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Agonistic antibodies in systemic sclerosis
Accepted Manuscript Title: Agonistic antibodies in systemic sclerosis Authors: Gianluca Moroncini, Silvia Svegliati Baroni, Armando Gabrielli PII: DOI: Reference:
S0165-2478(17)30364-4 https://doi.org/10.1016/j.imlet.2017.10.007 IMLET 6126
To appear in:
Immunology Letters
Received date: Revised date: Accepted date:
31-7-2017 5-10-2017 11-10-2017
Please cite this article as: Moroncini Gianluca, Baroni Silvia Svegliati, Gabrielli Armando.Agonistic antibodies in systemic sclerosis.Immunology Letters https://doi.org/10.1016/j.imlet.2017.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Agonistic antibodies in systemic sclerosis Gianluca Moroncini, Silvia Svegliati Baroni, Armando Gabrielli
Dipartimento di Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Ancona, 60126 Italy.
Correspondence: Armando Gabrielli, Dipartimento di Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Ancona, 60126 Italy. E-mail: [email protected]
Highlights • • • • •
Agonistic antibodies activate pathways leading to tissue and vascular damage in SSc Anti-endothelial cells antibodies (AECA) can induce either apoptosis or activation Antibodies targeting specific PDGFRα epitopes can induce skin fibrosis in vivo Anti-AT1R/ETaR antibodies are associated with severe SSc vascular manifestations Anti-muscarinic-3 receptor (M3R) antibodies may cause gastrointestinal dysfunction
Introduction Systemic sclerosis (SSc) is characterized by microangiopathy, excessive fibrosis, and the presence of circulating autoantibodies to several cellular and extracellular components. The role of autoimmunity in generating the clinical and pathologic phenotypes in SSc has been long debated and is still matter of controversy. Distinct specificities of antinuclear antibodies (ANAs) are selectively detected in SSc patients and are associated with unique disease manifestations, but do not have a proven pathogenic role. A new group of autoantibodies reactive with cell surface receptors have been identified in SSc patients. They have been shown to directly activate pathways that may contribute to tissue and vascular damage. As such, they are proposed to have
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a role as agonistic autoantibodies in SSc. According to Koch’s third postulate, the autoantibodies in question should cause disease when introduced into a healthy subject. Therefore, our review will focus on those autoantibodies for which agonistic activity has already been demonstrated not only in vitro, but, at least partly, also in vivo. These include the antibodies anti-endothelial cells (AECA), anti-Platelet-Derived Growth Factor Receptor (PDGFR), anti-Angiotensin II type 1 receptor (AT1R) and anti-endothelin-1 type A receptor (ETaR). In this review, we will discuss also a class of antagonistic autoantibodies, the anti-muscarinic-3 receptor (M3R) antibodies, since they seem to fulfill the aforementioned requirements.
Autoantibodies against endothelial cells (AECA) The original discovery. Anti-endothelial cells antibodies (AECA) were first identified in the early 1970s in sera of patients with different rheumatic diseases [1]. Later on, AECA have been described in other connective tissue diseases as well as in patients with diabetes mellitus, multiple sclerosis and pre-eclampsia [2]. Although AECA are not specific to SSc, they have been observed in a variable proportion of SSc patients, apparently defining patients with lung and vascular involvement [3,4].
The definition of the target antigen(s) and function in vitro. Many attempts to identify the target antigen(s) of AECA have failed, raising doubts on their role as pathogenetic agents in the induction of the endothelial dysfunction and damage observed in SSc. The target antigen of AECA was first reported in the study conducted by Lunardi et al [5]. They were able to identify in SSc patients circulating antibodies recognizing human cytomegalovirus (CMV) late protein UL94 that were capable to induce endothelial cell apoptosis in vitro by cross-reaction with the cell surface tetraspanin transmembrane 4 superfamily member 7 (TM4SF7 or Nag-2) molecule. Later on, the same group showed that Nag-2 is also expressed on dermal fibroblasts and that anti-Nag-2 antibodies, upon binding to fibroblasts, induce up-regulation of 989 transcripts including genes involved in extracellular matrix deposition and encoding growth factors, chemokines and cytokines
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Commented [LJv1]: Not necessarily animal.
[6]. These findings are of interest since i) they show a molecular target of AECA; ii) they indicate a possible link between a viral agent and SSc; iii) they suggest a novel pathogenic mechanism by which human CMV may cause vascular injury; iv) they support the molecular mimicry model of autoimmunity. Using a quantitative immunoblotting technique, it has been reported that the main target of AECA in patients with limited cutaneous SSc (lcSSc) is the nuclear centromeric protein B (CENP-B) [7]. This data is in agreement with the study of Hill et al [8] in patients with lcSSc. The functional relevance of this finding remains, however, elusive, given the nuclear localization of the antigen. In a more recent study aimed at defining the functionality of AECA in patients with connective tissue diseases, IgG from AECA-positive SSc patients induced human umbilical vein endothelial cell (HUVEC) activation in vitro, characterized by significantly higher expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and Eselectin, and production of IL-6, IL-8 and CCL2 compared to IgG from AECA-negative patients and normal donors [9]. Recently, Wolf et al [10], detected anti-ICAM-1 antibodies in 10/31 (32%) patients with diffuse cutaneous SSc and in 14/36 (39%) patients with lcSSc using ELISA. Interestingly, exposure of HUVEC to anti-ICAM-1 antibodies induced increased generation of reactive oxygen species (ROS) and VCAM-1 expression. These findings suggest that AECA include antibodies targeting ICAM-1 which cause pro-inflammatory activation of HUVEC, and thus may contribute to SSc vascular lesions.
The first evidence of agonistic activity in vivo. A potential pathogenic role of AECA has been suggested by their ability to induce apoptosis in human dermal microvascular endothelial cells, but not in HUVEC, in the presence of activated NK cells via the Fas pathway [11]. In vivo, transfer of AECA-positive serum from UCD-200 chickens, an animal model of human SSc, but not AECAnegative serum, resulted in induction of endothelial cellapoptosis in healthy chicken embryos [12]. Laplante et al [13] have proposed a general mechanism by which AECA may cause SSc lesions linking endothelial cell apoptosis and fibrosis. Apoptotic endothelial cells would release soluble mediators responsible for the induction of an anti-apoptotic phenotype in fibroblasts. Besides resistance to apoptosis, dermal fibroblasts would acquire a myofibroblast phenotype that
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Commented [LJv2]: HUVEC? Please specify.
constitutes the cellular basis of a persistent pro-fibrotic response. Interestingly, in the same study [13], human fibroblasts derived from SSc skin lesions were found to be more sensitive to the antiapoptotic activities of mediators produced by apoptotic endothelial cells than normal fibroblasts.
Commented [LJv3]: Add reference.
The molecular pattern of resistance to apoptosis in fibroblasts was reproduced by a synthetic
Commented [G4]: Same reference [13] as better indicated two lines above
peptide containing an endothelial growth factor (EGF) motif present on the C-terminal fragment of perlecan. Thus, persistent apoptosis of endothelial cells would induce and maintain fibrosis in SSc.
Autoantibodies against Platelet-Derived Growth Factor Receptor (PDGFR)
The background of the hypothesis and the original discovery. Platelet-Derived Growth Factor Receptor (PDGFR) can mediate the activation of cell types playing a fundamental role in SSc pathogenesis such as fibroblasts and smooth muscle cells. Yamakage et al [14] had reported a selective PDGFR up-regulation mediated by Transforming Growth Factor (TGF) β in SSc fibroblasts, making fibroblasts more sensitive to PDGF. Later on, it was demonstrated that PDGFR activation by PDGF triggers in normal fibroblasts an increased production of reactive oxygen species (ROS). ROS, in turn, activate the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway, which induces the viral Harvey rat sarcoma (Ha-Ras) gene. Activation of ERK1/2 and high ROS levels stabilize Ha-Ras protein, by inhibiting proteasomal degradation. As compared with normal cells, fibroblasts in SSc are characterized by an amplified and persistent ROS–ERK1/2– Ha-Ras signaling loop, which stimulates excessive collagen synthesis [15]. Importantly, constitutively elevated Ha-Ras protein levels in SSc fibroblasts can directly stimulate collagen I accumulation independently of TGFβ neo-synthesis and activation [16]. Thus, it was hypothesized that the presence of an external non-physiological factor, e.g. non-PDGF, non-TGFβ, might sustain
Commented [LJv5]: A bit vague this sentence, aren’t TGF and PDGF physiological then?
the profibrotic phenotype of SSc fibroblasts, like an anti-PDGFR stimulatory autoantibody. Such
Commented [G6]: By saying “non-PDGF, non-TGFβ” we meant indeed “non- physiological”. We can remove this sentence if it is misleading.
antibody was actually detected in whole IgG purified from serum of 46 SSc patients, which could
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immunoprecipitate PDGFR from human fibroblasts and stimulate ROS production and ROSERK1/2-Ha-Ras loop in mouse embryo fibroblasts expressing human PDGFR α (Fα) but not in PDGFRα negative cell conterpart (F-/-). Remarkably, IgG purified from serum of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Raynaud’s phenomenon (PRP), or interstitial lung disease (ILD) without SSc, did not display these binding and biological characteristics [17].
The controversy. Afterwards, some research groups tried to replicate this finding. However, even the best attempts failed to detect agonistic anti-PDGFR antibodies in serum of SSc patients [18,19]. This was mostly due to the use of different read-out methods, based on cell lines (32D myeloid cells and porcine aortic endothelial cells, respectively) much different from the Fα employed in the original study, as pointed out in the reply [20]. Nevertheless, replicating the original bioassay was too a difficult task and further assays providing evidence of PDGFR autoantibodies in SSc were rightly required by the scientific community[21].
The confirmation of the initial finding. In order to confirm the initial finding, the immune repertoire of one SSc patient was directly investigated. Thus, the memory B cells of this patient were isolated from peripheral blood mononuclear cells, seeded by limiting dilution in hundreds of cell culture plates, and the supernatant of each well was screened for the presence of anti-human PDGFRα IgG, using again the aforementioned Fα cells with F-/- as negative control. This methodology allowed the identification of a few B cell clones characterized by PDGFRα reactivity, from which antibody heavy and light chains were cloned and recombined in vitro to generate the
Commented [LJv7]: Why is this exciting? Would a monoclonal, functional antibody not be more excititng?
corresponding anti-PDGFRα monoclonal autoantibodies. It was quite exciting to observe that the 4
Commented [G8]: It is exciting to discover that anti-PDGFR autoimmune response is not consisting of a single antibody but a panel of antibodies directed toward different epitopes of the PDGFR, some relevant to disease phenotype, some not. This PDGFR epitope map was completely unknown before this study and shed light on the role of PDGFR in systemic sclerosis, explaining much of the controversial results on these autoantibodies: talking about anti-PDGFR autoantibodies is generic, whereas epitope-specific autoantibodies are diseasespecific. It was also exciting, from an immunological point of view, discovering that this different reactivity was based upon “light chain shuffling” whereas the heavy chain remains the same: this is related to the phenomenon known as ‘B cell receptor editing’. These considerations may be included in the text, eventually.
different monoclonals, differing only in the light chains, had distinct binding and functional features. In fact, binding and agonistic activities towards PDGFRα were dissociated in these autoantibodies, ranging from very low affinity binding without any agonistic activity towards fibroblasts to high affinity binding with induction of the ROS-ERK1/2-Ha-Ras loop and increased collagen gene transcription in human fibroblasts in vitro [22,23]. The use of a large conformational PDGFRα
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peptide library enabled the dissection of the PDGFRα epitope recognized by the nonagonistic antibody, i.e. one linear aminoacid sequence in the first extracellular PDGFRα domain, from the conformational epitope of the agonistic antibody, i.e. a discontinuous motif encompassing 3 distinct sequences across the second and third extracellular PDGFRα domains, largely overlapping with the PDGF binding region [22]. An epitope-based assay to detect only agonistic anti-PDGFRα autoantibodies directed towards this discontinuous motif has been designed and preliminarily tested [OP0031 Annual European Congress of Rheumatology EULAR 2017], and awaits validation. On the other hand, an ELISA detecting all serum anti-PDGFRα autoantibodies regardless of the different epitopes has already been developed. By this assay, total anti-PDGFRα antibodies were detected in 66 of 70 SSc patients (94.3%), 63 of 130 healthy controls (48.5%), 11 of 26 PRP patients (42.3%), 11 of 29 SLE patients (37.9%) (P < 0.0001 between SSc patients and the other groups). Importantly, IgG purified from ELISA-positive SSc serum samples turned out to be positive also in ROS bioassay, whereas IgG purified from ELISA-positive healthy controls serum samples did not. This indicates, again, that anti-PDGFRα antibodies directed toward non-
Commented [LJv9]: So what does this mean? Please add a sentence to explain to the uninitiated reader.
stimulatory epitopes of the receptor may be present in healthy controls, whereas anti-PDGFRα antibodies directed toward stimulatory epitopes are SSc-specific [22]. In addition, SSc serum antiPDGFRα antibodies with receptor affinity as high as the collagen-inducing monoclonal autoantibody can be detected by ELISA [23]. Overall, this scientific information may reconcile the controversial results concerning the existence of stimulatory anti-PDGFRα antibodies in SSc. In fact, these findings demonstrate that agonistic and nonagonistic anti-PDGFRα autoantibodies may coexist in the same SSc patient. While nonagonistic anti-PDGFRα autoantibodies can be detected also in healthy controls or patients affected by PRP or SLE,
agonistic anti-PDGFRα
autoantibodies are SSc-specific and recognize precise conformational epitopes, which must be preserved in binding assays to discriminate agonistic from total antibodies. Whereas the nonagonistic anti-PDGFRα autoantibodies detectable in subjects not affected from SSc can be considered as a product of the natural autoimmune repertoire[24], the agonistic anti-PDGFRα autoantibodies are part of the SSc-specific, deranged autoimmune response towards cellular antigens, possibly amplified by epitope spreading[25].
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Commented [LJv10]: Can you speculate how this difference can be explained?
The first evidence of agonistic activity in vivo. Besides confirming the existence of agonistic anti-PDGFRα autoantibodies in SSc patients, it was also mandatory to provide evidence of agonistic activity of these autoantibodies in vivo. The first, indirect evidence came from a small clinical study involving six SSc patients with severe skin fibrosis, unresponsive to canonical immunosuppressive therapies, who were treated with 375 mg/m2 per week of rituximab for a total of four doses. The good clinical response observed in all patients, evaluated as decrease of the skin score and improvement of the disability indices, was accompanied by a significant reduction of ROS stimulatory activity in vitro by IgG purified from serum of patients, and downregulation of the
Commented [LJv11]: Titer of? Please specify what you mean with reduction.
aforementioned intracellular signaling pathway and type I collagen gene expression in fibroblasts
Commented [G12]: Reduction of serum anti-PDGFRα antibodies in this study was measured by the cell-based functional bioassay (ROS bioassay) originally described in ref [17]. So, the term “titer” would not be appropriate because such assay does not use a serum titration but one standard concentration of IgG purified from serum. In order to avoid any ambiguity, I have rephrased this concept.
grown from skin biopsies performed at baseline and at months 3 and 6 post-treatment [26]. To obtain direct evidence, three-dimensional bioengineered skin samples containing human keratinocytes and fibroblasts isolated from skin biopsies of healthy donors were generated and grafted onto the back of SCID mice. The dermis of the skin grafts was then injected either with total IgG purified from serum of SSc patients (SSc IgG) or healthy controls (HC IgG), either with the agonistic, collagen-inducing anti-PDGFRα monoclonal antibody or with the nonagonistic one (both reported above). Unlike HC IgG, SSc IgG injection induced increase of human collagen deposition and fibroblast activation markers in healthy donor skin grafts. Replication of this scleroderma-like phenotype by injection of the agonistic monoclonal antibody, but not the nonagonistic one, directly demonstrated both the involvement of PDGFRα activation in the pathogenesis of skin fibrosis in vivo, and the profibrotic role in vivo of agonistic anti-PDGFRα autoantibodies [27].
Other cellular targets. Agonistic anti-PDGFRα autoantibodies (both total IgG and the collageninducing monoclonal antibody mentioned before) have been recently demonstrated to induce proliferation and migration of human pulmonary vascular smooth muscle cells in vitro [28]. This is an important finding further corroborating the agonistic activity of this class of autoantibodies and its contribution to the pathogenesis of SSc and potentially to SSc-related conditions such as pulmonary arterial hypertension (PAH), which awaits confirmation in vivo.
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Autoantibodies against angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETaR)
The background of the hypothesis and the original discovery. Angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETaR) are widely expressed on cells of the vascular system and on immune cells. Functional anti-AT1R autoantibodies have first been reported by Wallukat et al. in 1999 [29]. The autoantibodies were isolated from sera of preeclamptic women, and their agonistic activity was demonstrated by the chronotropic effect induced on cultured spontaneously beating neonatal rat cardiomyocytes, which was completely inhibited by the selective receptor antagonist losartan [30]. Subsequently, numerous studies have shown that antiAT1R autoantibodies stimulate AT1R on several cell types inducing biological responses relevant to the pathophysiology of vascular diseases, such as malignant hypertension, renovascular diseases, renal allograft rejection, in which AT1R aAbs were also detected [31,32]. Anti-ETAR autoantibodies have been first identified in sera from patients with idiopathic PAH [33]. In sera from SSc patients, anti-AT1R and anti-ETAR autoantibodies were first detected by Riemekasten et al by solid phase assay [34]. Anti-AT1R and anti-ETAR antibodies were found in about 85% of SSc patients, who have increased levels of anti-AT1R and anti-ETAR antibodies compared to healthy donors. In patients with SSc, the antibody levels strongly correlate with each other and show cross-reactivity for both receptors.
Clinical associations. Higher levels of anti-AT1R and anti-ETAR antibodies were associated with severe SSc vascular manifestations such as digital ulcers and PAH [34]. Becker et al [35] reported that anti-AT1R and anti-ETaR antibodies are more frequent in PAH associated to SSc or other connective tissue diseases compared with other forms of pulmonary hypertension and may serve as prognostic and predictive biomarkers (cardiovascular complications and mortality).
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In vitro agonistic activity. Interestingly, these autoantibodies induce ERK1/2 phosphorylation and increased TGFβ gene expression in human dermal microvascular endothelial cells [34]. Furthermore, when exposed to anti-AT1R and anti-ETaR antibodies, human microvascular endothelial cells (HMEC) show evidence of increased expression of IL-18 and vascular cell adhesion molecule-1 (VCAM-1) [36]. The activation of HMEC induced in such a way was responsible for increased neutrophil migration through an endothelial cell layer. More recently, AT1R and ETaR were also detected on human peripheral T cells, B cells, and monocytes, and their interaction with IgG from SSc patients was responsible for increased production of IL-8 and CC-chemokine ligand 18 (CCL18) [37].
In vivo agonistic activity. Healthy C57BL-6 mice were treated with repeated intravenous administrations of total SSc IgG testing positive for anti-AT1R and anti-ETAR antibodies. Histological analysis of the lungs, performed by hematoxylin and eosin (H&E) staining, showed thickening of airway vessels, and elevated cell density in interstitial tissue. However, no specific collagen staining was performed. Increased neutrophil count was found in bronchoalveolar lavage (BAL) of SSc IgG-treated mice as compared with HC IgG-treated mice, whereas no differences were observed for macrophages or lymphocytes [37]. In absence of human agonistic monoclonal anti-AT1R and anti-ETAR antibodies, these findings should be corroborated by administration of total SSc IgG fractions enriched or depleted of antiAT1R and anti-ETAR antibodies, in order to separate the specific effects of anti-AT1R and antiETAR antibodies from those of total SSc IgG, which may contain other agonistic antibody species such as anti-PDGFRα autoantibodies. Thus, it remains to be established how these autoantibodies are responsible for the development of SSc-specific vascular lesions.
Autoantibodies against muscarinic-3 receptor (M3R) The background of the hypothesis and the original discovery. Gastrointestinal (GI) involvement leading to dysmotility is frequent in SSc as a result of disturbance of cholinergic
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neurotransmission and smooth muscle atrophy. Acetylcholine secreted after stimulation of the muscarinic-3 receptor (M3R) is the principal excitatory mediator of GI tract motility acting on intrinsic neurons in the myenteric plexus. Antibodies blocking M3R would therefore inhibit excitatory enteric neurotransmission causing dysmotility. Following this hypothesis, several studies found a high incidence of anti-myenteric neuronal antibodies in the sera of SSc patients with GI symptoms [38] and demonstrated that passive transfer of these antibodies into a rat model significantly disrupted intestinal myoelectric activity [39], further supporting a neuropathic etiology to dysmotility in SSc patients. The precise targeted neuronal antigen in these studies, however, remained to be determined. Later, Goldblatt et al [40] showed that IgG fractions from 7/9 patients with SSc, 4/4 patients with primary Sjögren’s syndrome (SS), and 3/3 patients with secondary SS inhibited animal colonic smooth muscle contraction caused by carbachol-induced activation of M3R suggesting that anti-M3R autoantibodies contained in SSc IgG (but also in SS IgG) may lead to failure of the cholinergic neurotransmission and, in turn, result in GI motility dysfunction. Inhibition by patients’ IgG was concentration dependent, whereas HC IgG did not inhibit the M3Rmediated contractions.
The confirmation of the initial finding and further in vivo evidence. These results were confirmed and expanded by Kawaguchi et al [41] who used an enzyme immunoassay and found anti-M3R antibodies in 9/14 early-onset SSc patients with severe GI tract involvement compared to only 3 positive out of 62 early-onset SSc patients without severe GI tract involvement; and by Singh et al [42] who provided evidence that IgG from SSc patients attenuate the M3R activation in smooth muscle cells of a rat internal anal sphincter.
The controversy. Recently, Preuss et al [43] using a novel luminescence-based test to detect functionally active antibodies to M3R, could not find antibodies inhibiting carbachol-induced activation of M3R in a cohort of 47 SSc patients, but in 20/40 primary SS patients.
Commented [LJv13]: SS = Sjögren syndrome here? Commented [G14]: Yes, the abbreviation is indicated before, in the first paragraph of this chapter
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Conclusions and future perspectives The discovery in SSc patients of several types of serum autoantibodies carrying agonistic or antagonistic function towards different cell types involved in different aspects of disease pathogenesis may provide many advantages in the management of this heterogeneous condition lacking targeted therapies [44]. First, functional autoantibodies may help identify clinical variants of SSc. This task will require the screening of large, multicenter, well characterized patient populations with optimized assays based on selected epitopes of the target receptors rather than whole receptors, followed by correlation studies between serum reactivity profile and patient clinical features. For most autoantibodies reported in this review, this aim seems to be not too far away: Nag-2 and ICAM-1 for AECA, the well mapped epitope of the agonistic anti-PDGFRα monoclonal autoantibody, and the second extracellular loop of the M3R identified as a target of anti-M3R autoantibodies in patients with SS [45] may represent good candidates to define the serum reactivity profile of SSc patients cohorts. Identification of AT1R and ETaR epitopes will help keeping these receptors and the relative autoantibodies in the picture. Second, the identification of agonistic and antagonistic autoantibodies is very important to shed light into the pathogenesis of SSc: unraveling of the signaling cascade triggered or inhibited by the functional autoantibodies may in fact pave the way to novel therapeutic approaches. These therapeutic strategies should not rely on unselective block of total receptor activity, in order to avoid adverse effects, but on selective targeting of the receptors’ “hot spots” with small molecule inhibitors or selective capture of the agonistic/antagonistic antibodies with decoy mini-receptors or mimotopes, i.e. peptides mimicking the relevant epitopes bound by the functional autoantibodies. Alternative strategies such as anti-idiotype antibodies or tolerogenic peptides may also be rewarding. Clearly, all these preclinical therapeutic steps will require that robust animal models based on these functional autoantibodies reproduce consistent features of SSc. In sum, these observations provide an important step in the understanding of the mechanisms of interaction between autoimmunity and the different tissue/organ involvements in SSc, suggesting many potential avenues for future research activities in this field.
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References
[1]
Lindqvist KJ, Osterland CK. Human antibodies to vascular endothelium. Clin Exp Immunol 1971;9:753-760.
[2]
Belizna C, Tervaert JW. Specificity, pathogenecity, and clinical value of antiendothelial cell antibodies. Semin Arthritis Rheum 1997;27:98-109.
[3]
Salojin KV, Le Tonqueze M, Saraux A, Nassonov EL, Dueymes M, Piette JC, Youinou PY. Antiendothelial cell antibodies: useful markers of systemic sclerosis. Am J Med 1997;102:178-185.
[4]
Pignone A, Scaletti C, Matucci-Cerinic M, Vazquez-Abad D, Meroni PL, Del Papa N, Falcini F, Generini S, Rothfield N, Cagnoni M. Anti-endothelial cell antibodies in systemic sclerosis: significant association with vascular involvement and alveolocapillary impairment. Clin Exp Rheumatol 1998;16:527-532.
[5]
Lunardi C, Bason C, Navone R, Millo E, Damonte G, Corrocher R, Puccetti A. Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells. Nat Med 2000;6:11831186.
[6]
Lunardi C, Dolcino M, Peterlana D, Bason C, Navone R, Tamassia N, Beri R, Corrocher R, Puccetti A. Antibodies against human cytomegalovirus in the pathogenesis of systemic sclerosis: a gene array approach. PLoS Med 2006;3:e2.
[7]
Servettaz A, Tamby MC, Guilpain P, Reinbolt J, Garcia de la Pena-Lefebvre P, Allanore Y, Kahan A, Meyer O, Guillevin L, Mouthon L. Anti-endothelial cell antibodies from patients with limited cutaneous systemic sclerosis bind to centromeric protein B (CENP-B). Clin Immunol 2006;120:212-219. 12
[8]
Hill MB, Phipps JL, Cartwright RJ, Milford Ward A, Greaves M, Hughes P. Antibodies to membranes of endothelial cells and fibroblasts in scleroderma. Clin Exp Immunol 1996;106:491-497.
[9]
Arends SJ, Damoiseaux JG, Duijvestijn AM, Debrus-Palmans L, Vroomen M, Boomars KA, Brunner-La Rocca HP, Reutelingsperger CP, Cohen Tervaert JW, van Paassen P. Immunoglobulin G anti-endothelial cell antibodies: inducers of endothelial cell apoptosis in pulmonary arterial hypertension? Clin Exp Immunol;174:433-440.
[10]
Wolf SI, Howat S, Abraham DJ, Pearson JD, Lawson C. Agonistic anti-ICAM-1 antibodies in scleroderma: activation of endothelial pro-inflammatory cascades. Vascul Pharmacol;59:19-26.
[11]
Sgonc R, Gruschwitz MS, Boeck G, Sepp N, Gruber J, Wick G. Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95. Arthritis Rheum 2000;43:2550-2562.
[12]
Worda M, Sgonc R, Dietrich H, Niederegger H, Sundick RS, Gershwin ME, Wick G. In vivo analysis of the apoptosis-inducing effect of anti-endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay. Arthritis Rheum 2003;48:2605-2614.
[13]
Laplante P, Raymond MA, Gagnon G, Vigneault N, Sasseville AM, Langelier Y, Bernard M, Raymond Y, Hebert MJ. Novel fibrogenic pathways are activated in response to endothelial apoptosis: implications in the pathophysiology of systemic sclerosis. J Immunol 2005;174:5740-5749.
[14]
Yamakage A, Kikuchi K, Smith EA, LeRoy EC, Trojanowska M. Selective upregulation of platelet-derived growth factor alpha receptors by transforming growth factor beta in scleroderma fibroblasts. J Exp Med 1992;175:1227-1234.
13
[15]
Svegliati S, Cancello R, Sambo P, Luchetti M, Paroncini P, Orlandini G, Discepoli G, Paterno R, Santillo M, Cuozzo C, Cassano S, Avvedimento EV, Gabrielli A. Plateletderived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts. J Biol Chem 2005;280:36474-36482.
[16]
Smaldone S, Olivieri J, Gusella GL, Moroncini G, Gabrielli A, Ramirez F. Ha-Ras stabilization mediates pro-fibrotic signals in dermal fibroblasts. Fibrogenesis Tissue Repair 2011;4:8.
[17]
Baroni SS, Santillo M, Bevilacqua F, Luchetti M, Spadoni T, Mancini M, Fraticelli P, Sambo P, Funaro A, Kazlauskas A, Avvedimento EV, Gabrielli A. Stimulatory autoantibodies to the PDGF receptor in systemic sclerosis. N Engl J Med 2006;354:2667-2676.
[18]
Classen JF, Henrohn D, Rorsman F, Lennartsson J, Lauwerys BR, Wikstrom G, Rorsman C, Lenglez S, Franck-Larsson K, Tomasi JP, Kampe O, Vanthuyne M, Houssiau FA, Demoulin JB. Lack of evidence of stimulatory autoantibodies to platelet-derived growth factor receptor in patients with systemic sclerosis. Arthritis Rheum 2009;60:1137-1144.
[19]
Loizos N, Lariccia L, Weiner J, Griffith H, Boin F, Hummers L, Wigley F, Kussie P. Lack of detection of agonist activity by antibodies to platelet-derived growth factor receptor alpha in a subset of normal and systemic sclerosis patient sera. Arthritis Rheum 2009;60:1145-1151.
[20]
Gabrielli A, Moroncini G, Svegliati S, Avvedimento EV. Autoantibodies against the platelet-derived growth factor receptor in scleroderma: comment on the articles by Classen et al and Loizos et al. Arthritis Rheum 2009;60:3521-3522.
14
[21]
Dragun D, Distler JH, Riemekasten G, Distler O. Stimulatory autoantibodies to plateletderived growth factor receptors in systemic sclerosis: what functional autoimmunity could learn from receptor biology. Arthritis Rheum 2009;60:907-911.
[22]
Moroncini G, Grieco A, Nacci G, Paolini C, Tonnini C, Pozniak KN, Cuccioloni M, Mozzicafreddo M, Svegliati S, Angeletti M, Kazlauskas A, Avvedimento EV, Funaro A, Gabrielli A. Epitope Specificity Determines Pathogenicity and Detectability of AntiPlatelet-Derived Growth Factor Receptor alpha Autoantibodies in Systemic Sclerosis. Arthritis Rheumatol;67:1891-1903.
[23]
Moroncini G, Cuccioloni M, Mozzicafreddo M, Pozniak KN, Grieco A, Paolini C, Tonnini C, Spadoni T, Svegliati S, Funaro A, Angeletti M, Gabrielli A. Characterization of binding and quantification of human autoantibodies to PDGFRalpha using a biosensor-based approach. Anal Biochem;528:26-33.
[24]
Poletaev AB, Stepanyuk VL, Gershwin ME. Integrating immunity: the immunculus and self-reactivity. J Autoimmun 2008;30:68-73.
[25]
Cornaby C, Gibbons L, Mayhew V, Sloan CS, Welling A, Poole BD. B cell epitope spreading: mechanisms and contribution to autoimmune diseases. Immunol Lett 2015;163:56-68.
[26]
Fraticelli P, De Vita S, Franzolini N, Svegliati S, Scott CA, Tonnini C, Spadoni T, Gabrielli B, Pomponio G, Moroncini G, Gabrielli A. Reduced type I collagen gene expression by skin fibroblasts of patients with systemic sclerosis after one treatment course with rituximab. Clin Exp Rheumatol;33:S160-167.
[27]
Luchetti MM, Moroncini G, Jose Escamez M, Svegliati Baroni S, Spadoni T, Grieco A, Paolini C, Funaro A, Avvedimento EV, Larcher F, Del Rio M, Gabrielli A. Induction of Scleroderma Fibrosis in Skin-Humanized Mice by Administration of Anti-Platelet-
15
Derived
Growth
Factor
Receptor
Agonistic
Autoantibodies.
Arthritis
Rheumatol;68:2263-2273. [28]
Svegliati S, Amico D, Spadoni T, Fischetti C, Finke D, Moroncini G, Paolini C, Tonnini C, Grieco A, Rovinelli M, Funaro A, Gabrielli A. Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro. Front Immunol;8:75.
[29]
Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, Baur E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest 1999;103:945-952.
[30]
Xia Y, Kellems RE. Is preeclampsia an autoimmune disease? Clin Immunol 2009;133:112.
[31]
Fu ML, Herlitz H, Schulze W, Wallukat G, Micke P, Eftekhari P, Sjogren KG, Hjalmarson A, Muller-Esterl W, Hoebeke J. Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension. J Hypertens 2000;18:945-953.
[32]
Dragun D, Muller DN, Brasen JH, Fritsche L, Nieminen-Kelha M, Dechend R, Kintscher U, Rudolph B, Hoebeke J, Eckert D, Mazak I, Plehm R, Schonemann C, Unger T, Budde K, Neumayer HH, Luft FC, Wallukat G. Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection. N Engl J Med 2005;352:558-569.
[33]
Wallukat G, Schimke I. Agonistic autoantibodies directed against G-protein-coupled receptors
and
their
relationship
to
cardiovascular
diseases.
Semin
Immunopathol;36:351-363. [34]
Riemekasten G, Philippe A, Nather M, Slowinski T, Muller DN, Heidecke H, MatucciCerinic M, Czirjak L, Lukitsch I, Becker M, Kill A, van Laar JM, Catar R, Luft FC,
16
Burmester GR, Hegner B, Dragun D. Involvement of functional autoantibodies against vascular receptors in systemic sclerosis. Ann Rheum Dis 2011;70:530-536. [35]
Becker MO, Kill A, Kutsche M, Guenther J, Rose A, Tabeling C, Witzenrath M, Kuhl AA, Heidecke H, Ghofrani HA, Tiede H, Schermuly RT, Nickel N, Hoeper MM, Lukitsch I, Gollasch M, Kuebler WM, Bock S, Burmester GR, Dragun D, Riemekasten G. Vascular receptor autoantibodies in pulmonary arterial hypertension associated with systemic sclerosis. Am J Respir Crit Care Med 2014;190:808-817.
[36]
Gunther J, Kill A, Becker MO, Heidecke H, Rademacher J, Siegert E, Radic M, Burmester GR, Dragun D, Riemekasten G. Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients. Arthritis Res Ther;16:R65.
[37]
Kill A, Tabeling C, Undeutsch R, Kuhl AA, Gunther J, Radic M, Becker MO, Heidecke H, Worm M, Witzenrath M, Burmester GR, Dragun D, Riemekasten G. Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis. Arthritis Res Ther;16:R29.
[38]
Howe S, Eaker EY, Sallustio JE, Peebles C, Tan EM, Williams RC, Jr. Antimyenteric neuronal antibodies in scleroderma. J Clin Invest 1994;94:761-770.
[39]
Eaker EY, Kuldau JG, Verne GN, Ross SO, Sallustio JE. Myenteric neuronal antibodies in scleroderma: passive transfer evokes alterations in intestinal myoelectric activity in a rat model. J Lab Clin Med 1999;133:551-556.
[40]
Goldblatt F, Gordon TP, Waterman SA. Antibody-mediated gastrointestinal dysmotility in scleroderma. Gastroenterology 2002;123:1144-1150.
[41]
Kawaguchi Y, Nakamura Y, Matsumoto I, Nishimagi E, Satoh T, Kuwana M, Sumida T, Hara M. Muscarinic-3 acetylcholine receptor autoantibody in patients with systemic 17
sclerosis: contribution to severe gastrointestinal tract dysmotility. Ann Rheum Dis 2009;68:710-714. [42]
Singh J, Mehendiratta V, Del Galdo F, Jimenez SA, Cohen S, DiMarino AJ, Rattan S. Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 2009;297:G1206-1213.
[43]
Preuss B, Tunaru S, Henes J, Offermanns S, Klein R. A novel luminescence-based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients' sera. Clin Exp Immunol;177:179-189.
[44]
Kowal-Bielecka O, Fransen J, Avouac J, Becker M, Kulak A, Allanore Y, Distler O, Clements P, Cutolo M, Czirjak L, Damjanov N, Del Galdo F, Denton CP, Distler JHW, Foeldvari I, Figelstone K, Frerix M, Furst DE, Guiducci S, Hunzelmann N, Khanna D, Matucci-Cerinic M, Herrick AL, van den Hoogen F, van Laar JM, Riemekasten G, Silver R, Smith V, Sulli A, Tarner I, Tyndall A, Welling J, Wigley F, Valentini G, Walker UA, Zulian F, Muller-Ladner U. Update of EULAR recommendations for the treatment of systemic sclerosis. Ann Rheum Dis;76:1327-1339.
[45]
Cavill D, Waterman SA, Gordon TP. Antibodies raised against the second extracellular loop of the human muscarinic M3 receptor mimic functional autoantibodies in Sjogren's syndrome. Scand J Immunol 2004;59:261-266.
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S0165-2478(17)30364-4 https://doi.org/10.1016/j.imlet.2017.10.007 IMLET 6126
To appear in:
Immunology Letters
Received date: Revised date: Accepted date:
31-7-2017 5-10-2017 11-10-2017
Please cite this article as: Moroncini Gianluca, Baroni Silvia Svegliati, Gabrielli Armando.Agonistic antibodies in systemic sclerosis.Immunology Letters https://doi.org/10.1016/j.imlet.2017.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Agonistic antibodies in systemic sclerosis Gianluca Moroncini, Silvia Svegliati Baroni, Armando Gabrielli
Dipartimento di Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Ancona, 60126 Italy.
Correspondence: Armando Gabrielli, Dipartimento di Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Ancona, 60126 Italy. E-mail: [email protected]
Highlights • • • • •
Agonistic antibodies activate pathways leading to tissue and vascular damage in SSc Anti-endothelial cells antibodies (AECA) can induce either apoptosis or activation Antibodies targeting specific PDGFRα epitopes can induce skin fibrosis in vivo Anti-AT1R/ETaR antibodies are associated with severe SSc vascular manifestations Anti-muscarinic-3 receptor (M3R) antibodies may cause gastrointestinal dysfunction
Introduction Systemic sclerosis (SSc) is characterized by microangiopathy, excessive fibrosis, and the presence of circulating autoantibodies to several cellular and extracellular components. The role of autoimmunity in generating the clinical and pathologic phenotypes in SSc has been long debated and is still matter of controversy. Distinct specificities of antinuclear antibodies (ANAs) are selectively detected in SSc patients and are associated with unique disease manifestations, but do not have a proven pathogenic role. A new group of autoantibodies reactive with cell surface receptors have been identified in SSc patients. They have been shown to directly activate pathways that may contribute to tissue and vascular damage. As such, they are proposed to have
1
a role as agonistic autoantibodies in SSc. According to Koch’s third postulate, the autoantibodies in question should cause disease when introduced into a healthy subject. Therefore, our review will focus on those autoantibodies for which agonistic activity has already been demonstrated not only in vitro, but, at least partly, also in vivo. These include the antibodies anti-endothelial cells (AECA), anti-Platelet-Derived Growth Factor Receptor (PDGFR), anti-Angiotensin II type 1 receptor (AT1R) and anti-endothelin-1 type A receptor (ETaR). In this review, we will discuss also a class of antagonistic autoantibodies, the anti-muscarinic-3 receptor (M3R) antibodies, since they seem to fulfill the aforementioned requirements.
Autoantibodies against endothelial cells (AECA) The original discovery. Anti-endothelial cells antibodies (AECA) were first identified in the early 1970s in sera of patients with different rheumatic diseases [1]. Later on, AECA have been described in other connective tissue diseases as well as in patients with diabetes mellitus, multiple sclerosis and pre-eclampsia [2]. Although AECA are not specific to SSc, they have been observed in a variable proportion of SSc patients, apparently defining patients with lung and vascular involvement [3,4].
The definition of the target antigen(s) and function in vitro. Many attempts to identify the target antigen(s) of AECA have failed, raising doubts on their role as pathogenetic agents in the induction of the endothelial dysfunction and damage observed in SSc. The target antigen of AECA was first reported in the study conducted by Lunardi et al [5]. They were able to identify in SSc patients circulating antibodies recognizing human cytomegalovirus (CMV) late protein UL94 that were capable to induce endothelial cell apoptosis in vitro by cross-reaction with the cell surface tetraspanin transmembrane 4 superfamily member 7 (TM4SF7 or Nag-2) molecule. Later on, the same group showed that Nag-2 is also expressed on dermal fibroblasts and that anti-Nag-2 antibodies, upon binding to fibroblasts, induce up-regulation of 989 transcripts including genes involved in extracellular matrix deposition and encoding growth factors, chemokines and cytokines
2
Commented [LJv1]: Not necessarily animal.
[6]. These findings are of interest since i) they show a molecular target of AECA; ii) they indicate a possible link between a viral agent and SSc; iii) they suggest a novel pathogenic mechanism by which human CMV may cause vascular injury; iv) they support the molecular mimicry model of autoimmunity. Using a quantitative immunoblotting technique, it has been reported that the main target of AECA in patients with limited cutaneous SSc (lcSSc) is the nuclear centromeric protein B (CENP-B) [7]. This data is in agreement with the study of Hill et al [8] in patients with lcSSc. The functional relevance of this finding remains, however, elusive, given the nuclear localization of the antigen. In a more recent study aimed at defining the functionality of AECA in patients with connective tissue diseases, IgG from AECA-positive SSc patients induced human umbilical vein endothelial cell (HUVEC) activation in vitro, characterized by significantly higher expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and Eselectin, and production of IL-6, IL-8 and CCL2 compared to IgG from AECA-negative patients and normal donors [9]. Recently, Wolf et al [10], detected anti-ICAM-1 antibodies in 10/31 (32%) patients with diffuse cutaneous SSc and in 14/36 (39%) patients with lcSSc using ELISA. Interestingly, exposure of HUVEC to anti-ICAM-1 antibodies induced increased generation of reactive oxygen species (ROS) and VCAM-1 expression. These findings suggest that AECA include antibodies targeting ICAM-1 which cause pro-inflammatory activation of HUVEC, and thus may contribute to SSc vascular lesions.
The first evidence of agonistic activity in vivo. A potential pathogenic role of AECA has been suggested by their ability to induce apoptosis in human dermal microvascular endothelial cells, but not in HUVEC, in the presence of activated NK cells via the Fas pathway [11]. In vivo, transfer of AECA-positive serum from UCD-200 chickens, an animal model of human SSc, but not AECAnegative serum, resulted in induction of endothelial cellapoptosis in healthy chicken embryos [12]. Laplante et al [13] have proposed a general mechanism by which AECA may cause SSc lesions linking endothelial cell apoptosis and fibrosis. Apoptotic endothelial cells would release soluble mediators responsible for the induction of an anti-apoptotic phenotype in fibroblasts. Besides resistance to apoptosis, dermal fibroblasts would acquire a myofibroblast phenotype that
3
Commented [LJv2]: HUVEC? Please specify.
constitutes the cellular basis of a persistent pro-fibrotic response. Interestingly, in the same study [13], human fibroblasts derived from SSc skin lesions were found to be more sensitive to the antiapoptotic activities of mediators produced by apoptotic endothelial cells than normal fibroblasts.
Commented [LJv3]: Add reference.
The molecular pattern of resistance to apoptosis in fibroblasts was reproduced by a synthetic
Commented [G4]: Same reference [13] as better indicated two lines above
peptide containing an endothelial growth factor (EGF) motif present on the C-terminal fragment of perlecan. Thus, persistent apoptosis of endothelial cells would induce and maintain fibrosis in SSc.
Autoantibodies against Platelet-Derived Growth Factor Receptor (PDGFR)
The background of the hypothesis and the original discovery. Platelet-Derived Growth Factor Receptor (PDGFR) can mediate the activation of cell types playing a fundamental role in SSc pathogenesis such as fibroblasts and smooth muscle cells. Yamakage et al [14] had reported a selective PDGFR up-regulation mediated by Transforming Growth Factor (TGF) β in SSc fibroblasts, making fibroblasts more sensitive to PDGF. Later on, it was demonstrated that PDGFR activation by PDGF triggers in normal fibroblasts an increased production of reactive oxygen species (ROS). ROS, in turn, activate the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway, which induces the viral Harvey rat sarcoma (Ha-Ras) gene. Activation of ERK1/2 and high ROS levels stabilize Ha-Ras protein, by inhibiting proteasomal degradation. As compared with normal cells, fibroblasts in SSc are characterized by an amplified and persistent ROS–ERK1/2– Ha-Ras signaling loop, which stimulates excessive collagen synthesis [15]. Importantly, constitutively elevated Ha-Ras protein levels in SSc fibroblasts can directly stimulate collagen I accumulation independently of TGFβ neo-synthesis and activation [16]. Thus, it was hypothesized that the presence of an external non-physiological factor, e.g. non-PDGF, non-TGFβ, might sustain
Commented [LJv5]: A bit vague this sentence, aren’t TGF and PDGF physiological then?
the profibrotic phenotype of SSc fibroblasts, like an anti-PDGFR stimulatory autoantibody. Such
Commented [G6]: By saying “non-PDGF, non-TGFβ” we meant indeed “non- physiological”. We can remove this sentence if it is misleading.
antibody was actually detected in whole IgG purified from serum of 46 SSc patients, which could
4
immunoprecipitate PDGFR from human fibroblasts and stimulate ROS production and ROSERK1/2-Ha-Ras loop in mouse embryo fibroblasts expressing human PDGFR α (Fα) but not in PDGFRα negative cell conterpart (F-/-). Remarkably, IgG purified from serum of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Raynaud’s phenomenon (PRP), or interstitial lung disease (ILD) without SSc, did not display these binding and biological characteristics [17].
The controversy. Afterwards, some research groups tried to replicate this finding. However, even the best attempts failed to detect agonistic anti-PDGFR antibodies in serum of SSc patients [18,19]. This was mostly due to the use of different read-out methods, based on cell lines (32D myeloid cells and porcine aortic endothelial cells, respectively) much different from the Fα employed in the original study, as pointed out in the reply [20]. Nevertheless, replicating the original bioassay was too a difficult task and further assays providing evidence of PDGFR autoantibodies in SSc were rightly required by the scientific community[21].
The confirmation of the initial finding. In order to confirm the initial finding, the immune repertoire of one SSc patient was directly investigated. Thus, the memory B cells of this patient were isolated from peripheral blood mononuclear cells, seeded by limiting dilution in hundreds of cell culture plates, and the supernatant of each well was screened for the presence of anti-human PDGFRα IgG, using again the aforementioned Fα cells with F-/- as negative control. This methodology allowed the identification of a few B cell clones characterized by PDGFRα reactivity, from which antibody heavy and light chains were cloned and recombined in vitro to generate the
Commented [LJv7]: Why is this exciting? Would a monoclonal, functional antibody not be more excititng?
corresponding anti-PDGFRα monoclonal autoantibodies. It was quite exciting to observe that the 4
Commented [G8]: It is exciting to discover that anti-PDGFR autoimmune response is not consisting of a single antibody but a panel of antibodies directed toward different epitopes of the PDGFR, some relevant to disease phenotype, some not. This PDGFR epitope map was completely unknown before this study and shed light on the role of PDGFR in systemic sclerosis, explaining much of the controversial results on these autoantibodies: talking about anti-PDGFR autoantibodies is generic, whereas epitope-specific autoantibodies are diseasespecific. It was also exciting, from an immunological point of view, discovering that this different reactivity was based upon “light chain shuffling” whereas the heavy chain remains the same: this is related to the phenomenon known as ‘B cell receptor editing’. These considerations may be included in the text, eventually.
different monoclonals, differing only in the light chains, had distinct binding and functional features. In fact, binding and agonistic activities towards PDGFRα were dissociated in these autoantibodies, ranging from very low affinity binding without any agonistic activity towards fibroblasts to high affinity binding with induction of the ROS-ERK1/2-Ha-Ras loop and increased collagen gene transcription in human fibroblasts in vitro [22,23]. The use of a large conformational PDGFRα
5
peptide library enabled the dissection of the PDGFRα epitope recognized by the nonagonistic antibody, i.e. one linear aminoacid sequence in the first extracellular PDGFRα domain, from the conformational epitope of the agonistic antibody, i.e. a discontinuous motif encompassing 3 distinct sequences across the second and third extracellular PDGFRα domains, largely overlapping with the PDGF binding region [22]. An epitope-based assay to detect only agonistic anti-PDGFRα autoantibodies directed towards this discontinuous motif has been designed and preliminarily tested [OP0031 Annual European Congress of Rheumatology EULAR 2017], and awaits validation. On the other hand, an ELISA detecting all serum anti-PDGFRα autoantibodies regardless of the different epitopes has already been developed. By this assay, total anti-PDGFRα antibodies were detected in 66 of 70 SSc patients (94.3%), 63 of 130 healthy controls (48.5%), 11 of 26 PRP patients (42.3%), 11 of 29 SLE patients (37.9%) (P < 0.0001 between SSc patients and the other groups). Importantly, IgG purified from ELISA-positive SSc serum samples turned out to be positive also in ROS bioassay, whereas IgG purified from ELISA-positive healthy controls serum samples did not. This indicates, again, that anti-PDGFRα antibodies directed toward non-
Commented [LJv9]: So what does this mean? Please add a sentence to explain to the uninitiated reader.
stimulatory epitopes of the receptor may be present in healthy controls, whereas anti-PDGFRα antibodies directed toward stimulatory epitopes are SSc-specific [22]. In addition, SSc serum antiPDGFRα antibodies with receptor affinity as high as the collagen-inducing monoclonal autoantibody can be detected by ELISA [23]. Overall, this scientific information may reconcile the controversial results concerning the existence of stimulatory anti-PDGFRα antibodies in SSc. In fact, these findings demonstrate that agonistic and nonagonistic anti-PDGFRα autoantibodies may coexist in the same SSc patient. While nonagonistic anti-PDGFRα autoantibodies can be detected also in healthy controls or patients affected by PRP or SLE,
agonistic anti-PDGFRα
autoantibodies are SSc-specific and recognize precise conformational epitopes, which must be preserved in binding assays to discriminate agonistic from total antibodies. Whereas the nonagonistic anti-PDGFRα autoantibodies detectable in subjects not affected from SSc can be considered as a product of the natural autoimmune repertoire[24], the agonistic anti-PDGFRα autoantibodies are part of the SSc-specific, deranged autoimmune response towards cellular antigens, possibly amplified by epitope spreading[25].
6
Commented [LJv10]: Can you speculate how this difference can be explained?
The first evidence of agonistic activity in vivo. Besides confirming the existence of agonistic anti-PDGFRα autoantibodies in SSc patients, it was also mandatory to provide evidence of agonistic activity of these autoantibodies in vivo. The first, indirect evidence came from a small clinical study involving six SSc patients with severe skin fibrosis, unresponsive to canonical immunosuppressive therapies, who were treated with 375 mg/m2 per week of rituximab for a total of four doses. The good clinical response observed in all patients, evaluated as decrease of the skin score and improvement of the disability indices, was accompanied by a significant reduction of ROS stimulatory activity in vitro by IgG purified from serum of patients, and downregulation of the
Commented [LJv11]: Titer of? Please specify what you mean with reduction.
aforementioned intracellular signaling pathway and type I collagen gene expression in fibroblasts
Commented [G12]: Reduction of serum anti-PDGFRα antibodies in this study was measured by the cell-based functional bioassay (ROS bioassay) originally described in ref [17]. So, the term “titer” would not be appropriate because such assay does not use a serum titration but one standard concentration of IgG purified from serum. In order to avoid any ambiguity, I have rephrased this concept.
grown from skin biopsies performed at baseline and at months 3 and 6 post-treatment [26]. To obtain direct evidence, three-dimensional bioengineered skin samples containing human keratinocytes and fibroblasts isolated from skin biopsies of healthy donors were generated and grafted onto the back of SCID mice. The dermis of the skin grafts was then injected either with total IgG purified from serum of SSc patients (SSc IgG) or healthy controls (HC IgG), either with the agonistic, collagen-inducing anti-PDGFRα monoclonal antibody or with the nonagonistic one (both reported above). Unlike HC IgG, SSc IgG injection induced increase of human collagen deposition and fibroblast activation markers in healthy donor skin grafts. Replication of this scleroderma-like phenotype by injection of the agonistic monoclonal antibody, but not the nonagonistic one, directly demonstrated both the involvement of PDGFRα activation in the pathogenesis of skin fibrosis in vivo, and the profibrotic role in vivo of agonistic anti-PDGFRα autoantibodies [27].
Other cellular targets. Agonistic anti-PDGFRα autoantibodies (both total IgG and the collageninducing monoclonal antibody mentioned before) have been recently demonstrated to induce proliferation and migration of human pulmonary vascular smooth muscle cells in vitro [28]. This is an important finding further corroborating the agonistic activity of this class of autoantibodies and its contribution to the pathogenesis of SSc and potentially to SSc-related conditions such as pulmonary arterial hypertension (PAH), which awaits confirmation in vivo.
7
Autoantibodies against angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETaR)
The background of the hypothesis and the original discovery. Angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETaR) are widely expressed on cells of the vascular system and on immune cells. Functional anti-AT1R autoantibodies have first been reported by Wallukat et al. in 1999 [29]. The autoantibodies were isolated from sera of preeclamptic women, and their agonistic activity was demonstrated by the chronotropic effect induced on cultured spontaneously beating neonatal rat cardiomyocytes, which was completely inhibited by the selective receptor antagonist losartan [30]. Subsequently, numerous studies have shown that antiAT1R autoantibodies stimulate AT1R on several cell types inducing biological responses relevant to the pathophysiology of vascular diseases, such as malignant hypertension, renovascular diseases, renal allograft rejection, in which AT1R aAbs were also detected [31,32]. Anti-ETAR autoantibodies have been first identified in sera from patients with idiopathic PAH [33]. In sera from SSc patients, anti-AT1R and anti-ETAR autoantibodies were first detected by Riemekasten et al by solid phase assay [34]. Anti-AT1R and anti-ETAR antibodies were found in about 85% of SSc patients, who have increased levels of anti-AT1R and anti-ETAR antibodies compared to healthy donors. In patients with SSc, the antibody levels strongly correlate with each other and show cross-reactivity for both receptors.
Clinical associations. Higher levels of anti-AT1R and anti-ETAR antibodies were associated with severe SSc vascular manifestations such as digital ulcers and PAH [34]. Becker et al [35] reported that anti-AT1R and anti-ETaR antibodies are more frequent in PAH associated to SSc or other connective tissue diseases compared with other forms of pulmonary hypertension and may serve as prognostic and predictive biomarkers (cardiovascular complications and mortality).
8
In vitro agonistic activity. Interestingly, these autoantibodies induce ERK1/2 phosphorylation and increased TGFβ gene expression in human dermal microvascular endothelial cells [34]. Furthermore, when exposed to anti-AT1R and anti-ETaR antibodies, human microvascular endothelial cells (HMEC) show evidence of increased expression of IL-18 and vascular cell adhesion molecule-1 (VCAM-1) [36]. The activation of HMEC induced in such a way was responsible for increased neutrophil migration through an endothelial cell layer. More recently, AT1R and ETaR were also detected on human peripheral T cells, B cells, and monocytes, and their interaction with IgG from SSc patients was responsible for increased production of IL-8 and CC-chemokine ligand 18 (CCL18) [37].
In vivo agonistic activity. Healthy C57BL-6 mice were treated with repeated intravenous administrations of total SSc IgG testing positive for anti-AT1R and anti-ETAR antibodies. Histological analysis of the lungs, performed by hematoxylin and eosin (H&E) staining, showed thickening of airway vessels, and elevated cell density in interstitial tissue. However, no specific collagen staining was performed. Increased neutrophil count was found in bronchoalveolar lavage (BAL) of SSc IgG-treated mice as compared with HC IgG-treated mice, whereas no differences were observed for macrophages or lymphocytes [37]. In absence of human agonistic monoclonal anti-AT1R and anti-ETAR antibodies, these findings should be corroborated by administration of total SSc IgG fractions enriched or depleted of antiAT1R and anti-ETAR antibodies, in order to separate the specific effects of anti-AT1R and antiETAR antibodies from those of total SSc IgG, which may contain other agonistic antibody species such as anti-PDGFRα autoantibodies. Thus, it remains to be established how these autoantibodies are responsible for the development of SSc-specific vascular lesions.
Autoantibodies against muscarinic-3 receptor (M3R) The background of the hypothesis and the original discovery. Gastrointestinal (GI) involvement leading to dysmotility is frequent in SSc as a result of disturbance of cholinergic
9
neurotransmission and smooth muscle atrophy. Acetylcholine secreted after stimulation of the muscarinic-3 receptor (M3R) is the principal excitatory mediator of GI tract motility acting on intrinsic neurons in the myenteric plexus. Antibodies blocking M3R would therefore inhibit excitatory enteric neurotransmission causing dysmotility. Following this hypothesis, several studies found a high incidence of anti-myenteric neuronal antibodies in the sera of SSc patients with GI symptoms [38] and demonstrated that passive transfer of these antibodies into a rat model significantly disrupted intestinal myoelectric activity [39], further supporting a neuropathic etiology to dysmotility in SSc patients. The precise targeted neuronal antigen in these studies, however, remained to be determined. Later, Goldblatt et al [40] showed that IgG fractions from 7/9 patients with SSc, 4/4 patients with primary Sjögren’s syndrome (SS), and 3/3 patients with secondary SS inhibited animal colonic smooth muscle contraction caused by carbachol-induced activation of M3R suggesting that anti-M3R autoantibodies contained in SSc IgG (but also in SS IgG) may lead to failure of the cholinergic neurotransmission and, in turn, result in GI motility dysfunction. Inhibition by patients’ IgG was concentration dependent, whereas HC IgG did not inhibit the M3Rmediated contractions.
The confirmation of the initial finding and further in vivo evidence. These results were confirmed and expanded by Kawaguchi et al [41] who used an enzyme immunoassay and found anti-M3R antibodies in 9/14 early-onset SSc patients with severe GI tract involvement compared to only 3 positive out of 62 early-onset SSc patients without severe GI tract involvement; and by Singh et al [42] who provided evidence that IgG from SSc patients attenuate the M3R activation in smooth muscle cells of a rat internal anal sphincter.
The controversy. Recently, Preuss et al [43] using a novel luminescence-based test to detect functionally active antibodies to M3R, could not find antibodies inhibiting carbachol-induced activation of M3R in a cohort of 47 SSc patients, but in 20/40 primary SS patients.
Commented [LJv13]: SS = Sjögren syndrome here? Commented [G14]: Yes, the abbreviation is indicated before, in the first paragraph of this chapter
10
Conclusions and future perspectives The discovery in SSc patients of several types of serum autoantibodies carrying agonistic or antagonistic function towards different cell types involved in different aspects of disease pathogenesis may provide many advantages in the management of this heterogeneous condition lacking targeted therapies [44]. First, functional autoantibodies may help identify clinical variants of SSc. This task will require the screening of large, multicenter, well characterized patient populations with optimized assays based on selected epitopes of the target receptors rather than whole receptors, followed by correlation studies between serum reactivity profile and patient clinical features. For most autoantibodies reported in this review, this aim seems to be not too far away: Nag-2 and ICAM-1 for AECA, the well mapped epitope of the agonistic anti-PDGFRα monoclonal autoantibody, and the second extracellular loop of the M3R identified as a target of anti-M3R autoantibodies in patients with SS [45] may represent good candidates to define the serum reactivity profile of SSc patients cohorts. Identification of AT1R and ETaR epitopes will help keeping these receptors and the relative autoantibodies in the picture. Second, the identification of agonistic and antagonistic autoantibodies is very important to shed light into the pathogenesis of SSc: unraveling of the signaling cascade triggered or inhibited by the functional autoantibodies may in fact pave the way to novel therapeutic approaches. These therapeutic strategies should not rely on unselective block of total receptor activity, in order to avoid adverse effects, but on selective targeting of the receptors’ “hot spots” with small molecule inhibitors or selective capture of the agonistic/antagonistic antibodies with decoy mini-receptors or mimotopes, i.e. peptides mimicking the relevant epitopes bound by the functional autoantibodies. Alternative strategies such as anti-idiotype antibodies or tolerogenic peptides may also be rewarding. Clearly, all these preclinical therapeutic steps will require that robust animal models based on these functional autoantibodies reproduce consistent features of SSc. In sum, these observations provide an important step in the understanding of the mechanisms of interaction between autoimmunity and the different tissue/organ involvements in SSc, suggesting many potential avenues for future research activities in this field.
11
References
[1]
Lindqvist KJ, Osterland CK. Human antibodies to vascular endothelium. Clin Exp Immunol 1971;9:753-760.
[2]
Belizna C, Tervaert JW. Specificity, pathogenecity, and clinical value of antiendothelial cell antibodies. Semin Arthritis Rheum 1997;27:98-109.
[3]
Salojin KV, Le Tonqueze M, Saraux A, Nassonov EL, Dueymes M, Piette JC, Youinou PY. Antiendothelial cell antibodies: useful markers of systemic sclerosis. Am J Med 1997;102:178-185.
[4]
Pignone A, Scaletti C, Matucci-Cerinic M, Vazquez-Abad D, Meroni PL, Del Papa N, Falcini F, Generini S, Rothfield N, Cagnoni M. Anti-endothelial cell antibodies in systemic sclerosis: significant association with vascular involvement and alveolocapillary impairment. Clin Exp Rheumatol 1998;16:527-532.
[5]
Lunardi C, Bason C, Navone R, Millo E, Damonte G, Corrocher R, Puccetti A. Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells. Nat Med 2000;6:11831186.
[6]
Lunardi C, Dolcino M, Peterlana D, Bason C, Navone R, Tamassia N, Beri R, Corrocher R, Puccetti A. Antibodies against human cytomegalovirus in the pathogenesis of systemic sclerosis: a gene array approach. PLoS Med 2006;3:e2.
[7]
Servettaz A, Tamby MC, Guilpain P, Reinbolt J, Garcia de la Pena-Lefebvre P, Allanore Y, Kahan A, Meyer O, Guillevin L, Mouthon L. Anti-endothelial cell antibodies from patients with limited cutaneous systemic sclerosis bind to centromeric protein B (CENP-B). Clin Immunol 2006;120:212-219. 12
[8]
Hill MB, Phipps JL, Cartwright RJ, Milford Ward A, Greaves M, Hughes P. Antibodies to membranes of endothelial cells and fibroblasts in scleroderma. Clin Exp Immunol 1996;106:491-497.
[9]
Arends SJ, Damoiseaux JG, Duijvestijn AM, Debrus-Palmans L, Vroomen M, Boomars KA, Brunner-La Rocca HP, Reutelingsperger CP, Cohen Tervaert JW, van Paassen P. Immunoglobulin G anti-endothelial cell antibodies: inducers of endothelial cell apoptosis in pulmonary arterial hypertension? Clin Exp Immunol;174:433-440.
[10]
Wolf SI, Howat S, Abraham DJ, Pearson JD, Lawson C. Agonistic anti-ICAM-1 antibodies in scleroderma: activation of endothelial pro-inflammatory cascades. Vascul Pharmacol;59:19-26.
[11]
Sgonc R, Gruschwitz MS, Boeck G, Sepp N, Gruber J, Wick G. Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95. Arthritis Rheum 2000;43:2550-2562.
[12]
Worda M, Sgonc R, Dietrich H, Niederegger H, Sundick RS, Gershwin ME, Wick G. In vivo analysis of the apoptosis-inducing effect of anti-endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay. Arthritis Rheum 2003;48:2605-2614.
[13]
Laplante P, Raymond MA, Gagnon G, Vigneault N, Sasseville AM, Langelier Y, Bernard M, Raymond Y, Hebert MJ. Novel fibrogenic pathways are activated in response to endothelial apoptosis: implications in the pathophysiology of systemic sclerosis. J Immunol 2005;174:5740-5749.
[14]
Yamakage A, Kikuchi K, Smith EA, LeRoy EC, Trojanowska M. Selective upregulation of platelet-derived growth factor alpha receptors by transforming growth factor beta in scleroderma fibroblasts. J Exp Med 1992;175:1227-1234.
13
[15]
Svegliati S, Cancello R, Sambo P, Luchetti M, Paroncini P, Orlandini G, Discepoli G, Paterno R, Santillo M, Cuozzo C, Cassano S, Avvedimento EV, Gabrielli A. Plateletderived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts. J Biol Chem 2005;280:36474-36482.
[16]
Smaldone S, Olivieri J, Gusella GL, Moroncini G, Gabrielli A, Ramirez F. Ha-Ras stabilization mediates pro-fibrotic signals in dermal fibroblasts. Fibrogenesis Tissue Repair 2011;4:8.
[17]
Baroni SS, Santillo M, Bevilacqua F, Luchetti M, Spadoni T, Mancini M, Fraticelli P, Sambo P, Funaro A, Kazlauskas A, Avvedimento EV, Gabrielli A. Stimulatory autoantibodies to the PDGF receptor in systemic sclerosis. N Engl J Med 2006;354:2667-2676.
[18]
Classen JF, Henrohn D, Rorsman F, Lennartsson J, Lauwerys BR, Wikstrom G, Rorsman C, Lenglez S, Franck-Larsson K, Tomasi JP, Kampe O, Vanthuyne M, Houssiau FA, Demoulin JB. Lack of evidence of stimulatory autoantibodies to platelet-derived growth factor receptor in patients with systemic sclerosis. Arthritis Rheum 2009;60:1137-1144.
[19]
Loizos N, Lariccia L, Weiner J, Griffith H, Boin F, Hummers L, Wigley F, Kussie P. Lack of detection of agonist activity by antibodies to platelet-derived growth factor receptor alpha in a subset of normal and systemic sclerosis patient sera. Arthritis Rheum 2009;60:1145-1151.
[20]
Gabrielli A, Moroncini G, Svegliati S, Avvedimento EV. Autoantibodies against the platelet-derived growth factor receptor in scleroderma: comment on the articles by Classen et al and Loizos et al. Arthritis Rheum 2009;60:3521-3522.
14
[21]
Dragun D, Distler JH, Riemekasten G, Distler O. Stimulatory autoantibodies to plateletderived growth factor receptors in systemic sclerosis: what functional autoimmunity could learn from receptor biology. Arthritis Rheum 2009;60:907-911.
[22]
Moroncini G, Grieco A, Nacci G, Paolini C, Tonnini C, Pozniak KN, Cuccioloni M, Mozzicafreddo M, Svegliati S, Angeletti M, Kazlauskas A, Avvedimento EV, Funaro A, Gabrielli A. Epitope Specificity Determines Pathogenicity and Detectability of AntiPlatelet-Derived Growth Factor Receptor alpha Autoantibodies in Systemic Sclerosis. Arthritis Rheumatol;67:1891-1903.
[23]
Moroncini G, Cuccioloni M, Mozzicafreddo M, Pozniak KN, Grieco A, Paolini C, Tonnini C, Spadoni T, Svegliati S, Funaro A, Angeletti M, Gabrielli A. Characterization of binding and quantification of human autoantibodies to PDGFRalpha using a biosensor-based approach. Anal Biochem;528:26-33.
[24]
Poletaev AB, Stepanyuk VL, Gershwin ME. Integrating immunity: the immunculus and self-reactivity. J Autoimmun 2008;30:68-73.
[25]
Cornaby C, Gibbons L, Mayhew V, Sloan CS, Welling A, Poole BD. B cell epitope spreading: mechanisms and contribution to autoimmune diseases. Immunol Lett 2015;163:56-68.
[26]
Fraticelli P, De Vita S, Franzolini N, Svegliati S, Scott CA, Tonnini C, Spadoni T, Gabrielli B, Pomponio G, Moroncini G, Gabrielli A. Reduced type I collagen gene expression by skin fibroblasts of patients with systemic sclerosis after one treatment course with rituximab. Clin Exp Rheumatol;33:S160-167.
[27]
Luchetti MM, Moroncini G, Jose Escamez M, Svegliati Baroni S, Spadoni T, Grieco A, Paolini C, Funaro A, Avvedimento EV, Larcher F, Del Rio M, Gabrielli A. Induction of Scleroderma Fibrosis in Skin-Humanized Mice by Administration of Anti-Platelet-
15
Derived
Growth
Factor
Receptor
Agonistic
Autoantibodies.
Arthritis
Rheumatol;68:2263-2273. [28]
Svegliati S, Amico D, Spadoni T, Fischetti C, Finke D, Moroncini G, Paolini C, Tonnini C, Grieco A, Rovinelli M, Funaro A, Gabrielli A. Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro. Front Immunol;8:75.
[29]
Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, Baur E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC. Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest 1999;103:945-952.
[30]
Xia Y, Kellems RE. Is preeclampsia an autoimmune disease? Clin Immunol 2009;133:112.
[31]
Fu ML, Herlitz H, Schulze W, Wallukat G, Micke P, Eftekhari P, Sjogren KG, Hjalmarson A, Muller-Esterl W, Hoebeke J. Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension. J Hypertens 2000;18:945-953.
[32]
Dragun D, Muller DN, Brasen JH, Fritsche L, Nieminen-Kelha M, Dechend R, Kintscher U, Rudolph B, Hoebeke J, Eckert D, Mazak I, Plehm R, Schonemann C, Unger T, Budde K, Neumayer HH, Luft FC, Wallukat G. Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection. N Engl J Med 2005;352:558-569.
[33]
Wallukat G, Schimke I. Agonistic autoantibodies directed against G-protein-coupled receptors
and
their
relationship
to
cardiovascular
diseases.
Semin
Immunopathol;36:351-363. [34]
Riemekasten G, Philippe A, Nather M, Slowinski T, Muller DN, Heidecke H, MatucciCerinic M, Czirjak L, Lukitsch I, Becker M, Kill A, van Laar JM, Catar R, Luft FC,
16
Burmester GR, Hegner B, Dragun D. Involvement of functional autoantibodies against vascular receptors in systemic sclerosis. Ann Rheum Dis 2011;70:530-536. [35]
Becker MO, Kill A, Kutsche M, Guenther J, Rose A, Tabeling C, Witzenrath M, Kuhl AA, Heidecke H, Ghofrani HA, Tiede H, Schermuly RT, Nickel N, Hoeper MM, Lukitsch I, Gollasch M, Kuebler WM, Bock S, Burmester GR, Dragun D, Riemekasten G. Vascular receptor autoantibodies in pulmonary arterial hypertension associated with systemic sclerosis. Am J Respir Crit Care Med 2014;190:808-817.
[36]
Gunther J, Kill A, Becker MO, Heidecke H, Rademacher J, Siegert E, Radic M, Burmester GR, Dragun D, Riemekasten G. Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients. Arthritis Res Ther;16:R65.
[37]
Kill A, Tabeling C, Undeutsch R, Kuhl AA, Gunther J, Radic M, Becker MO, Heidecke H, Worm M, Witzenrath M, Burmester GR, Dragun D, Riemekasten G. Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis. Arthritis Res Ther;16:R29.
[38]
Howe S, Eaker EY, Sallustio JE, Peebles C, Tan EM, Williams RC, Jr. Antimyenteric neuronal antibodies in scleroderma. J Clin Invest 1994;94:761-770.
[39]
Eaker EY, Kuldau JG, Verne GN, Ross SO, Sallustio JE. Myenteric neuronal antibodies in scleroderma: passive transfer evokes alterations in intestinal myoelectric activity in a rat model. J Lab Clin Med 1999;133:551-556.
[40]
Goldblatt F, Gordon TP, Waterman SA. Antibody-mediated gastrointestinal dysmotility in scleroderma. Gastroenterology 2002;123:1144-1150.
[41]
Kawaguchi Y, Nakamura Y, Matsumoto I, Nishimagi E, Satoh T, Kuwana M, Sumida T, Hara M. Muscarinic-3 acetylcholine receptor autoantibody in patients with systemic 17
sclerosis: contribution to severe gastrointestinal tract dysmotility. Ann Rheum Dis 2009;68:710-714. [42]
Singh J, Mehendiratta V, Del Galdo F, Jimenez SA, Cohen S, DiMarino AJ, Rattan S. Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 2009;297:G1206-1213.
[43]
Preuss B, Tunaru S, Henes J, Offermanns S, Klein R. A novel luminescence-based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients' sera. Clin Exp Immunol;177:179-189.
[44]
Kowal-Bielecka O, Fransen J, Avouac J, Becker M, Kulak A, Allanore Y, Distler O, Clements P, Cutolo M, Czirjak L, Damjanov N, Del Galdo F, Denton CP, Distler JHW, Foeldvari I, Figelstone K, Frerix M, Furst DE, Guiducci S, Hunzelmann N, Khanna D, Matucci-Cerinic M, Herrick AL, van den Hoogen F, van Laar JM, Riemekasten G, Silver R, Smith V, Sulli A, Tarner I, Tyndall A, Welling J, Wigley F, Valentini G, Walker UA, Zulian F, Muller-Ladner U. Update of EULAR recommendations for the treatment of systemic sclerosis. Ann Rheum Dis;76:1327-1339.
[45]
Cavill D, Waterman SA, Gordon TP. Antibodies raised against the second extracellular loop of the human muscarinic M3 receptor mimic functional autoantibodies in Sjogren's syndrome. Scand J Immunol 2004;59:261-266.
18