotinamide adenosine dinucleotide phosphate oxidase expression, and nuclear factor-kappa activation.22 We investigated human vascular smooth muscle cells and trophoblasts, as well as placentas. We found that the antibodies
activated nuclear factor-kappa, stimulated ROS release, caused vascular smooth muscle cells to produce tissue factor, and convinced ourselves that the antibodies could elucidate pathological effects, at least in vivo. Stringent reviewers demanded successful coimmunoprecipitation experiments to verify specificity, which we provided. Pregnant patients should not be treated with angiotensin-converting enzyme inhibitors or AT1 receptor blockers under any circumstances. As a result, therapeutic options other than antibody removal in terms of providing proof are limited in this disease. Earlier, we had confirmed the work of Japanese investigators showing that transgenic rodents for the human renin and angiotensinogen genes are a practicable pre-eclampsia model.23 When dams (mice or rats) harboring the human angiotensinogen gene are mated with sires carrying the human renin gene, the dams develop severe hypertension, proteinuria, and target organ damage resembling preeclampsia in the last half of their 22-day pregnancy. We showed that this rat model also develops agonistic antibodies directed at the AT1 receptor during the last half of pregnancy.24 The epitope is highly conserved and is shown in the Figure. Encouraging information for us were independent, confirmatory, and expanding data from Xia et al.25 They also found agonistic AT1 receptor antibodies and showed that the antibodies caused a human trophoblast cell line to produce plasminogen activator inhibitor-1 via a nuclear factor of activated T cells pathway. They followed this report with another study showing that the antibodies can induce calcium signaling, and stimulate mesangial cells to produce interleukin-6 and plasminogen activator inhibitor-1.26,27 The group has subsequently shown that Ang II can stimulate the production of soluble feline leukemia virus (Fms)-like tyrosine kinase receptor (sFlt-1) in a trophoblast cell line.28 sFlt-1 interferes with angiogenesis and has been implicated in the pathogenesis of preeclampsia. Similarly, the same investigators have reported in abstract form that agonistic AT1 receptor antibodies can do likewise (Kellems laboratory, Houston, TX, unpublished data, 2007). Hubel et al compared the presence of sFlt-1, a splice variant of the corresponding vascular endothelial growth factor – placental growth factor receptor, and agonistic AT1 receptor antibodies in the plasma of 29 previously preeclamptic women, 18 months after their pregnancies.29 Seventeen percent of these women still had circulating agonistic AT1 receptor antibodies. Women who did, also had elevated circulating levels of sFlt-1. Herse et al examined sera and placenta from pre-eclamptic women and women with normal pregnancies.30 They also found agonistic AT1 receptor antibodies in the sera of the pre-eclamptic women, but not in the normal women. In the placentas of the preeclamptic women, the AT1 receptor was increased in terms of expression in the deciduas, compared with placentas from normal pregnancies. The renin gene was upregulated in the placentas from both groups.
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Figure. View of the second AT1 receptor extracellular loop and the binding site (amino acid sequence) of agonistic AT1 receptor antibodies are shown.
Walther et al studied agonistic AT1 receptor antibodies in women with second-trimester pregnancies.31 They monitored uterine perfusion with ultrasonography and found that the antibodies arose prior to overt disease development. The antibodies were also found in some women who did not develop preeclamspia. Only women with abnormal Doppler examinations developed antibodies. The authors concluded that the antibodies were associated with impaired placental development. In a subsequent report, the group studied agonistic AT1 receptor antibodies in pregnancy and incorporated sFlt-1 measurements in their studies. They found that autoantibody bioactivity and sFlt1 concentrations did not necessarily correlate, were not mutually dependent, and thus probably involved distinct pathogenic mechanisms.32 The authors concluded that the two factors in combination may not be causative for the early impaired trophoblast invasion and pathological uterine perfusion. A vexing question is how these agonistic AT1 receptor antibodies might arise. The epitope recognized by the antibodies, shown in the figure, also occurs on parvovirus B19 capsid proteins. Stepan et al pursued the hypothesis that women developing the antibodies might also have a high prevalence of parvovirus B19 infection.33 However, they found no evidence to support this hypothesis. Thus, this possible epitope mimicry has not been proved and is not supported by current published evidence. However, the possibility remains under investigation. Dragun et al made further serendipitous but elucidating observations.34 A patient with an unusual form of C4d-negative humoral rejection after her kidney transplant developed
profound hypertension. The patient also related having had preeclampsia years earlier. The investigators then focused on patients that developed C4d-negative humoral rejection after renal transplantation. Most humoral rejection after transplant involves antibodies against human leukocyte antigen (HLA) moieties and causes a characteristic C4d complement fraction deposition along peritubular capillaries in the transplanted kidney. However, their patients had C4d-negative humoral rejection; they compared these patients with those with C4d-positive humoral rejection or other rejection forms. Agonistic AT1 receptor antibodies were identified in the patients with C4dnegative humoral rejection. The antibodies addressed two different epitopes. In a rat transplant model, the antibodies were passively transferred to transplanted rats and an increase in blood pressure was reproduced. The data suggest that the malignant hypertension occurring with acute humoral rejection may involve stimulating antibodies directed against the AT1 receptor. Patients after transplantation can readily be treated with AT1 receptor blockers. We have preliminary clinical inference that AT1 receptor blockers are helpful in patients with C4d-negative humoral rejection. However, no controlled studies in this regard have been performed.
Activating Antibodies Against the PDGF Receptor Systemic sclerosis (scleroderma) is severe connective tissue disease characterized by immunologic abnormalities, injury of endothelial cells, and fibrosis. Abnormal oxidative
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stress has been documented in scleroderma and linked to fibroblast activation. PDGF stimulates ROS production. Furthermore, IgG from patients with scleroderma reacts with human fibroblasts. These events caused Baroni et al to test the hypothesis that patients with scleroderma might have serum autoantibodies that target the PDGF receptor, thereby activating collagen gene expression.35 They analyzed serum from 46 patients with scleroderma and 75 control subjects, including patients with other autoimmune diseases, for stimulatory autoantibodies to the PDGF receptor. They incubated mouse embryo fibroblasts carrying either inactive or active copies of PDGF receptor ␣ or  chains with purified IgG from the patients and measured ROS production. The antibodies were characterized by immunoprecipitation, immunoblotting, and absorption experiments. Baroni et al found stimulatory antibodies to the PDGF receptor in all the patients with scleroderma.35 The antibodies recognized the native PDGF receptor ␣ chain. Moreover, the antibodies induced tyrosine phosphorylation and ROS accumulation. The autoantibody activity was abolished by preincubation of antibodies with cells expressing the PDGF receptor ␣ chain, with recombinant PDGF receptor, or with PDGF receptor tyrosine kinase inhibitors. Baroni et al also found that stimulatory PDGF receptor antibodies selectively induced the Ha-Ras-ExtracellularSignal Regulated Kinase (ERK)1/2 and ROS cascades and stimulated type I collagen gene expression and myofibroblast phenotype conversion in normal human primary fibroblasts.35 Thus, stimulatory autoantibodies against the PDGF receptor ␣ chain appear to be a specific hallmark of scleroderma. Their biologic activity on fibroblasts strongly suggests that the antibodies have a causal role in the pathogenesis of the disease. Notably, since this report, Svegliati et al have reported the presence of the same antibodies in patients with graft-versus-host disease that can cause similar connective tissue fibrosis.36
Genesis of Antibodies Directed Against Receptors The mechanisms leading to antibodies directed against receptors are unknown. We have already speculated on the VP2 surface protein of parvovirus and the implication of molecular mimicry.33 In Graves’ disease, viral infections may cause stimulation of cytokine production that results in thyroid cells expressing certain surface class II molecules of specific HLA-DR types, which present fragments of the TSH receptor to T lymphocytes.37 These T lymphocytes would then stimulate B cells to produce TSH receptor antibodies. The susceptibility is strongly influenced by genetic variance, coupled with environmental stressors, such as iodide exposure, viral or bacterial infections, and physical and emotional stress. Autoantibodies directed against receptors are not necessarily stimulatory, but also inhibitory. Examples include autoantibodies that cause decreased sensitivity of 2 receptor functions involved in asthma38 and
against the ganglionic acetylcholine receptor resulting in complete autonomic failure.39 Another feature is the very low actual concentrations of antibodies involved in these syndromes. The nicotinic acetylcholine receptor antibody titers in the patient we described earlier were about 0.4 nmol/L.39 The AT1 receptor-activating antibodies detected in preeclamptic patients were present in low titers allowing their detection only with a bioassay.20 Our efforts to develop an enzyme-linked immunosorbent assay for AT1 receptor activating antibodies have not yet been successful. Nikolaev et al used a fluorescence resonance energy transfer technique to detect functional beta1-adrenergic receptor antibodies in heart failure patients.40 The fluorescence resonance energy transfer technique assay detected antibody concentrations of 0.001 nmol/L to 0.2 nmol/L. Finally, we would like to raise a novel hypothesis outlined by Pendergraft et al that we have not yet explored.41 These investigators point out that auto-immunity affects more than 5% of the general population. Their theory proposes that the initiator is not the self-antigen (in our case, the AT1 receptor), but rather a protein that is complementary in surface structure to the autoantigen. Such a protein would be homologous or identical to the amino acid sequence of translated antisense RNA from the noncoding strand of the autoantigen gene. This idea is novel and has support from studies of antineutrophil cytoplasmic autoantibody-mediated vasculitis.
Perspectives Numerous groups have published a comprehensive series of reports regarding the existence of agonistic antibodies directed against G protein-coupled and tyrosine kinase receptors. Compelling evidence suggests that these antibodies are of pathogenic importance in terms of producing disease. In any event, the antibodies could play an ancillary accelerating role. In most instances, bioassay results have been required to demonstrate the relevance of agonistic antibodies. One reason includes the very low circulating concentrations of the antibodies, a fact that does not detract from their potential pathogenic relevance. Technical problems have precluded the establishment of enzyme-linked immunosorbent assays in some cases. Numerous loose ends exist. The soundest data to date involve the agonistic antibodies directed against the 1adrenergic receptor. The meticulous studies by Jahns et al12 largely fulfilled Koch’s postulates in terms of establishing these antibodies as a cause for a disease. The evidence concerning agonistic antibodies directed against the AT1 receptor is compelling but not quite as convincing. Furthermore, recent evidence from other sources implicating sFlt-1 and soluble endoglin in pre-eclampsia development provide alternative explanations.42 Antibodies to the AT1 receptor in post-transplant patients has greatly increased interest in C4d-negative humoral rejection and the strong association
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between C4d-positive rejection and post-transplant HLA antibodies.43 Much more work needs to be done in this area. Finally, do agonistic antibodies lead us further to novel therapeutic options? Affinity columns or less specifically plasmapheresis are available to remove autoantibodies. These techniques clearly have potential for diseases such as scleroderma or after kidney transplantation. For preeclampsia, antibody removal procedures have proved exceptionally difficult because of logistical problems. Randomized controlled double-blind trials, the gold standard studies, are a nightmare in this area. A closed treatment room with arterial and venous lines coming through holes in the wall with “blinded” machine operators that are never allowed to see the patients, coupled with care givers also totally blinded as to the treatments, would be necessary. With tablets, these strategies are straightforward; with machinery, they are considerably more difficult. Nonetheless, other possibilities exist. Our exploratory studies to develop a fusion protein failed because of numerous technical problems. However, aptamers and other small molecules of similar ilk could offer an alternative.44 Xia, Ramin, and Kellems have outlined these strategies elsewhere.45
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32. Stepan H, Faber R, Wessel N, Wallukat G, Schultheiss HP, Walther T. Relation between circulating angiotensin II type 1 receptor agonistic autoantibodies and soluble fms-like tyrosine kinase 1 in the pathogenesis of preeclampsia. J Clin Endocrinol Metab 2006;91: 2424 –7. 33. Stepan H, Wallukat G, Schultheiss HP, Faber R, Walther T. Is parvovirus B19 the cause for autoimmunity against the angiotensin II type receptor? J Reprod Immunol 2007;73:130 – 4. 34. Dragun D, Müller DN, Brasen JH, Fritsche L, Nieminen-Kelha M, Dechend R, et al. Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection. N Engl J Med 2005;352:558 – 69. 35. Baroni SS, Santillo M, Bevilacqua F, Luchetti M, Spadoni T, Mancini M, et al. Stimulatory autoantibodies to the PDGF receptor in systemic sclerosis. N Engl J Med 2006;354:2667–76. 36. Svegliati S, Olivieri A, Campelli N, Luchetti M, Poloni A, Trappolini S, et al. Stimulatory autoantibodies to PDGF receptor in patients with extensive chronic graftversus-host disease. Blood 2007;110:237– 41. 37. Collins J, Gough S. Autoimmunity in thyroid disease. Eur J Nucl Med Mol Imaging 2002;29 Suppl 2:S417– 24. 38. Venter JC, Fraser CM. Beta-adrenergic receptor structure, synthesis, antibodies and human disease. Bull Eur Physiopathol Respir 1985;21:13S–18S. 39. Schroeder C, Vernino S, Birkenfeld AL, Tank J, Heusser K, Lipp A, et al. Plasma exchange for primary autoimmune autonomic failure. N Engl J Med 2005; 353:1585–90. 40. Nikolaev VO, Boivin V, Stork S, Angermann CE, Ertl G, Lohse MJ, et al. A novel fluorescence method for the rapid detection of functional beta1-adrenergic receptor autoantibodies in heart failure. J Am Coll Cardiol 2007; 50:423–31. 41. Pendergraft WF III, Pressler BM, Jennette JC, Falk RJ, Presont GA. Autoantigen complementarity: a new theory implicating complementary proteins as initiators of autoimmune disease. J Mol Med 2005;83:12–25. 42. Widmer M, Villar J, Benigni A, Conde-Agudelo A, Karumanchi SA, Lindheimer M. Mapping the theories of pre-eclampsia and the role of angiogenic factors: a systematic review. Obstet Gynecol 2007;109:168 – 80. 43. Scornik JC, Guerra G, Schold JD, Srinivas TR, Dragun D, Meier-Kriesche HU. Value of post-transplant antibody testing in the evaluation of patients with renal graft dysfunction. Am J Transplant 2007;7:1808 –14. 44. Tachikawa K, Briggs SP. Targeting the human genome. Curr Opin Biotechnol 2006;17:659 – 65. 45. Xia Y, Ramin SM, Kellems RE. Potential roles of angiotensin receptor-activating antibody in the pathophysiology of pre-eclampsia. Hypertension 2007;50: 269 –75.
Review Article
Agonistic antibodies directed at cell surface receptors and cardiovascular disease Friedrich C. Luft, MD*, Ralf Dechend, MD, Duska Dragun, MD, Dominik N. Müller, PhD, and Gerd Wallukat, PhD Experimental and Clinical Research Center, Medical Faculty of the Charité, Max-Delbrück Center for Molecular Medicine, HELIOSKlinikum, Berlin, Germany Manuscript received June 20, 2007 and accepted August 20, 2007
Abstract Antibodies directed at receptors can block or stimulate them. Hallmark example of the latter action is Graves’ disease where antibodies directed at the thyroid-stimulating hormone receptor exert an agonistic action. Recently, compelling evidence has been presented regarding agonistic antibodies directed against the alpha-adrenergic receptor, the beta-adrenergic receptors, the angiotensin II AT1 receptor, and the platelet-derived growth factor-alpha receptor. The antibodies could play a pathogenic role in various cardiovascular diseases, including hypertension, cardiomyopathy, pre-eclampsia, acute humoral rejection, and connective tissue disease. The mechanisms that result in the production of these antibodies are unclear, automated assays to determine their presence are beset with technical difficulties, and the therapeutic implications are uncertain. Nevertheless, the signaling phenomena resulting from these antibodies are well established and mechanistic studies are being intensively pursued. The discovery of agonistic antibodies may provide additional therapeutic avenues. © 2008 American Society of Hypertension. All rights reserved. Keywords : Adrenergic receptors; AT1 receptor; antibodies; angiotensin.
Introduction Agonistic antibodies that stimulate G protein-coupled receptors are accepted as causing diseases of the thyroid gland. Agonistic antithyrotropin receptor antibodies (TSAb) that mimic the action of thyrotropin (TSH) characterize Graves’ disease. Nonetheless, the immunological mechanisms of TSAb production remain obscure. Recently, KimSaijo et al produced a transgenic mouse that expresses a patient-derived TSAb.1 Their model may prove useful to explain Graves’ disease. Autoantibodies directed against specific epitopes in the insulin receptor are rarely the cause of either recurrent hypoglycemia or a severe form of insulin resistance (type B insulin resistance).2 In cancer chemotherapy, antibodies against the death receptors DR4 and DR5 of The Deutsche Forschungsgemeinschaft has supported the work of Drs. Luft and Müller. Further support for this work has been provided by the Bundesministerium für Bildung und Forschung. Conflict of interest: none. *Corresponding author: Friedrich C. Luft, MD, Experimental and Clinical Research Center, Lindenbergerweg 80, 13125 Berlin, Germany. Tel: 49 30 9406 4249; fax: 49 30 9406 4220. E-mail: [email protected]
tumor necrosis factor-alpha-related apoptosis-inducing ligand are interesting therapeutic targets, because agonistic antibodies against DR5 and DR2 induce apoptosis in cancer cells.3 Furthermore, agonistic anti-CD40 antibodies profoundly suppress the immune response to infection with lymphocytic choriomeningitis virus.4 Thus agonistic antibodies directed against cell-surface receptors appear to have an in vivo functional capacity. The purpose of this article is to critically review the notion that such antibodies can cause or amplify cardiovascular disease. In terms of targets, we will focus on the ␣-adrenergic receptor, the -adrenergic receptor, the angiotensin (Ang) II AT1 receptor, and the platelet-derived growth factor (PDGF) receptor. The focus of our report is directed at the question: “What is the evidence that these antibodies actually cause or augment any cardiovascular disease?”
Alpha 1-adrenergic Receptor Antibodies in Patients with Hypertension Fu et al first described and characterized agonistic antibodies directed against an extracellular immunogenic epitope on the human ␣1-adrenergic receptor in the heart.5
1933-1711/08/$ – see front matter © 2008 American Society of Hypertension. All rights reserved. doi:10.1016/j.jash.2007.08.005
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The group subsequently studied patients with malignant hypertension and found these antibodies in 7 of 11 patients.6 The antibodies were also found in normotensive subjects, drawing question as to their relevance. Luther et al investigated the incidence of auto-antibodies against the ␣1adrenoceptor in patients with primary hypertension.7 They examined the immunoglobulin fractions of sera from 54 patients with primary hypertension and 26 normotensive control subjects. They relied on the same bioassay used by Fu et al5,6 to assess the spontaneously beating rate of neonatal rat cardiomyocytes subjected to sera or immune globulin fractions from affected patients. Sera from 24 patients (44%) and three subjects (12%) were positive for agonistic antibodies directed at the ␣1-adrenergic receptor. The authors then performed an epitope analysis of 16 autoantibody-positive immunoglobulin fractions and found that in two thirds of cases, the antibodies were directed against the first extracellular loop of the ␣1-adrenoceptor. In one-third of cases, the antibody was directed against the second extraceullar loop. The autoantibodies exerted a positive chronotropic effect on isolated neonatal rat cardiomyocytes, an effect that was blocked pharmacologically by ␣1-adrenergic antagonists. The authors concluded that because the functional characteristics of the autoantibodies showed no desensitization phenomena, they could play a role in elevating peripheral vascular resistance and promoting cardiac hypertrophy in patients with primary hypertension. Unfortunately, there are no animal models to help clarify these mechanisms. Thus, the role of agonistic ␣1-adrenergic receptor antibodies in human disease remains unproved.
Beta-adrenergic Receptor Antibodies in Patients with Heart Disease Wallukat and Wollenberger identified antibodies directed at -adrenergic receptors in the serum of asthmatic patients and patients with dilative cardiomyopathy.8 In the asthmatic patients, the antibodies were inhibitory and decreased the spontaneous beating rate of neonatal rat cardiomyocytes. In the cardiomyopathy patients, the antibodies exerted a stimulating effect and increased the spontaneous beating rate of neonatal rat cardiomyocytes. The authors attributed this chronotropic effect to a putative agonistic autoantibody directed at -adrenergic receptors. Thus, the asthmatic patients appeared to produce inhibitory antibodies to the 2adrenergic receptor, while the cardiomyopathy patients produced an agonistic antibody to the 1-adrenergic receptor. The group subsequently demonstrated that the latter antibody epitope was confined to the second extracellular loop of the 1-adrenergic receptor, although an epitope on the first extracellular loop was also found.9 Details on the precise amino acid sequence addressed by the antibodies were worked out in a subsequent study.10 Trypanosomiasis is perhaps the most common infectious cause of cardiomyopathy worldwide. A particularly novel observation was
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that Trypanosoma cruzi is able to induce a functional autoimmune response against the cardiovascular human 1adrenergic receptor through a molecular mimicry mechanism.11 Jahns et al provided direct evidence that an autoimmune attack directed against the cardiac 1-adrenergic receptor plays a causal role in dilative cardiomyopathy.12 They immunized inbred rats against the second extracellular 1adrenergic receptor loop that has 100% sequence identity between human and rat every month over the course of a year. The rats developed receptor-stimulating anti-1-adrenergic receptor autoantibodies and after 9 months, progressive severe left ventricular dilatation and dysfunction were apparent. Next, they transferred sera from anti-1adrenergic receptor antibody-positive and antibody-negative animals every month to healthy rats of the same strain. All anti-1-adrenergic receptor antibody-positive-transferred rats also developed a similar cardiomyopathy. These experiments in essence satisfied Koch’s postulate. The disease was produced. A suitable mechanism was demonstrated. This mechanism was then transferred to healthy animals that subsequently contracted the disease. Störk et al followed 65 patients with dilative cardiomyopathy and 40 patients with ischemic cardiomyopathy for 10.7 ⫾ 2.5 years.13 One-quarter of the former (17/65) and 13% of the latter (5/40) harbored agonistic antibodies. In univariate and multivariable Cox regression analysis, the presence of stimulating anti-1-adrenergic receptor was associated with increased all-cause and cardiovascular mortality risk in dilative cardiomyopathy, but not in ischemic cardiomyopathy. Similar data were reported by Staudt et al.14 They reported that in the majority of dilative cardiomyopathy patients, disturbances of humoral immunity with production of cardiodepressant antibodies played a functional role. They then presented evidence that cardiodepressant antibodies predict hemodynamic benefits following immunoabsorption. Immunoglobulin absorption has been suggested as a potential therapy for cardiomyopathy associated with agonistic anti-1-adrenergic receptor antibody-induced cardiomyopathy. Non-randomized, non-blinded observational studies are encouraging.15,16 A randomized study is in progress and preliminary data are available. Staudt et al randomized 22 patients with ejection fractions less than 35% to two treatments.17 Eleven patients received a course of four immunoadsorptions, while another 11 patients received only 1 immunoabsorption session. All sessions consisted of five consecutive days of treatment. After 3 and 6 months, there were no significant differences between the two groups with respect to ejection fraction and all measured hemodynamic parameters. Because no patient was randomized to “no immunoabsorption,” conclusions from this study are limited. Another question is whether or not pharmacological beta blockade is an adequate treatment for patients with agonistic antibodies directed against the 1-adrenergic re-
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ceptor. In vitro studies showed that bisoprolol influenced 1-adrenergic receptor expression, which was otherwise down-regulated by anti-1-adrenergic receptor autoantibodies, suggesting that beta blockade does indeed influence the signaling induced by the autoantibodies.16 However, a careful clinical evaluation has not been performed. Thus, beta blockers are surely indicated for any patient with congestive heart failure. But whether or not beta blockade is specifically helpful in patients with agonistic autoantibodies or whether or not such a therapy supplants therapies aimed at removal of agonistic autoantibodies is currently unknown.
Agonistic Anti-AT1 Receptor Antibodies Fu et al produced antibodies against a synthetic peptide corresponding to amino acids 165 to 191 of the second extraceullar loop of the Ang II, AT1 receptor.18 The authors found that the antibodies did not interfere with ligand binding properties of the receptor, but did exert an agonist effect. They next immunized rats with the AT1 receptor peptides for 3 months.19 The rats developed circulating antibodies directed at the AT1 receptor that exerted a chronotropic effect in the neonatal rat cardiomyocyte bioassay. However, the rats did not develop hypertension by tail-cuff measurement and had no evidence for target organ damage. The studies may have been incomplete. Radiotelemetric blood pressure measurements would be considered standard today. Furthermore, Jahns et al required almost a year before their immunization experiments caused a demonstrable effect.12 We managed a 36-year-old woman with malignant hypertension who related having had pre-eclampsia 17 years earlier. The Wallukat laboratory (Max Delbrück Center, Berlin-Buch, Germany) tested her serum for agonistic AT1 receptor antibodies with a positive result. We next began a study of preeclamptic (de novo hypertension and proteinuria after 20 weeks pregnancy) patients. We found that women with preeclampsia regularly develop agonistic AT1 receptor antibodies. In a single patient, we serendipitously had serum samples available for antibody testing prior to the development of overt disease. The antibodies developed with the syndrome but nonetheless regressed about 4 to 6 weeks after pregnancy in all the patients tested. The report was greeted with some skepticism; however, we were able to show that the IgG3 fraction from the patients functioned as well in a Western blot as any available commercial antibody.20 In the meantime, Fu et al published a report about the presence of the antibodies in patients with malignant hypertension.21 We next showed that the antibodies from the patients exert a signaling effect. We studied the effects of AT1 receptor agonistic antibodies on reactive oxygen species (ROS), nicotinamide adenosine dinucleotide phosphate oxidase expression, and nuclear factor-kappa activation.22 We investigated human vascular smooth muscle cells and trophoblasts, as well as placentas. We found that the antibodies
activated nuclear factor-kappa, stimulated ROS release, caused vascular smooth muscle cells to produce tissue factor, and convinced ourselves that the antibodies could elucidate pathological effects, at least in vivo. Stringent reviewers demanded successful coimmunoprecipitation experiments to verify specificity, which we provided. Pregnant patients should not be treated with angiotensin-converting enzyme inhibitors or AT1 receptor blockers under any circumstances. As a result, therapeutic options other than antibody removal in terms of providing proof are limited in this disease. Earlier, we had confirmed the work of Japanese investigators showing that transgenic rodents for the human renin and angiotensinogen genes are a practicable pre-eclampsia model.23 When dams (mice or rats) harboring the human angiotensinogen gene are mated with sires carrying the human renin gene, the dams develop severe hypertension, proteinuria, and target organ damage resembling preeclampsia in the last half of their 22-day pregnancy. We showed that this rat model also develops agonistic antibodies directed at the AT1 receptor during the last half of pregnancy.24 The epitope is highly conserved and is shown in the Figure. Encouraging information for us were independent, confirmatory, and expanding data from Xia et al.25 They also found agonistic AT1 receptor antibodies and showed that the antibodies caused a human trophoblast cell line to produce plasminogen activator inhibitor-1 via a nuclear factor of activated T cells pathway. They followed this report with another study showing that the antibodies can induce calcium signaling, and stimulate mesangial cells to produce interleukin-6 and plasminogen activator inhibitor-1.26,27 The group has subsequently shown that Ang II can stimulate the production of soluble feline leukemia virus (Fms)-like tyrosine kinase receptor (sFlt-1) in a trophoblast cell line.28 sFlt-1 interferes with angiogenesis and has been implicated in the pathogenesis of preeclampsia. Similarly, the same investigators have reported in abstract form that agonistic AT1 receptor antibodies can do likewise (Kellems laboratory, Houston, TX, unpublished data, 2007). Hubel et al compared the presence of sFlt-1, a splice variant of the corresponding vascular endothelial growth factor – placental growth factor receptor, and agonistic AT1 receptor antibodies in the plasma of 29 previously preeclamptic women, 18 months after their pregnancies.29 Seventeen percent of these women still had circulating agonistic AT1 receptor antibodies. Women who did, also had elevated circulating levels of sFlt-1. Herse et al examined sera and placenta from pre-eclamptic women and women with normal pregnancies.30 They also found agonistic AT1 receptor antibodies in the sera of the pre-eclamptic women, but not in the normal women. In the placentas of the preeclamptic women, the AT1 receptor was increased in terms of expression in the deciduas, compared with placentas from normal pregnancies. The renin gene was upregulated in the placentas from both groups.
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Figure. View of the second AT1 receptor extracellular loop and the binding site (amino acid sequence) of agonistic AT1 receptor antibodies are shown.
Walther et al studied agonistic AT1 receptor antibodies in women with second-trimester pregnancies.31 They monitored uterine perfusion with ultrasonography and found that the antibodies arose prior to overt disease development. The antibodies were also found in some women who did not develop preeclamspia. Only women with abnormal Doppler examinations developed antibodies. The authors concluded that the antibodies were associated with impaired placental development. In a subsequent report, the group studied agonistic AT1 receptor antibodies in pregnancy and incorporated sFlt-1 measurements in their studies. They found that autoantibody bioactivity and sFlt1 concentrations did not necessarily correlate, were not mutually dependent, and thus probably involved distinct pathogenic mechanisms.32 The authors concluded that the two factors in combination may not be causative for the early impaired trophoblast invasion and pathological uterine perfusion. A vexing question is how these agonistic AT1 receptor antibodies might arise. The epitope recognized by the antibodies, shown in the figure, also occurs on parvovirus B19 capsid proteins. Stepan et al pursued the hypothesis that women developing the antibodies might also have a high prevalence of parvovirus B19 infection.33 However, they found no evidence to support this hypothesis. Thus, this possible epitope mimicry has not been proved and is not supported by current published evidence. However, the possibility remains under investigation. Dragun et al made further serendipitous but elucidating observations.34 A patient with an unusual form of C4d-negative humoral rejection after her kidney transplant developed
profound hypertension. The patient also related having had preeclampsia years earlier. The investigators then focused on patients that developed C4d-negative humoral rejection after renal transplantation. Most humoral rejection after transplant involves antibodies against human leukocyte antigen (HLA) moieties and causes a characteristic C4d complement fraction deposition along peritubular capillaries in the transplanted kidney. However, their patients had C4d-negative humoral rejection; they compared these patients with those with C4d-positive humoral rejection or other rejection forms. Agonistic AT1 receptor antibodies were identified in the patients with C4dnegative humoral rejection. The antibodies addressed two different epitopes. In a rat transplant model, the antibodies were passively transferred to transplanted rats and an increase in blood pressure was reproduced. The data suggest that the malignant hypertension occurring with acute humoral rejection may involve stimulating antibodies directed against the AT1 receptor. Patients after transplantation can readily be treated with AT1 receptor blockers. We have preliminary clinical inference that AT1 receptor blockers are helpful in patients with C4d-negative humoral rejection. However, no controlled studies in this regard have been performed.
Activating Antibodies Against the PDGF Receptor Systemic sclerosis (scleroderma) is severe connective tissue disease characterized by immunologic abnormalities, injury of endothelial cells, and fibrosis. Abnormal oxidative
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stress has been documented in scleroderma and linked to fibroblast activation. PDGF stimulates ROS production. Furthermore, IgG from patients with scleroderma reacts with human fibroblasts. These events caused Baroni et al to test the hypothesis that patients with scleroderma might have serum autoantibodies that target the PDGF receptor, thereby activating collagen gene expression.35 They analyzed serum from 46 patients with scleroderma and 75 control subjects, including patients with other autoimmune diseases, for stimulatory autoantibodies to the PDGF receptor. They incubated mouse embryo fibroblasts carrying either inactive or active copies of PDGF receptor ␣ or  chains with purified IgG from the patients and measured ROS production. The antibodies were characterized by immunoprecipitation, immunoblotting, and absorption experiments. Baroni et al found stimulatory antibodies to the PDGF receptor in all the patients with scleroderma.35 The antibodies recognized the native PDGF receptor ␣ chain. Moreover, the antibodies induced tyrosine phosphorylation and ROS accumulation. The autoantibody activity was abolished by preincubation of antibodies with cells expressing the PDGF receptor ␣ chain, with recombinant PDGF receptor, or with PDGF receptor tyrosine kinase inhibitors. Baroni et al also found that stimulatory PDGF receptor antibodies selectively induced the Ha-Ras-ExtracellularSignal Regulated Kinase (ERK)1/2 and ROS cascades and stimulated type I collagen gene expression and myofibroblast phenotype conversion in normal human primary fibroblasts.35 Thus, stimulatory autoantibodies against the PDGF receptor ␣ chain appear to be a specific hallmark of scleroderma. Their biologic activity on fibroblasts strongly suggests that the antibodies have a causal role in the pathogenesis of the disease. Notably, since this report, Svegliati et al have reported the presence of the same antibodies in patients with graft-versus-host disease that can cause similar connective tissue fibrosis.36
Genesis of Antibodies Directed Against Receptors The mechanisms leading to antibodies directed against receptors are unknown. We have already speculated on the VP2 surface protein of parvovirus and the implication of molecular mimicry.33 In Graves’ disease, viral infections may cause stimulation of cytokine production that results in thyroid cells expressing certain surface class II molecules of specific HLA-DR types, which present fragments of the TSH receptor to T lymphocytes.37 These T lymphocytes would then stimulate B cells to produce TSH receptor antibodies. The susceptibility is strongly influenced by genetic variance, coupled with environmental stressors, such as iodide exposure, viral or bacterial infections, and physical and emotional stress. Autoantibodies directed against receptors are not necessarily stimulatory, but also inhibitory. Examples include autoantibodies that cause decreased sensitivity of 2 receptor functions involved in asthma38 and
against the ganglionic acetylcholine receptor resulting in complete autonomic failure.39 Another feature is the very low actual concentrations of antibodies involved in these syndromes. The nicotinic acetylcholine receptor antibody titers in the patient we described earlier were about 0.4 nmol/L.39 The AT1 receptor-activating antibodies detected in preeclamptic patients were present in low titers allowing their detection only with a bioassay.20 Our efforts to develop an enzyme-linked immunosorbent assay for AT1 receptor activating antibodies have not yet been successful. Nikolaev et al used a fluorescence resonance energy transfer technique to detect functional beta1-adrenergic receptor antibodies in heart failure patients.40 The fluorescence resonance energy transfer technique assay detected antibody concentrations of 0.001 nmol/L to 0.2 nmol/L. Finally, we would like to raise a novel hypothesis outlined by Pendergraft et al that we have not yet explored.41 These investigators point out that auto-immunity affects more than 5% of the general population. Their theory proposes that the initiator is not the self-antigen (in our case, the AT1 receptor), but rather a protein that is complementary in surface structure to the autoantigen. Such a protein would be homologous or identical to the amino acid sequence of translated antisense RNA from the noncoding strand of the autoantigen gene. This idea is novel and has support from studies of antineutrophil cytoplasmic autoantibody-mediated vasculitis.
Perspectives Numerous groups have published a comprehensive series of reports regarding the existence of agonistic antibodies directed against G protein-coupled and tyrosine kinase receptors. Compelling evidence suggests that these antibodies are of pathogenic importance in terms of producing disease. In any event, the antibodies could play an ancillary accelerating role. In most instances, bioassay results have been required to demonstrate the relevance of agonistic antibodies. One reason includes the very low circulating concentrations of the antibodies, a fact that does not detract from their potential pathogenic relevance. Technical problems have precluded the establishment of enzyme-linked immunosorbent assays in some cases. Numerous loose ends exist. The soundest data to date involve the agonistic antibodies directed against the 1adrenergic receptor. The meticulous studies by Jahns et al12 largely fulfilled Koch’s postulates in terms of establishing these antibodies as a cause for a disease. The evidence concerning agonistic antibodies directed against the AT1 receptor is compelling but not quite as convincing. Furthermore, recent evidence from other sources implicating sFlt-1 and soluble endoglin in pre-eclampsia development provide alternative explanations.42 Antibodies to the AT1 receptor in post-transplant patients has greatly increased interest in C4d-negative humoral rejection and the strong association
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between C4d-positive rejection and post-transplant HLA antibodies.43 Much more work needs to be done in this area. Finally, do agonistic antibodies lead us further to novel therapeutic options? Affinity columns or less specifically plasmapheresis are available to remove autoantibodies. These techniques clearly have potential for diseases such as scleroderma or after kidney transplantation. For preeclampsia, antibody removal procedures have proved exceptionally difficult because of logistical problems. Randomized controlled double-blind trials, the gold standard studies, are a nightmare in this area. A closed treatment room with arterial and venous lines coming through holes in the wall with “blinded” machine operators that are never allowed to see the patients, coupled with care givers also totally blinded as to the treatments, would be necessary. With tablets, these strategies are straightforward; with machinery, they are considerably more difficult. Nonetheless, other possibilities exist. Our exploratory studies to develop a fusion protein failed because of numerous technical problems. However, aptamers and other small molecules of similar ilk could offer an alternative.44 Xia, Ramin, and Kellems have outlined these strategies elsewhere.45
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