Dig Dis Sci DOI 10.1007/s10620-014-3089-3
ORIGINAL ARTICLE
Proinflammatory Progranulin Antibodies in Inflammatory Bowel Diseases Lorenz Thurner • Elisabeth Sto¨ger • Natalie Fadle • Philipp Klemm • Evi Regitz • Maria Kemele • Birgit Bette • Gerhard Held • Marc Dauer • Frank Lammert • Klaus-Dieter Preuss • Vincent Zimmer • Michael Pfreundschuh
Received: 20 July 2013 / Accepted: 19 February 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Recently, we identified neutralizing autoantibodies against progranulin (PGRN) in a wide spectrum of rheumatic diseases including cases with enteropathic spondylarthritis. PGRN is a secreted protein with strong anti-inflammatory effects, believed to be mediated by the direct inhibition of TNF receptors 1&2. Given the central role of TNF-a as proinflammatory cytokine, a neutralizing
Electronic supplementary material The online version of this article (doi:10.1007/s10620-014-3089-3) contains supplementary material, which is available to authorized users. L. Thurner E. Sto¨ger N. Fadle P. Klemm E. Regitz M. Kemele B. Bette G. Held K.-D. Preuss M. Pfreundschuh Jose´ Carreras Center for Immuno- and Gene Therapy and Internal Medicine I, Saarland University Medical School, Homburg/Saar, Germany e-mail: [email protected] L. Thurner (&) Department of Internal Medicine I, University Hospital Homburg/Saar, Kirrberger Str., 66421 Homburg/Saar, Germany e-mail: [email protected]; [email protected] Present Address: M. Dauer Department of Medicine, St. Elisabeth Kliniken, Neuburg/ Donau, Germany F. Lammert V. Zimmer Department of Internal Medicine II, Saarland University Medical Center, Homburg/Saar, Germany e-mail: [email protected] V. Zimmer e-mail: [email protected]
antibody directed against its physiologic antagonist PGRN might entertain a proinflammatory environment. Objective The aim of the present study was to investigate a possible occurrence of PGRN-antibodies (PGRN-Abs) in inflammatory bowel disease (IBD), and to investigate a possible pathogenic effect. Materials and Methods Sera samples of 141 patients with Crohn’s disease (CD) and of 71 patients with ulcerative colitis (UC) were tested for PGRN-Abs by ELISA. PGRN plasma levels were detected by ELISA. Proinflammatory effects of progranulin-antibodies were analyzed by TNF-amediated cytotoxicity assays using HT29 cells and by examination of possible effects of PGRN and of PGRNantibodies on TNF-a-induced downmodulation of FOXP3 expression in CD4?CD25hi Tregs. Results PGRN-Abs were found in sera of 23/141 (16.31 %) patients with CD, and 15/71 (21.13 %) patients with UC. PGRN-Abs were more frequent than anti-neutrophil cytoplasmic autoantibodies (ANCAs) in UC, but less frequent than anti-Saccharomyces cerevisiae antibodies (ASCAs) in CD. PGRN-Abs belonged mostly to IgG1 (71.1 %) and IgA (26.3 %). They occurred in relevant titres and had significant neutralizing effects on PGRN plasma levels. Cytotoxicity assays comparing PGRN-antibody-positive sera with negative sera from matched patients with IBD showed a proinflammatory effect of PGRN-Abs on HT29 cells. Moreover, PGRN-antibodies led to an increase of TNF-a-induced downmodulation of FOXP3 in CD4?CD25hi Tregs. Conclusion The results suggest that PGRN-Abs occur frequently in CD and UC, and have a proinflammatory effect. Keywords Progranulin Inhibitor of TNFR1&2 Neutralizing proinflammatory progranulin-antibody Crohn’s disease Ulcerative colitis
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Introduction Inflammatory bowel disease (IBD) encompasses two clinically, histopathologically, and pathogenetically distinct diseases, namely ulcerative colitis (UC) and Crohn’s disease (CD). In both diseases, alterations of the innate and adaptive immune system eventually result in systemic and/ or local increases of proinflammatory cytokine levels such as TNF-a, IL-6, IL-1b, and IFN-c, the latter particularly in CD. We recently discovered progranulin antibodies (PGRNAbs) in various primary vasculitides using a protein macroarray-based approach, and these antibodies were subsequently also found in several other rheumatic autoimmune diseases with varying frequency. So far, PGRN-Abs have been found in several primary vasculitides: Takayasu arteriitis, giant cell arteriitis, Churg-Strauss syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, panarteritis nodosa, Behcet’s disease, polymyalgia rheumatica, and apart from primary vasculitides, in systemic lupus erythematosus and rheumatoid arthritis. In contrast, PGRN-Abs were rarely found in healthy controls and in patients with sepsis or melanoma [1]. Progranulin (PGRN) is a secreted precursor protein of 88 kDa, with a signal peptide and seven granulin motives, each containing 12 cysteins and six disulfide bonds [2]. PGRN has several functions, amongst others a strong antiinflammatory effect [3]. This anti-inflammatory action of PGRN was proposed to be mediated by the direct inhibition of the TNF receptors 1&2 (TNFR1&2) [4], while PGRN is regarded as a physiological endogenous TNF-blocker, and recombinant human PGRN or its synthetically constructed derivative molecule, ATSTTRIN, have been proposed as next-generation TNF-blockers [5]. However, this proposed mechanism of the known anti-inflammatory effect of PGRN was challenged recently [6] without a denial of the anti-inflammatory effect of PGRN [3]. In detail, Chen et al. could not reproduce the interaction of PGRN binding to TNFR1&2 as reported by Tang et al. [4]. Moreover, Etemadi et al. [7] could not reproduce any antagonistic effects of recombinant human PGRN on TNFa-induced cytotoxicity and on TNF-a-induced downstream signal cascade activation via TNFR1. Etemadi et al. used recombinant PGRN from different commercial sources (R&D and Adipogen). The authors responded in a letter to the editor, suggesting that the murine PGRN utilized by Chen et al. might be improperly folded. Furthermore, a validation of PGRN functionality based only on its C-terminal binding to Sortilin would be insufficient. Following this, they demonstrated that PGRN binds similarily to the way TNF-a joins to CRD2 and CRD3 of TNFR, and that proper folding of PGRN is required for this binding. Moreover, DTT
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treatment of PGRN, which had been performed by Chen et al., abolishes the binding of PGRN to TNFR, but enhances its binding to Sortilin [8]. In the meantime, the binding of PGRN to TNFR and its inhibitory effect on TNF-a-induced effects were recently reproduced independently by two other groups. In vitro PGRN suppressed TNF-a-induced neutrophil chemotaxis and ICAM-1 expression in endothelial cells, and in vivo recombinant PGRN treatment suppressed neutrophil recruitment into the ischemia/reperfusion region of the brain via middle cerebral artery occlusion in a mouse-model of cerebral ischemia–reperfusion (I/R) injury, resulting in a reduction of NF-jB and MMP-9 activation [9]. Recombinant PGRN suppressed TNF-a-induced expression of adhesion molecules ICAM-1 and VCAM-1 in HUVECs [10]. PGRN-Abs have a neutralizing effect on plasma levels of secreted PGRN, suggesting a proinflammatory effect of PGRN-Abs by neutralizing this physiologic TNF-a antagonist [1], comparable to the effects of anti-drug antibodies that can arise after the repeated administration of therapeutic TNFblockers [11]. This hypothesis is supported by the fact that the presence of PGRN-Abs was associated with the active disease state in granulomatosis with polyangiitis [1]. Furthermore, we recently reported the presence of proinflammatory PGRN-Abs in psoriatic arthritis. Psoriatic arthritis was believed to be a seronegative disease. PGRNAb containing sera of patients with psoriatic arthritis resulted in an increased sensitivity of HT-1080 cells to the effects of TNF-a compared to the administration of sera from patients with psoriatic arthritis without PGRN-Abs [12]. IBD are often associated with rheumatic diseases. Both IBD and several rheumatic autoimmune diseases respond to therapeutically-administrated anti-TNF-a-antibodies. In addition, the ‘‘shared’’ presence of perinuclear anti-neutrophil cytoplasmic autoantibodies (pANCAs) as autoantibodies in IBD and small-vessel vasculitides, and the occurrence of PGRN-Abs in patients with enteropathic spondylarthritis prompted us to investigate the presence of PGRN-Abs in the serum of patients with IBD.
Materials and Methods Study Participants This study was approved by the local ethical review board ¨ rztekammer des Saarlandes’’ (‘‘Ethikkommission der A N242/11) and conducted according to the guidelines laid down in the Declaration of Helsinki. Plasma and sera samples of patients with UC and CD were obtained at the outpatient IBD clinic of Saarland University Medical Center in Homburg/Saar during routine diagnostics. From
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some individual patients, more than one serum sample had been obtained at different points of time during the course of their disease. Blood samples from healthy controls were obtained with written informed consent at the Saarland University Medical Center in Homburg/Saar. Blood samples were stored at -80 °C until use. All 141 patients with CD and 71 patients with UC were classified according to the Montreal Classification Criteria [13]. In addition, data on the prescribed medication, anti-neutrophil cytoplasmic autoantibody (ANCA) status, and anti-Saccharomyces cerevisiae antibody (ASCA) status were obtained, whenever available.
(PCR) using a proofreading taq. Mutations were excluded by sequencing. Subsequently, the proteins were recombinantly expressed with a C-terminal FLAG tag in HEK293 cells under the control of a CMV promoter (pSFI). Chromatographic purification of FLAG-tagged PGRN was performed with an anti-FLAG affinity matrix. In short, crude whole-cell lysates were clarified by centrifugation. To pull down the Flag-tagged PGRN, 50 ll of anti-FLAG gel suspension were coincubated with 2 ml of cell lysates for 30 min at 4 °C. Afterwards, 100 ll FLAG-Peptid solution 0.1 mg/ml was used for non-denaturing elution of FLAG-tagged PGRN. The resin was washed in PBS. Final concentration was 90 lg/ml.
Progranulin-Antibody ELISA Cytotoxicity Assay A number of enzyme-linked immunosorbent assays (ELISAs) for PRGN-Abs and for the determination of immunoglobulin class and IgG subclasses were performed as previously described [1]. The average of the optical density (OD) values of the negative samples plus three standard deviations was applied as the cut off for positivity. From several patients, more than one serum sample had been obtained during their course of disease. If at least one of these serum samples was positive for PGRN-Abs, this patient was regarded as positive for PGRN-Abs in the course of disease. Progranulin ELISA Progranulin plasma levels were determined by a commercially available ELISA kit (AdipoGen, South Korea) according to the manufacturer’s instructions. The median PGRN plasma level of healthy controls was set at 100 %, as described before [1]. Selection of Anti-PGRN-Fabs Specific Against the N-Terminus The selection of Fabs (antigen binding fragment) with specific reactivity against the N-terminus of PGRN, was performed as described before [14, 15]. As a phagemid library, a non-immune, semi-synthetic human Fab repertoire containing 3.7 9 1010 different possible antibody fragments was used [16]. For selection, an N-terminally biotinylated PGRN AA70-91 peptide was used. To obtain specific Fabs against the N-terminus of PGRN, five rounds of selection were necessary. Expression and Purification of Recombinant Human PGRN and SLP2 The coding sequences of PGRN and SLP2 were amplified from a human cDNA library by polymerase chain reaction
A non-radioactive cytotoxicity assay (EZ4U, Biomedica) was performed according to the manufacturer’s instructions. As target cells for this TNF-a-induced cytotoxicity assay, the human colorectal adenocarcinoma HT29 cell-line was used. In short, 4 9 104 HT29 cells were seeded in 200-ll cell cultures at 37 °C and at 5 % CO2. To detect possible differences between added sera of UC patients with and without PGRN-Abs, or of CD patients with and without PGRN-Abs and of healthy controls, the serum of a PGRNAb positive patient, serum of a matched PGRN-Ab-negative patient, and serum of a healthy control were administered in dilutions from 1:4 to 1:512 to the cultured HT29 cells, followed by the administration of TNF-a (10 lg/ml). Serum samples from gender-, age-, disease- and therapy modalitymatched patients were chosen. Serum samples from patients receiving TNF-blockers or other biological agents were excluded. As the 20-min half-life of TNF-a [17] is negligibly short compared to the 40-h half-life of PGRN [4], the effects of the intrinsic TNF-a in the sera of patients and controls were neglected. HT29 cells without the addition of TNF-a and serum, or solely with the addition of TNF-a (10 lg/ml) were used as positive and negative controls. After 24 h of incubation at 37 °C, 20 ll of chromophore substrate were added to each well. This chromophore substrate is only converted by vital cells. The adsorbance of the product was measured at an OD of 450 nm. To exclude possible influences on the TNF-a-induced cytotoxicity assay by any ingredient of the administrated sera other than PGRN, the assay was repeated without sera of PGRN-Ab positive or negative patients, but with recombinant human PGRN expressed in HEK293 cells at a concentration of 250 ng/ml, with recombinant human SLP2 at a concentration of 250 ng/ml expressed in the same HEK293 expression system, and with recombinant anti-human-PGRN-Fab (starting concentration 2 lg/ml) in dilutions from 1:2 to 1:128 directed against the N-terminus of PGRN. To investigate whether commercially available
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recombinant PGRN expressed in HEK293 cells (Adipogen) has comparable TNF-antagonizing effects to the recombinant PGRN expressed in our laboratory, the cytotoxicity assay was repeated with 4 9 104 WEHI-S cells/ml, the administration of 100 pg/ml TNF-a, and of either 250 ng/ml commercially available recombinant PGRN (Adipogen, Lot Nr. K110517) or recombinant PGRN, which had been synthesized in our laboratory. After 24 h incubation at 37 °C, 20 ll of chromophore substrate were added to each well. This chromophore substrate is only converted by vital cells. The adsorbance of the product was measured at an OD of 450 nm. Effect of PGRN-Abs on FOXP3 Expression of CD4?CD25hi Regulatory T Cells PBMCs were isolated from the venous blood of healthy controls in tubes containing EDTA by Ficoll density gradient centrifugation. Subsequently, CD4?CD25hi Tregs were isolated (Milteny Biotech, Bergisch Gladbach, Germany). In each well, CD4?CD25hi regulatory T cells at a concentration of 5 9 104/ml were cultured and treated either with Etanercept 1 lg/ml, with 250 ng/ml recombinant human PGRN, with 250 ng/ml recombinant human SLP2, with 250 ng/ml recombinant PGRN and 1 lg/ml recombinant anti-PGRN-Ab, with 250 ng/ml recombinant SLP2 and 1 lg/ml recombinant anti-PGRN-Ab, and with 250 ng/ml recombinant PGRN and 1 lg/ml recombinant anti-SLP2-Ab for 30 min at 37 °C. The mentioned mixtures of recombinant proteins and monoclonal antibodies had been preincubated for 30 min at 4 °C, before they were administrated to the CD4?CD25hi Tregs. Subsequently, 50 ng/ml TNF-a were administrated to each well and the Tregs were cultured overnight at 37 °C. Following this, FOXP3 expression was determined by Western blot analysis real-time PCR was not performed. Etanercept was chosen for this experiment, despite its lower efficiency in IBD compared to anti-TNF-antibodies. By choosing Etanercept, the direct targeting of T cells by anti-TNF-antibodies via membranous TNF-a should be avoided. Equivalent amounts of Treg cell lysates were loaded and separated in a 10 % SDS-PAGE, transferred to PVDF membrane using a transblot semidry transfer cell (Bio Rad). Transferred proteins were incubated with mouse antihuman FOXP3 antibody at a dilution of 1:1,000 (Exbio) followed by incubation with a goat HRP labeled antimouse IgG antibody at a dilution of 1:3,000 (Bio Rad). For the actin control, a rabbit anti-human Actin antibody was incubated at a dilution of 1:1,000 (Sigma) followed by incubation with a goat HRP-labeled anti-rabbit IgG antibody at a dilution of 1:3,000 (Bio Rad). ECL reagent (NEB) was utilized for immunoblot detection.
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ANCA and ASCA Status Detection of perinuclear anti-neutrophil cytoplasmic autoantibodies (pANCA) and cytoplasmic anti-neutrophil cytoplasmic autoantibodies (cANCA) had already been performed during clinical routine diagnostics, according to standardized laboratory procedures, by initial indirect immunofluorescence screening for perinuclear or cytoplasmic ANCA patterns, followed in positive or borderline cases by ELISA (Varelisa-Kit) for MPO-ANCA and Pr3ANCA at the autoimmune diagnostic laboratory of the Department of Internal Medicine I at the Saarland University Medical School. The cut off for positive MPOANCA and Pr3-ANCA was set according to the manufacturer’s instructions. ASCA status had been determined in subgroups of patients with CD and UC by a commercial laboratory. Statistical Analysis Different frequencies of PRGN-Abs in subgroups of patients with CD and UC classified by clinical characteristics, prescribed medication, and ANCA- and ASCA-status were tested by v2 test, and, respectively, Fisher’s exact test when indicated. Differences of PGRN serum levels between PGRN-Ab-positive patients with IBD, PGRN-Abnegative patients with IBD, and healthy controls were compared utilizing the Mann–Whitney U test. A p value of \0.05 was considered as statistically significant.
Results Patient Characteristics Tables 1 and 2 in the supplementary materials summarize the clinical characteristics of patients with UC and CD, respectively. In seven of 71 (9.9 %) patients with UC and in 12 of 141 (8.5 %) patients with CD, more than one serum sample was tested for PGRN-Abs. Frequency, Titres, and Immunoglobulin Class of Progranulin-Antibodies Overall, PGRN-Abs were detected in 23 of 141 (16.31 %) patients with CD and in 15 of 71 (20.13 %) patients with UC. In contrast to this, PGRN-Abs had only been detected in a single individual among 97 healthy controls (1.03 %) (Fig. 1a) with low antibody-titres. Moreover, PGRN-Ab titres were determined in randomly chosen PGRN-Abpositive patients and ranged from 1:800 to 1:3,200 (Fig. 1b). The analysis of immunoglobulin classes of
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Occurence of PGRN-Ab during course of disease [%]
(a)
b Fig. 1 a Frequency of PGRN-Abs in IBDs. Columns represent the 24 22 20 18
23/141 = 16.31%
16 14 12 10 8 6 4
1/97 =1.03%
2 0 Chron's disease
(b)
frequency of PGRN-Ab during the course of disease in CD, UC, and healthy controls. b Titres of PGRN antibodies in IBDs. Each curve indicates the ODs obtained for sera from an individual patient or a healthy control at different dilution steps. c Frequency of different immunoglobulin classes of PGRN-Ab in IBDs. Columns represent the relative frequency of each IgG subclass and IgA or IgM class of PGRN-Abs in CD and UC. d Frequency of PGRN-Abs and ANCAs in UC. Columns represent the relative frequency of PGRN-Abs, pANCAs, and cANCAs in UC
15/71 =21.13%
Ulcerative Colitis
Healthy Controls
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Adsorption (OD ad 490nm)
Negative Control
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Ulcerative colitis 2 Chron's disease 1
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100 90 80
27/38 71.1%
70 60 50 40
10/38 26.3%
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IgG1
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Association of PGRN-Ab Status with Clinical Characteristics, ANCA, and ASCA
Ulcerative colitis 1
0.0
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(26.1 %). In each of the PGRN-Ab-positive sera samples, PGRN-Abs belonged only to a single immunoglobulin class, i.e., either IgG1, IgG2, or IgA could be identified.
IgG2
IgA
IgM
21.13%
13.85%
15 10.77%
10 6.15%
5
0 PGRN-Ab
pANCA
cANCA
pANCA and/or cANCA
PGRN-Abs revealed IgG1 in 27/38 (71.1 %), IgA in 10/38 (26.3 %) and IgG2 in 1/38 (2.6 %) (Fig. 1c). PGRN-Abs of IgA class were detected with similar frequencies in patients with UC 4/15 (26.7 %) and patients with CD 6/23
Furthermore, the presence of PGRN-Abs was analyzed for associations with clinical characteristics, and if available with ANCA or ASCA status and the class of prescribed medication. Using the v2 tests, no statistically significant associations between the presence of PGRN-Abs and clinical characteristics, prescribed medication, or ANCA and ASCA status were observed for UC or for CD. Regarding UC, of the seven individual patients in which more than one serum sample was obtained, three of these patients were positive during the course of disease compared to 64 patients with a single tested sample and 12 positives [3/7 (42.9 %) vs. 12/64 (18.75 %); p = 0.158]. Regarding Crohn’s disease, PGRN-Abs were more often observed in a given patient if more than one serum sample taken at different time points during her/his disease was available for testing [6/12 (50 %) vs. 17/129 (13.2 %); p = 0.005]. Moreover, in UC, PGRN-Abs were more frequent (15 of 71; 20.13 %) than pANCA (four of 65; 6.2 %; p = 0.0135), more frequent than cANCA (seven of 65; 9.9 %; p = 0.101) and more frequent than pANCA and/or cANCA (nine of 65; 13.8 %; p = 0.266; Fig. 1d and supplementary material Table 1). There was no statistically significant association between positive serostatus for ANCA and positive serostatus for PGRN-Ab. In CD, PGRN-Abs were more frequent (23 of 141; 16.31 %) than pANCA (one of 127; 0.8 %; p \ 0.001) and cANCA (zero of 127; 0.0 %; p \ 0.001), but less frequent than ASCA (14 of 29; 48.3 %; p \ 0.001). Only a minor proportion of patients with CD had been tested in the context of clinical routine diagnostics for ASCA (29/ 141 or 20.57 %; supplementary Table 2). Of the 29 tested patients for ASCA, 14 (48.3 %) were positive. There was no statistically significant association for ASCA-positive patients to be PGRN-Ab-positive. Progranulin ELISA Using a commercially available ELISA, we observed that PGRN levels were significantly decreased in plasma of
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recombinant human SLP-2 as a control, and recombinant anti-PGRN-Fab (directed against the N-terminus) confirmed the TNF-a antagonism of PGRN, which was reversed by the additional administration of anti-PGRNFab (Fig. 3c). Commercially available recombinant PGRN had an TNFantagonizing effect as well. This effect was weaker than the effect of the recombinant PGRN expressed and purified in our laboratory, but the effect of commercially available PGRN was still significant (supplementary material).
Fig. 2 The neutralizing effect of PGRN-Abs on PGRN plasma levels in IBD. Lane a healthy controls (n = 7); lane b PGRN-Ab-negative patients with IBD (n = 7); lane c PGRN-Ab-positive patients with IBD (n = 7). Statistical analysis revealed highly significant differences in the PGRN plasma levels of PGRN-Ab-positive patients with IBD compared to PGRN-Ab-negative patients with IBD (Mann– Whitney test: p \ 0.001) and to healthy controls (Mann–Whitney test: p \ 0.001). No significant differences were detected between PGRN-Ab-negative patients with Chron’s disease or UC and the healthy control group (p = 0.456). The median PGRN serum level obtained of the healthy controls was set at 100 %
PGRN-Ab-positive patients with IBD (n = 7) compared to PGRN-Ab-negative patients with IBD (n = 7; Mann– Whitney U test: p \ 0.001) and compared to healthy controls (n = 7; Mann–Whitney U test: p \ 0.001). There was no significant difference in the PGRN plasma levels of PGRN-Ab-negative patients with IBD compared to healthy controls (Mann–Whitney U test: p = 0.456; Fig. 2). The median PGRN plasma level of healthy controls was set at 100 %, as described before [1]. Cytotoxicity Assay For the TNF-a-induced cytotoxicity assays, the protective effect of serum from healthy controls and from patients with UC and CD with and without PGRN-Ab was analyzed. The addition of serum from patients with UC without PGRN-Ab and from healthy controls reduced TNF-a-induced cytotoxicity of HT29 cells to a significantly higher degree compared to PGRN-Ab containing serum of patients with UC. This difference was significant up to a dilution of serum of 1:64 (Fig. 3a). In analogy to this, the addition of serum from patients with CD without PGRN-Ab and from healthy controls reduced the TNF-ainduced cytotoxicity of HT29 cells to a significantly higher degree compared to serum containing PGRN-Ab from patients with CD. This difference was also significant up to a serum dilution of 1:64 (Fig. 3b). The TNF-a-induced cytotoxicity assay of HT29 cells with administrated recombinant human PGRN,
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Effect of PGRN-Abs on TNF-a-Induced Downregulation of Foxp3 Expression in CD4?CD25hi T Cells To study the role of PGRN and PGRN-Abs on the function of inflammatory cells, we analyzed the TNF-a-induced downmodulation of FOXP3 expression in CD4?CD25hi regulatory T cells in the presence of recombinant PGRN, recombinant SLP2, Etanercept, recombinant SLP2-Fabs, and recombinant PGRN-Fabs. Western blot showed that the administration of recombinant PGRN antagonized the TNF-a-induced downmodulation of FOXP3 expression to a similar extent as the administration of Etanercept. The administrated recombinant control protein SLP-2, which had been expressed in the same cell system and purified by the same means as recombinant PGRN, didn’t show any antagonistic effect on TNF-a. The TNF-a-inhibiting effect of PGRN was blocked by the administration of anti-PGRNFab.
Discussion The present study firstly demonstrates the important point that PGRN-Abs are not restricted to rheumatic autoimmune diseases such as primary vasculitides, SLE, rheumatoid arthritis [1], or psoriatic arthritis [12], but can also be encountered in a subgroup of patients with UC and CD. In detail, 16.31 % of patients with CD and 20.13 % of patients with UC were positive for PGRN-Abs (Fig. 1a). Both in UC and in CD, PGRN-Abs had relevant titres (Fig. 1b) that were comparable to the titres found previously in rheumatic autoimmune diseases [1]. Moreover, the neutralizing effect of PGRN-Abs on the levels of circulating PGRN, which was already shown for rheumatic autoimmune diseases [1], could be confirmed for IBD (Fig. 2). This is of particular interest because PGRN displays anti-inflammatory effects [3], which were reported to be mediated by the direct inhibition of TNFR 1 and 2 [4], which was challenged recently by Chen et al. In detail, Chen et al. were not able to reproduce the interaction of PGRN with TNFR1&2, as reported by Tang et al [4].
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Cell viability [OD 450nm]
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Fig. 3 TNF-a-mediated cytotoxicity and PGRN-Ab status. a Proinflammatory effect of PGRN-Abs in UC: the adsorbance of colored Formazan, which is a marker for cell viability, was detected at 450 nm in HT29 cells after the administration of TNF-a, as well as serum of UC patients with or without PGRN-Abs and the serum of a healthy control. b Proinflammatory effect of PGRN-Abs in CD. Administration of TNF-a and of serum of CD patients with or without PGRN-Abs and of serum from a healthy control. c Anti-inflammatory effect of recombinant PGRN and proinflammatory effect of recombinant PGRN-Fabs. Administration of recombinant human PGRN, recombinant human SLP-2, monoclonal anti-PGRN-Fab (directed against the N-terminus), and TNF-a
In this respect, the present study demonstrates in vitro a proinflammatory effect of PGRN-Abs in the sera of patients with Crohn’s disease and UC. In TNF-a-mediated
cytotoxicity assays, the loss of the protective effects of PGRN in the presence of sera containing PGRN-Ab from patients with UC or with CD was demonstrated (Fig. 3a, b). Similar results were reported for psoriatic arthritis [12]. These results confirm the reported antagonistic action of PGRN on the effects of TNF-a, and the observation that human PGRN protects cells in vitro from the cytotoxic effects of TNF-a [4]. The results of the cytotoxicity assays with the administration of TNF-a and serum from PGRN-Ab-positive patients or matched PGRN-Ab-negative patients and healthy controls to HT29 cells demonstrate the significant proinflammatory effects of the serum samples containing neutralizing PGRN-Abs (Fig. 3a, b), and thus having lower PGRN levels (Fig. 2). A further TNF-a-induced cytotoxicity assay performed without serum samples but with recombinant PGRN and monoclonal anti-PGRN-Fabs excluded the possible influences of other ingredients of serum, and clearly confirmed the TNF-a antagonism by PGRN, which could be reversed by the anti-PGRN-Fab (Fig. 3c). Moreover, the present study confirmed the finding that PGRN inhibits the TNF-a-induced downmodulation of FOXP3 expression in CD4?CD25hi regulatory T cells in a similar manner as Etanercept [4] (Fig. 4). The proinflammatory effect of PGRN-Abs was demonstrated by the enhancement of the TNF-a-induced downmodulation of FOXP3 expression in CD4?CD25hi regulatory T cells. In this experiment, Etanercept was chosen as the established TNF-inhibitor, despite its therapeutic inferiority in IBD compared to monoclonal anti-TNF-antibodies, to avoid a possible bias evoked by the induction of apoptosis of CD4?CD25hi Tregs by anti-TNF-antibodies, since a subset of CD4?CD25hi Tregs does express membranebound TNF-a [18]. In the context of the recent controversy following publications that had challenged the interaction of PGRN and TNFR1&2 [6, 7], the present study confirms independently the anti-inflammatory effect of PGRN and demonstrates that this effect is at least partly due to antagonism of TNF-a. Whether or not this antagonism of TNF-a by PGRN is mediated by the direct inhibition of TNFR 1 and 2, as proposed by Tang et al., was not investigated in this study. Theoretically, it could be mediated as well by the inhibition of other members of the TNF-a receptor super family. Moreover, the TNF-a antagonizing effect of PGRN was demonstrated as well for commercially available PGRN (supplementary material). Considering these results, the neutralizing antibody directed against the physiological TNF-inhibitor PGRN could entertain an inflammatory environment and thus participate in the pathogenesis of IBDs. TNF-a plays a key role as a proinflammatory cytokine in IBD [19–21] and in most other rheumatic autoimmune
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Fig. 4 Enhanced TNF-a-induced downmodulation of FOXP3 of CD4?CD25hi Tregs by administration of PGRN-Ab. Cultured CD4?CD25hi regulatory T cells at a concentration of 5 9 104/ml were treated I with nothing, II with TNF-a (50 ng/ml), III with TNF-a and Etanercept (1 lg/ml), IV with TNF-a and recombinant human PGRN (250 ng/ml), V with TNF-a and recombinant human SLP2 (250 ng/ml), VI with TNF-a, recombinant PGRN and recombinant anti-PGRN-Ab (1 lg/ml), VII with TNF-a, recombinant SLP2 and recombinant anti-PGRN-Ab, or VIII with TNF-a, recombinant PGRN and recombinant anti-SLP2-Ab (1 lg/ml). Subsequently, the Tregs were cultured overnight at 37 °C. Following this, FOXP3 expression was determined by Western blot
diseases, which is supported by the overall clinical response rates to therapeutic antibodies against TNF-a in IBD and the great majority of rheumatic autoimmune diseases. However, the well-known major differences of the efficacy of the receptor fusion protein TNF-blocker Etanercept and the anti-TNF-a antibodies Inflximab and Adalimumab in CD, indicate that it is not the neutralizing effect on soluble TNF-a plasma levels that is relevant in CD, but rather the direct targeting of membranous TNF-a, which induces apoptosis of activated T-lymphocytes within the gut tissue [22]. The mechanism of neutralizing PGRN-Abs might be comparable to the well-known effects of neutralizing antidrug antibodies to therapeutic TNF-blockers [11]. Moreover, the pathomechanism of PGRN-Abs might also be comparable to anti-cytokine antibodies, e.g., autoantibodies against IFN-y, which have a contrary effect and result in adult-onset immunodeficiency characterized by different opportunistic infections despite normal CD4? Th cell counts [23]. Moreover, PGRN was shown to be strongly expressed in the gastrointestinal tract of rodents [24], so besides the neutralizing effect on the plasma level of secreted PGRN, PGRN-Abs might directly target the gut tissue in IBD. In general, a possible pathogenic role of B lymphocytes in IBD has been under debate, as the clinically established antibodies seem not to be pathogenically relevant. Moreover, case reports described exacerbations of UC after B-cell depletion therapy [25, 26]. However, a randomized case-control study about the safety and efficacy of B-cell depletion in UC showed a good tolerability, but only possible short-term responses [27]. Most experimental studies on B lymphocytes in IBD have analyzed B lymphocytes
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infiltrating inflamed gut tissue [28, 29]. The presence of frequently detectable antibodies against microbial antigens, such as anti-Saccharomyces cerevisiae antibodies [30] and anti-Flagellin-antibodies [31] in CD, suggest that the reactivity of at least a part of these tissue-infiltrating B lymphocytes is directed against local microbes. Antigens of enteric bacteria have also been suggested as the drivers of pANCAs in UC by a process of molecular mimicry of bacterial components and human myeloperoxidase [32]. Remarkable in UC is a disproportionally increased serum fraction of the IgG1 subclass [33]. However, it is not yet clear whether this increased fraction is a mere epiphenomenon or if it indicates a pathogenic involvement of B lymphocytes. As reported for tropomyosin antibodies in UC, PGRN-Abs in primary vasculitides belong predominantly to the IgG1 subclass (96.3 %) [1, 34]. A lower, but still disproportionate overrepresentation of the IgG1 subclass for PGRN-Abs was observed for IBD (27/38; 71.1 %) (Fig. 1c). The presence of PGRN-Abs of the IgA-class in IBD (10/38; 26.3 %) could indicate an involvement of B cells located in the MALT. In both CD and UC, the presence of PGRN-Abs showed no association with either clinical parameters or with particular classes of prescribed medication. This might be explained by the relatively small absolute numbers of PGRN-Ab-positive patients and by the patient population included in this study with a relatively high proportion of patients receiving immunosuppression and having severe courses. Moreover, for CD a statistically significant association between multiple tested serum samples of an individual patient and positivity for PGRN-Abs in the course of disease was observed (p = 0.005), suggesting that PGRN-Abs occur transiently. Furthermore, there was neither a statistically significant association of the presence of PGRN-Abs with positive ASCA status in CD nor with positive ANCA status in UC. As mentioned before, pANCA is the only established antibody associated with UC, despite its unspecificity due to the wide spectrum of coexisting pANCA-associated diseases. In this respect, it might be of particular interest that PGRN-Abs were more frequently detected in UC (15/71; 20.13 %) than were pANCAs (4/65; 6.2 %; p = 0.012), cANCAs (7/65; 9.9 %; p = 0.101) and pANCAs and cANCAs together (9/65; 13.8 %, p = 0.266) (Fig. 1d). Surprisingly, in the present study cANCA/Pr3ANCA were slightly more frequent than pANCA/MPOANCA in patients with UC without any signs of vasculitis. The frequent presence of cANCA/Pr3-ANCA in UC was also reported recently [35]. In CD, PGRN-Abs were less frequent than ASCAs. Although the high percentage of missing data limits the validity of this analysis, ASCAs certainly have a higher
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specificity for CD than PGRN-Abs. However, it should be kept in mind that ASCAs also frequently occur in intestinal tuberculosis [36]. In conclusion, this study shows that IBD shares with several rheumatic autoimmune diseases the presence of PGRN-Abs, suggesting that these autoantibodies represent a common finding in autoimmune diseases affecting different organs. Paying attention to the obtained in-vitro data of the present study, PGRN-Abs presumably participate actively in the pathogenesis of the seropositive subgroup of patients with IBDs by their neutralizing effect on the plasma level of secreted anti-inflammatory PGRN. Nevertheless, the data on the frequency of PGRN-Abs in the course of IBD have to be regarded as preliminary, since multiple serum samples obtained at different points of time during the course of disease could only be tested for PGRN-Abs from 8.5 % of patients with CD (Table 2, supplementary material) and from 9.9 % of patients with UC (Table 1, supplementary material). Despite the low percentage of serial serum samples available for this study, the observed seroconversions suggest that PGRN-Abs can be found in a considerably higher percentage of patients at some time points during the course of their disease. Future studies could investigate possible family clustering of PGRN-Abs similar to those reported for anti-tropomyosin antibodies [37]. Furthermore, such studies should also investigate in vivo the suspected pathogenicity of PGRN-Abs. In vivo studies could combine either a passive transfer of murinized neutralizing anti-murine PGRN antibodies or an active immunization approach by the administration of N-terminal immunogenic fragments of PGRN with established mouse models for IBDs. Furthermore, future studies should focus on identifying the reason for the breakdown of self-tolerance against PGRN. Acknowledgments We are grateful to Jutta Conigliaro, Christian Schorpp, Ralph Hastenteufel, Gabi Carbon, the outpatient IBD clinic of Saarland University Medical Center in Homburg/Saar, and Diana Thurner and Bernhard Thurner, who helped with the collection of blood samples from IBD patients or gave helpful advice. The study was supported by a Saarland University Fellowship to L.T. and the HOMFOR program of the Saarland University Medical School to V.Z. Conflict of interest Saarland University, L.T., K.D.P., and M.P. filed 61/730,772, which covers the means and methods for detecting autoimmune disorders in which progranulin may be involved.
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30. Main J, McKenzie H, Yeaman GR, et al. Antibody to Saccharomyces cerevisiae (bakers’ yeast) in Crohn’s disease. BMJ. 1988;297:1105–1106. 31. Lodes MJ, Cong Y, Elson CO, et al. Bacterial flagellin is a dominant antigen in Crohn disease. J Clin Invest. 2004;113: 1296–1306. 32. Seibold F, Brandwein S, Simpson S, Terhorst C, Elson CO. pANCA represents a cross-reactivity to enteric bacterial antigens. J Clin Immunol. 1998;18:153–160. 33. MacDermott RP, Nash GS, Auer IO, et al. Alterations in serum immunoglobulin G subclasses in patients with ulcerative colitis and Crohn’s disease. Gastroenterology. 1989;96:764–768. 34. Geng X, Biancone L, Dai HH, et al. Tropomyosin isoforms in intestinal mucosa: production of autoantibodies to tropomyosin isoforms in ulcerative colitis. Gastroenterology. 1998;114:912–922. 35. Arias-Loste MT, Bonilla G, Moraleja I, Mahler M, Mieses MA, Castro B, Rivero M, Crespo J, Lopez-Hoyos M. Presence of Antiproteinase 3 Antineutrophil Cytoplasmic Antibodies (Anti-PR3 ANCA) as Serologic Markers in Inflammatory Bowel Disease. Clin Rev Allergy Immunol. 2013;45:109–116. 36. Makharia GK, Sachdev V, Gupta R, Lal S, Pandey RM. AntiSaccharomyces cerevisiae antibody does not differentiate between Crohn’s disease and intestinal tuberculosis. Dig Dis Sci. 2007;52:33–39. 37. Biancone L, Monteleone G, Marasco R, Pallone F. Autoimmunity to tropomyosin isoforms in ulcerative colitis (UC) patients and unaffected relatives. Clin Exp Immunol. 1998;113:198–205.
ORIGINAL ARTICLE
Proinflammatory Progranulin Antibodies in Inflammatory Bowel Diseases Lorenz Thurner • Elisabeth Sto¨ger • Natalie Fadle • Philipp Klemm • Evi Regitz • Maria Kemele • Birgit Bette • Gerhard Held • Marc Dauer • Frank Lammert • Klaus-Dieter Preuss • Vincent Zimmer • Michael Pfreundschuh
Received: 20 July 2013 / Accepted: 19 February 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Recently, we identified neutralizing autoantibodies against progranulin (PGRN) in a wide spectrum of rheumatic diseases including cases with enteropathic spondylarthritis. PGRN is a secreted protein with strong anti-inflammatory effects, believed to be mediated by the direct inhibition of TNF receptors 1&2. Given the central role of TNF-a as proinflammatory cytokine, a neutralizing
Electronic supplementary material The online version of this article (doi:10.1007/s10620-014-3089-3) contains supplementary material, which is available to authorized users. L. Thurner E. Sto¨ger N. Fadle P. Klemm E. Regitz M. Kemele B. Bette G. Held K.-D. Preuss M. Pfreundschuh Jose´ Carreras Center for Immuno- and Gene Therapy and Internal Medicine I, Saarland University Medical School, Homburg/Saar, Germany e-mail: [email protected] L. Thurner (&) Department of Internal Medicine I, University Hospital Homburg/Saar, Kirrberger Str., 66421 Homburg/Saar, Germany e-mail: [email protected]; [email protected] Present Address: M. Dauer Department of Medicine, St. Elisabeth Kliniken, Neuburg/ Donau, Germany F. Lammert V. Zimmer Department of Internal Medicine II, Saarland University Medical Center, Homburg/Saar, Germany e-mail: [email protected] V. Zimmer e-mail: [email protected]
antibody directed against its physiologic antagonist PGRN might entertain a proinflammatory environment. Objective The aim of the present study was to investigate a possible occurrence of PGRN-antibodies (PGRN-Abs) in inflammatory bowel disease (IBD), and to investigate a possible pathogenic effect. Materials and Methods Sera samples of 141 patients with Crohn’s disease (CD) and of 71 patients with ulcerative colitis (UC) were tested for PGRN-Abs by ELISA. PGRN plasma levels were detected by ELISA. Proinflammatory effects of progranulin-antibodies were analyzed by TNF-amediated cytotoxicity assays using HT29 cells and by examination of possible effects of PGRN and of PGRNantibodies on TNF-a-induced downmodulation of FOXP3 expression in CD4?CD25hi Tregs. Results PGRN-Abs were found in sera of 23/141 (16.31 %) patients with CD, and 15/71 (21.13 %) patients with UC. PGRN-Abs were more frequent than anti-neutrophil cytoplasmic autoantibodies (ANCAs) in UC, but less frequent than anti-Saccharomyces cerevisiae antibodies (ASCAs) in CD. PGRN-Abs belonged mostly to IgG1 (71.1 %) and IgA (26.3 %). They occurred in relevant titres and had significant neutralizing effects on PGRN plasma levels. Cytotoxicity assays comparing PGRN-antibody-positive sera with negative sera from matched patients with IBD showed a proinflammatory effect of PGRN-Abs on HT29 cells. Moreover, PGRN-antibodies led to an increase of TNF-a-induced downmodulation of FOXP3 in CD4?CD25hi Tregs. Conclusion The results suggest that PGRN-Abs occur frequently in CD and UC, and have a proinflammatory effect. Keywords Progranulin Inhibitor of TNFR1&2 Neutralizing proinflammatory progranulin-antibody Crohn’s disease Ulcerative colitis
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Introduction Inflammatory bowel disease (IBD) encompasses two clinically, histopathologically, and pathogenetically distinct diseases, namely ulcerative colitis (UC) and Crohn’s disease (CD). In both diseases, alterations of the innate and adaptive immune system eventually result in systemic and/ or local increases of proinflammatory cytokine levels such as TNF-a, IL-6, IL-1b, and IFN-c, the latter particularly in CD. We recently discovered progranulin antibodies (PGRNAbs) in various primary vasculitides using a protein macroarray-based approach, and these antibodies were subsequently also found in several other rheumatic autoimmune diseases with varying frequency. So far, PGRN-Abs have been found in several primary vasculitides: Takayasu arteriitis, giant cell arteriitis, Churg-Strauss syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, panarteritis nodosa, Behcet’s disease, polymyalgia rheumatica, and apart from primary vasculitides, in systemic lupus erythematosus and rheumatoid arthritis. In contrast, PGRN-Abs were rarely found in healthy controls and in patients with sepsis or melanoma [1]. Progranulin (PGRN) is a secreted precursor protein of 88 kDa, with a signal peptide and seven granulin motives, each containing 12 cysteins and six disulfide bonds [2]. PGRN has several functions, amongst others a strong antiinflammatory effect [3]. This anti-inflammatory action of PGRN was proposed to be mediated by the direct inhibition of the TNF receptors 1&2 (TNFR1&2) [4], while PGRN is regarded as a physiological endogenous TNF-blocker, and recombinant human PGRN or its synthetically constructed derivative molecule, ATSTTRIN, have been proposed as next-generation TNF-blockers [5]. However, this proposed mechanism of the known anti-inflammatory effect of PGRN was challenged recently [6] without a denial of the anti-inflammatory effect of PGRN [3]. In detail, Chen et al. could not reproduce the interaction of PGRN binding to TNFR1&2 as reported by Tang et al. [4]. Moreover, Etemadi et al. [7] could not reproduce any antagonistic effects of recombinant human PGRN on TNFa-induced cytotoxicity and on TNF-a-induced downstream signal cascade activation via TNFR1. Etemadi et al. used recombinant PGRN from different commercial sources (R&D and Adipogen). The authors responded in a letter to the editor, suggesting that the murine PGRN utilized by Chen et al. might be improperly folded. Furthermore, a validation of PGRN functionality based only on its C-terminal binding to Sortilin would be insufficient. Following this, they demonstrated that PGRN binds similarily to the way TNF-a joins to CRD2 and CRD3 of TNFR, and that proper folding of PGRN is required for this binding. Moreover, DTT
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treatment of PGRN, which had been performed by Chen et al., abolishes the binding of PGRN to TNFR, but enhances its binding to Sortilin [8]. In the meantime, the binding of PGRN to TNFR and its inhibitory effect on TNF-a-induced effects were recently reproduced independently by two other groups. In vitro PGRN suppressed TNF-a-induced neutrophil chemotaxis and ICAM-1 expression in endothelial cells, and in vivo recombinant PGRN treatment suppressed neutrophil recruitment into the ischemia/reperfusion region of the brain via middle cerebral artery occlusion in a mouse-model of cerebral ischemia–reperfusion (I/R) injury, resulting in a reduction of NF-jB and MMP-9 activation [9]. Recombinant PGRN suppressed TNF-a-induced expression of adhesion molecules ICAM-1 and VCAM-1 in HUVECs [10]. PGRN-Abs have a neutralizing effect on plasma levels of secreted PGRN, suggesting a proinflammatory effect of PGRN-Abs by neutralizing this physiologic TNF-a antagonist [1], comparable to the effects of anti-drug antibodies that can arise after the repeated administration of therapeutic TNFblockers [11]. This hypothesis is supported by the fact that the presence of PGRN-Abs was associated with the active disease state in granulomatosis with polyangiitis [1]. Furthermore, we recently reported the presence of proinflammatory PGRN-Abs in psoriatic arthritis. Psoriatic arthritis was believed to be a seronegative disease. PGRNAb containing sera of patients with psoriatic arthritis resulted in an increased sensitivity of HT-1080 cells to the effects of TNF-a compared to the administration of sera from patients with psoriatic arthritis without PGRN-Abs [12]. IBD are often associated with rheumatic diseases. Both IBD and several rheumatic autoimmune diseases respond to therapeutically-administrated anti-TNF-a-antibodies. In addition, the ‘‘shared’’ presence of perinuclear anti-neutrophil cytoplasmic autoantibodies (pANCAs) as autoantibodies in IBD and small-vessel vasculitides, and the occurrence of PGRN-Abs in patients with enteropathic spondylarthritis prompted us to investigate the presence of PGRN-Abs in the serum of patients with IBD.
Materials and Methods Study Participants This study was approved by the local ethical review board ¨ rztekammer des Saarlandes’’ (‘‘Ethikkommission der A N242/11) and conducted according to the guidelines laid down in the Declaration of Helsinki. Plasma and sera samples of patients with UC and CD were obtained at the outpatient IBD clinic of Saarland University Medical Center in Homburg/Saar during routine diagnostics. From
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some individual patients, more than one serum sample had been obtained at different points of time during the course of their disease. Blood samples from healthy controls were obtained with written informed consent at the Saarland University Medical Center in Homburg/Saar. Blood samples were stored at -80 °C until use. All 141 patients with CD and 71 patients with UC were classified according to the Montreal Classification Criteria [13]. In addition, data on the prescribed medication, anti-neutrophil cytoplasmic autoantibody (ANCA) status, and anti-Saccharomyces cerevisiae antibody (ASCA) status were obtained, whenever available.
(PCR) using a proofreading taq. Mutations were excluded by sequencing. Subsequently, the proteins were recombinantly expressed with a C-terminal FLAG tag in HEK293 cells under the control of a CMV promoter (pSFI). Chromatographic purification of FLAG-tagged PGRN was performed with an anti-FLAG affinity matrix. In short, crude whole-cell lysates were clarified by centrifugation. To pull down the Flag-tagged PGRN, 50 ll of anti-FLAG gel suspension were coincubated with 2 ml of cell lysates for 30 min at 4 °C. Afterwards, 100 ll FLAG-Peptid solution 0.1 mg/ml was used for non-denaturing elution of FLAG-tagged PGRN. The resin was washed in PBS. Final concentration was 90 lg/ml.
Progranulin-Antibody ELISA Cytotoxicity Assay A number of enzyme-linked immunosorbent assays (ELISAs) for PRGN-Abs and for the determination of immunoglobulin class and IgG subclasses were performed as previously described [1]. The average of the optical density (OD) values of the negative samples plus three standard deviations was applied as the cut off for positivity. From several patients, more than one serum sample had been obtained during their course of disease. If at least one of these serum samples was positive for PGRN-Abs, this patient was regarded as positive for PGRN-Abs in the course of disease. Progranulin ELISA Progranulin plasma levels were determined by a commercially available ELISA kit (AdipoGen, South Korea) according to the manufacturer’s instructions. The median PGRN plasma level of healthy controls was set at 100 %, as described before [1]. Selection of Anti-PGRN-Fabs Specific Against the N-Terminus The selection of Fabs (antigen binding fragment) with specific reactivity against the N-terminus of PGRN, was performed as described before [14, 15]. As a phagemid library, a non-immune, semi-synthetic human Fab repertoire containing 3.7 9 1010 different possible antibody fragments was used [16]. For selection, an N-terminally biotinylated PGRN AA70-91 peptide was used. To obtain specific Fabs against the N-terminus of PGRN, five rounds of selection were necessary. Expression and Purification of Recombinant Human PGRN and SLP2 The coding sequences of PGRN and SLP2 were amplified from a human cDNA library by polymerase chain reaction
A non-radioactive cytotoxicity assay (EZ4U, Biomedica) was performed according to the manufacturer’s instructions. As target cells for this TNF-a-induced cytotoxicity assay, the human colorectal adenocarcinoma HT29 cell-line was used. In short, 4 9 104 HT29 cells were seeded in 200-ll cell cultures at 37 °C and at 5 % CO2. To detect possible differences between added sera of UC patients with and without PGRN-Abs, or of CD patients with and without PGRN-Abs and of healthy controls, the serum of a PGRNAb positive patient, serum of a matched PGRN-Ab-negative patient, and serum of a healthy control were administered in dilutions from 1:4 to 1:512 to the cultured HT29 cells, followed by the administration of TNF-a (10 lg/ml). Serum samples from gender-, age-, disease- and therapy modalitymatched patients were chosen. Serum samples from patients receiving TNF-blockers or other biological agents were excluded. As the 20-min half-life of TNF-a [17] is negligibly short compared to the 40-h half-life of PGRN [4], the effects of the intrinsic TNF-a in the sera of patients and controls were neglected. HT29 cells without the addition of TNF-a and serum, or solely with the addition of TNF-a (10 lg/ml) were used as positive and negative controls. After 24 h of incubation at 37 °C, 20 ll of chromophore substrate were added to each well. This chromophore substrate is only converted by vital cells. The adsorbance of the product was measured at an OD of 450 nm. To exclude possible influences on the TNF-a-induced cytotoxicity assay by any ingredient of the administrated sera other than PGRN, the assay was repeated without sera of PGRN-Ab positive or negative patients, but with recombinant human PGRN expressed in HEK293 cells at a concentration of 250 ng/ml, with recombinant human SLP2 at a concentration of 250 ng/ml expressed in the same HEK293 expression system, and with recombinant anti-human-PGRN-Fab (starting concentration 2 lg/ml) in dilutions from 1:2 to 1:128 directed against the N-terminus of PGRN. To investigate whether commercially available
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recombinant PGRN expressed in HEK293 cells (Adipogen) has comparable TNF-antagonizing effects to the recombinant PGRN expressed in our laboratory, the cytotoxicity assay was repeated with 4 9 104 WEHI-S cells/ml, the administration of 100 pg/ml TNF-a, and of either 250 ng/ml commercially available recombinant PGRN (Adipogen, Lot Nr. K110517) or recombinant PGRN, which had been synthesized in our laboratory. After 24 h incubation at 37 °C, 20 ll of chromophore substrate were added to each well. This chromophore substrate is only converted by vital cells. The adsorbance of the product was measured at an OD of 450 nm. Effect of PGRN-Abs on FOXP3 Expression of CD4?CD25hi Regulatory T Cells PBMCs were isolated from the venous blood of healthy controls in tubes containing EDTA by Ficoll density gradient centrifugation. Subsequently, CD4?CD25hi Tregs were isolated (Milteny Biotech, Bergisch Gladbach, Germany). In each well, CD4?CD25hi regulatory T cells at a concentration of 5 9 104/ml were cultured and treated either with Etanercept 1 lg/ml, with 250 ng/ml recombinant human PGRN, with 250 ng/ml recombinant human SLP2, with 250 ng/ml recombinant PGRN and 1 lg/ml recombinant anti-PGRN-Ab, with 250 ng/ml recombinant SLP2 and 1 lg/ml recombinant anti-PGRN-Ab, and with 250 ng/ml recombinant PGRN and 1 lg/ml recombinant anti-SLP2-Ab for 30 min at 37 °C. The mentioned mixtures of recombinant proteins and monoclonal antibodies had been preincubated for 30 min at 4 °C, before they were administrated to the CD4?CD25hi Tregs. Subsequently, 50 ng/ml TNF-a were administrated to each well and the Tregs were cultured overnight at 37 °C. Following this, FOXP3 expression was determined by Western blot analysis real-time PCR was not performed. Etanercept was chosen for this experiment, despite its lower efficiency in IBD compared to anti-TNF-antibodies. By choosing Etanercept, the direct targeting of T cells by anti-TNF-antibodies via membranous TNF-a should be avoided. Equivalent amounts of Treg cell lysates were loaded and separated in a 10 % SDS-PAGE, transferred to PVDF membrane using a transblot semidry transfer cell (Bio Rad). Transferred proteins were incubated with mouse antihuman FOXP3 antibody at a dilution of 1:1,000 (Exbio) followed by incubation with a goat HRP labeled antimouse IgG antibody at a dilution of 1:3,000 (Bio Rad). For the actin control, a rabbit anti-human Actin antibody was incubated at a dilution of 1:1,000 (Sigma) followed by incubation with a goat HRP-labeled anti-rabbit IgG antibody at a dilution of 1:3,000 (Bio Rad). ECL reagent (NEB) was utilized for immunoblot detection.
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ANCA and ASCA Status Detection of perinuclear anti-neutrophil cytoplasmic autoantibodies (pANCA) and cytoplasmic anti-neutrophil cytoplasmic autoantibodies (cANCA) had already been performed during clinical routine diagnostics, according to standardized laboratory procedures, by initial indirect immunofluorescence screening for perinuclear or cytoplasmic ANCA patterns, followed in positive or borderline cases by ELISA (Varelisa-Kit) for MPO-ANCA and Pr3ANCA at the autoimmune diagnostic laboratory of the Department of Internal Medicine I at the Saarland University Medical School. The cut off for positive MPOANCA and Pr3-ANCA was set according to the manufacturer’s instructions. ASCA status had been determined in subgroups of patients with CD and UC by a commercial laboratory. Statistical Analysis Different frequencies of PRGN-Abs in subgroups of patients with CD and UC classified by clinical characteristics, prescribed medication, and ANCA- and ASCA-status were tested by v2 test, and, respectively, Fisher’s exact test when indicated. Differences of PGRN serum levels between PGRN-Ab-positive patients with IBD, PGRN-Abnegative patients with IBD, and healthy controls were compared utilizing the Mann–Whitney U test. A p value of \0.05 was considered as statistically significant.
Results Patient Characteristics Tables 1 and 2 in the supplementary materials summarize the clinical characteristics of patients with UC and CD, respectively. In seven of 71 (9.9 %) patients with UC and in 12 of 141 (8.5 %) patients with CD, more than one serum sample was tested for PGRN-Abs. Frequency, Titres, and Immunoglobulin Class of Progranulin-Antibodies Overall, PGRN-Abs were detected in 23 of 141 (16.31 %) patients with CD and in 15 of 71 (20.13 %) patients with UC. In contrast to this, PGRN-Abs had only been detected in a single individual among 97 healthy controls (1.03 %) (Fig. 1a) with low antibody-titres. Moreover, PGRN-Ab titres were determined in randomly chosen PGRN-Abpositive patients and ranged from 1:800 to 1:3,200 (Fig. 1b). The analysis of immunoglobulin classes of
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Occurence of PGRN-Ab during course of disease [%]
(a)
b Fig. 1 a Frequency of PGRN-Abs in IBDs. Columns represent the 24 22 20 18
23/141 = 16.31%
16 14 12 10 8 6 4
1/97 =1.03%
2 0 Chron's disease
(b)
frequency of PGRN-Ab during the course of disease in CD, UC, and healthy controls. b Titres of PGRN antibodies in IBDs. Each curve indicates the ODs obtained for sera from an individual patient or a healthy control at different dilution steps. c Frequency of different immunoglobulin classes of PGRN-Ab in IBDs. Columns represent the relative frequency of each IgG subclass and IgA or IgM class of PGRN-Abs in CD and UC. d Frequency of PGRN-Abs and ANCAs in UC. Columns represent the relative frequency of PGRN-Abs, pANCAs, and cANCAs in UC
15/71 =21.13%
Ulcerative Colitis
Healthy Controls
2.5
Adsorption (OD ad 490nm)
Negative Control
2.0
Ulcerative colitis 2 Chron's disease 1
1.5
Chron's disease 2 Chron's disease 3
1.0
0.5
1:100
Immunoglobulin classes of PGRN-Abs in IBD [%]
1:200
1:400
1:800
1:1600
1:3200
1:6400
100 90 80
27/38 71.1%
70 60 50 40
10/38 26.3%
30 20 1/38 2.6%
10
0/38 0.0%
0/38 0.0%
IgG3
IgG4
0/38 0.0%
0
25
Occurence during course of disease [%]
IgG1
(d)
20
Association of PGRN-Ab Status with Clinical Characteristics, ANCA, and ASCA
Ulcerative colitis 1
0.0
(c)
(26.1 %). In each of the PGRN-Ab-positive sera samples, PGRN-Abs belonged only to a single immunoglobulin class, i.e., either IgG1, IgG2, or IgA could be identified.
IgG2
IgA
IgM
21.13%
13.85%
15 10.77%
10 6.15%
5
0 PGRN-Ab
pANCA
cANCA
pANCA and/or cANCA
PGRN-Abs revealed IgG1 in 27/38 (71.1 %), IgA in 10/38 (26.3 %) and IgG2 in 1/38 (2.6 %) (Fig. 1c). PGRN-Abs of IgA class were detected with similar frequencies in patients with UC 4/15 (26.7 %) and patients with CD 6/23
Furthermore, the presence of PGRN-Abs was analyzed for associations with clinical characteristics, and if available with ANCA or ASCA status and the class of prescribed medication. Using the v2 tests, no statistically significant associations between the presence of PGRN-Abs and clinical characteristics, prescribed medication, or ANCA and ASCA status were observed for UC or for CD. Regarding UC, of the seven individual patients in which more than one serum sample was obtained, three of these patients were positive during the course of disease compared to 64 patients with a single tested sample and 12 positives [3/7 (42.9 %) vs. 12/64 (18.75 %); p = 0.158]. Regarding Crohn’s disease, PGRN-Abs were more often observed in a given patient if more than one serum sample taken at different time points during her/his disease was available for testing [6/12 (50 %) vs. 17/129 (13.2 %); p = 0.005]. Moreover, in UC, PGRN-Abs were more frequent (15 of 71; 20.13 %) than pANCA (four of 65; 6.2 %; p = 0.0135), more frequent than cANCA (seven of 65; 9.9 %; p = 0.101) and more frequent than pANCA and/or cANCA (nine of 65; 13.8 %; p = 0.266; Fig. 1d and supplementary material Table 1). There was no statistically significant association between positive serostatus for ANCA and positive serostatus for PGRN-Ab. In CD, PGRN-Abs were more frequent (23 of 141; 16.31 %) than pANCA (one of 127; 0.8 %; p \ 0.001) and cANCA (zero of 127; 0.0 %; p \ 0.001), but less frequent than ASCA (14 of 29; 48.3 %; p \ 0.001). Only a minor proportion of patients with CD had been tested in the context of clinical routine diagnostics for ASCA (29/ 141 or 20.57 %; supplementary Table 2). Of the 29 tested patients for ASCA, 14 (48.3 %) were positive. There was no statistically significant association for ASCA-positive patients to be PGRN-Ab-positive. Progranulin ELISA Using a commercially available ELISA, we observed that PGRN levels were significantly decreased in plasma of
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recombinant human SLP-2 as a control, and recombinant anti-PGRN-Fab (directed against the N-terminus) confirmed the TNF-a antagonism of PGRN, which was reversed by the additional administration of anti-PGRNFab (Fig. 3c). Commercially available recombinant PGRN had an TNFantagonizing effect as well. This effect was weaker than the effect of the recombinant PGRN expressed and purified in our laboratory, but the effect of commercially available PGRN was still significant (supplementary material).
Fig. 2 The neutralizing effect of PGRN-Abs on PGRN plasma levels in IBD. Lane a healthy controls (n = 7); lane b PGRN-Ab-negative patients with IBD (n = 7); lane c PGRN-Ab-positive patients with IBD (n = 7). Statistical analysis revealed highly significant differences in the PGRN plasma levels of PGRN-Ab-positive patients with IBD compared to PGRN-Ab-negative patients with IBD (Mann– Whitney test: p \ 0.001) and to healthy controls (Mann–Whitney test: p \ 0.001). No significant differences were detected between PGRN-Ab-negative patients with Chron’s disease or UC and the healthy control group (p = 0.456). The median PGRN serum level obtained of the healthy controls was set at 100 %
PGRN-Ab-positive patients with IBD (n = 7) compared to PGRN-Ab-negative patients with IBD (n = 7; Mann– Whitney U test: p \ 0.001) and compared to healthy controls (n = 7; Mann–Whitney U test: p \ 0.001). There was no significant difference in the PGRN plasma levels of PGRN-Ab-negative patients with IBD compared to healthy controls (Mann–Whitney U test: p = 0.456; Fig. 2). The median PGRN plasma level of healthy controls was set at 100 %, as described before [1]. Cytotoxicity Assay For the TNF-a-induced cytotoxicity assays, the protective effect of serum from healthy controls and from patients with UC and CD with and without PGRN-Ab was analyzed. The addition of serum from patients with UC without PGRN-Ab and from healthy controls reduced TNF-a-induced cytotoxicity of HT29 cells to a significantly higher degree compared to PGRN-Ab containing serum of patients with UC. This difference was significant up to a dilution of serum of 1:64 (Fig. 3a). In analogy to this, the addition of serum from patients with CD without PGRN-Ab and from healthy controls reduced the TNF-ainduced cytotoxicity of HT29 cells to a significantly higher degree compared to serum containing PGRN-Ab from patients with CD. This difference was also significant up to a serum dilution of 1:64 (Fig. 3b). The TNF-a-induced cytotoxicity assay of HT29 cells with administrated recombinant human PGRN,
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Effect of PGRN-Abs on TNF-a-Induced Downregulation of Foxp3 Expression in CD4?CD25hi T Cells To study the role of PGRN and PGRN-Abs on the function of inflammatory cells, we analyzed the TNF-a-induced downmodulation of FOXP3 expression in CD4?CD25hi regulatory T cells in the presence of recombinant PGRN, recombinant SLP2, Etanercept, recombinant SLP2-Fabs, and recombinant PGRN-Fabs. Western blot showed that the administration of recombinant PGRN antagonized the TNF-a-induced downmodulation of FOXP3 expression to a similar extent as the administration of Etanercept. The administrated recombinant control protein SLP-2, which had been expressed in the same cell system and purified by the same means as recombinant PGRN, didn’t show any antagonistic effect on TNF-a. The TNF-a-inhibiting effect of PGRN was blocked by the administration of anti-PGRNFab.
Discussion The present study firstly demonstrates the important point that PGRN-Abs are not restricted to rheumatic autoimmune diseases such as primary vasculitides, SLE, rheumatoid arthritis [1], or psoriatic arthritis [12], but can also be encountered in a subgroup of patients with UC and CD. In detail, 16.31 % of patients with CD and 20.13 % of patients with UC were positive for PGRN-Abs (Fig. 1a). Both in UC and in CD, PGRN-Abs had relevant titres (Fig. 1b) that were comparable to the titres found previously in rheumatic autoimmune diseases [1]. Moreover, the neutralizing effect of PGRN-Abs on the levels of circulating PGRN, which was already shown for rheumatic autoimmune diseases [1], could be confirmed for IBD (Fig. 2). This is of particular interest because PGRN displays anti-inflammatory effects [3], which were reported to be mediated by the direct inhibition of TNFR 1 and 2 [4], which was challenged recently by Chen et al. In detail, Chen et al. were not able to reproduce the interaction of PGRN with TNFR1&2, as reported by Tang et al [4].
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Cell viability [OD 450nm]
(a)
1.4 1.2 1.0
neither serum nor TNF-alpha only TNF-alpha
0.8 serum of healthy control + TNF-alpha serum of PGRN-Ab negative ulcerative colitis + TNF-alpha
0.6
serum of PGRN-Ab positive ulcerative colitis + TNF-alpha
0.4 0.2 0.0 1:4
1:8
1:16
1:32
1:64
1:128
dilution of administrated sera
Cell viability [OD 450nm]
(b)
1.2
1.0 neither serum nor TNF-alpha
0.8
only TNF-alpha serum of healthy control + TNF-alpha
0.6
serum of PGRN-Ab positive Crohn's disease + TNF-alpha Serum of PGRN-Ab negative Crohn's disease + TNF-alpha
0.4
0.2
0.0 1:4
1:8
1:16
1:32
1:64
1:128
(c)
1,4
Cell viability [OD at 450 nm]
dilution of administrated sera
1,2
only TNF
1,0 TNF + rec. SLP2
0,8 TNF + rec. PGRN + anti-PGRN-Fab
0,6
TNF + rec. PGRN without anti-PGRNFab
0,4 0,2
1:128
1:64
1:32
1:16
1:8
1:4
1:2
1:1
0,0
dilution of administrated Anti-PGRN-Fab
Fig. 3 TNF-a-mediated cytotoxicity and PGRN-Ab status. a Proinflammatory effect of PGRN-Abs in UC: the adsorbance of colored Formazan, which is a marker for cell viability, was detected at 450 nm in HT29 cells after the administration of TNF-a, as well as serum of UC patients with or without PGRN-Abs and the serum of a healthy control. b Proinflammatory effect of PGRN-Abs in CD. Administration of TNF-a and of serum of CD patients with or without PGRN-Abs and of serum from a healthy control. c Anti-inflammatory effect of recombinant PGRN and proinflammatory effect of recombinant PGRN-Fabs. Administration of recombinant human PGRN, recombinant human SLP-2, monoclonal anti-PGRN-Fab (directed against the N-terminus), and TNF-a
In this respect, the present study demonstrates in vitro a proinflammatory effect of PGRN-Abs in the sera of patients with Crohn’s disease and UC. In TNF-a-mediated
cytotoxicity assays, the loss of the protective effects of PGRN in the presence of sera containing PGRN-Ab from patients with UC or with CD was demonstrated (Fig. 3a, b). Similar results were reported for psoriatic arthritis [12]. These results confirm the reported antagonistic action of PGRN on the effects of TNF-a, and the observation that human PGRN protects cells in vitro from the cytotoxic effects of TNF-a [4]. The results of the cytotoxicity assays with the administration of TNF-a and serum from PGRN-Ab-positive patients or matched PGRN-Ab-negative patients and healthy controls to HT29 cells demonstrate the significant proinflammatory effects of the serum samples containing neutralizing PGRN-Abs (Fig. 3a, b), and thus having lower PGRN levels (Fig. 2). A further TNF-a-induced cytotoxicity assay performed without serum samples but with recombinant PGRN and monoclonal anti-PGRN-Fabs excluded the possible influences of other ingredients of serum, and clearly confirmed the TNF-a antagonism by PGRN, which could be reversed by the anti-PGRN-Fab (Fig. 3c). Moreover, the present study confirmed the finding that PGRN inhibits the TNF-a-induced downmodulation of FOXP3 expression in CD4?CD25hi regulatory T cells in a similar manner as Etanercept [4] (Fig. 4). The proinflammatory effect of PGRN-Abs was demonstrated by the enhancement of the TNF-a-induced downmodulation of FOXP3 expression in CD4?CD25hi regulatory T cells. In this experiment, Etanercept was chosen as the established TNF-inhibitor, despite its therapeutic inferiority in IBD compared to monoclonal anti-TNF-antibodies, to avoid a possible bias evoked by the induction of apoptosis of CD4?CD25hi Tregs by anti-TNF-antibodies, since a subset of CD4?CD25hi Tregs does express membranebound TNF-a [18]. In the context of the recent controversy following publications that had challenged the interaction of PGRN and TNFR1&2 [6, 7], the present study confirms independently the anti-inflammatory effect of PGRN and demonstrates that this effect is at least partly due to antagonism of TNF-a. Whether or not this antagonism of TNF-a by PGRN is mediated by the direct inhibition of TNFR 1 and 2, as proposed by Tang et al., was not investigated in this study. Theoretically, it could be mediated as well by the inhibition of other members of the TNF-a receptor super family. Moreover, the TNF-a antagonizing effect of PGRN was demonstrated as well for commercially available PGRN (supplementary material). Considering these results, the neutralizing antibody directed against the physiological TNF-inhibitor PGRN could entertain an inflammatory environment and thus participate in the pathogenesis of IBDs. TNF-a plays a key role as a proinflammatory cytokine in IBD [19–21] and in most other rheumatic autoimmune
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Fig. 4 Enhanced TNF-a-induced downmodulation of FOXP3 of CD4?CD25hi Tregs by administration of PGRN-Ab. Cultured CD4?CD25hi regulatory T cells at a concentration of 5 9 104/ml were treated I with nothing, II with TNF-a (50 ng/ml), III with TNF-a and Etanercept (1 lg/ml), IV with TNF-a and recombinant human PGRN (250 ng/ml), V with TNF-a and recombinant human SLP2 (250 ng/ml), VI with TNF-a, recombinant PGRN and recombinant anti-PGRN-Ab (1 lg/ml), VII with TNF-a, recombinant SLP2 and recombinant anti-PGRN-Ab, or VIII with TNF-a, recombinant PGRN and recombinant anti-SLP2-Ab (1 lg/ml). Subsequently, the Tregs were cultured overnight at 37 °C. Following this, FOXP3 expression was determined by Western blot
diseases, which is supported by the overall clinical response rates to therapeutic antibodies against TNF-a in IBD and the great majority of rheumatic autoimmune diseases. However, the well-known major differences of the efficacy of the receptor fusion protein TNF-blocker Etanercept and the anti-TNF-a antibodies Inflximab and Adalimumab in CD, indicate that it is not the neutralizing effect on soluble TNF-a plasma levels that is relevant in CD, but rather the direct targeting of membranous TNF-a, which induces apoptosis of activated T-lymphocytes within the gut tissue [22]. The mechanism of neutralizing PGRN-Abs might be comparable to the well-known effects of neutralizing antidrug antibodies to therapeutic TNF-blockers [11]. Moreover, the pathomechanism of PGRN-Abs might also be comparable to anti-cytokine antibodies, e.g., autoantibodies against IFN-y, which have a contrary effect and result in adult-onset immunodeficiency characterized by different opportunistic infections despite normal CD4? Th cell counts [23]. Moreover, PGRN was shown to be strongly expressed in the gastrointestinal tract of rodents [24], so besides the neutralizing effect on the plasma level of secreted PGRN, PGRN-Abs might directly target the gut tissue in IBD. In general, a possible pathogenic role of B lymphocytes in IBD has been under debate, as the clinically established antibodies seem not to be pathogenically relevant. Moreover, case reports described exacerbations of UC after B-cell depletion therapy [25, 26]. However, a randomized case-control study about the safety and efficacy of B-cell depletion in UC showed a good tolerability, but only possible short-term responses [27]. Most experimental studies on B lymphocytes in IBD have analyzed B lymphocytes
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infiltrating inflamed gut tissue [28, 29]. The presence of frequently detectable antibodies against microbial antigens, such as anti-Saccharomyces cerevisiae antibodies [30] and anti-Flagellin-antibodies [31] in CD, suggest that the reactivity of at least a part of these tissue-infiltrating B lymphocytes is directed against local microbes. Antigens of enteric bacteria have also been suggested as the drivers of pANCAs in UC by a process of molecular mimicry of bacterial components and human myeloperoxidase [32]. Remarkable in UC is a disproportionally increased serum fraction of the IgG1 subclass [33]. However, it is not yet clear whether this increased fraction is a mere epiphenomenon or if it indicates a pathogenic involvement of B lymphocytes. As reported for tropomyosin antibodies in UC, PGRN-Abs in primary vasculitides belong predominantly to the IgG1 subclass (96.3 %) [1, 34]. A lower, but still disproportionate overrepresentation of the IgG1 subclass for PGRN-Abs was observed for IBD (27/38; 71.1 %) (Fig. 1c). The presence of PGRN-Abs of the IgA-class in IBD (10/38; 26.3 %) could indicate an involvement of B cells located in the MALT. In both CD and UC, the presence of PGRN-Abs showed no association with either clinical parameters or with particular classes of prescribed medication. This might be explained by the relatively small absolute numbers of PGRN-Ab-positive patients and by the patient population included in this study with a relatively high proportion of patients receiving immunosuppression and having severe courses. Moreover, for CD a statistically significant association between multiple tested serum samples of an individual patient and positivity for PGRN-Abs in the course of disease was observed (p = 0.005), suggesting that PGRN-Abs occur transiently. Furthermore, there was neither a statistically significant association of the presence of PGRN-Abs with positive ASCA status in CD nor with positive ANCA status in UC. As mentioned before, pANCA is the only established antibody associated with UC, despite its unspecificity due to the wide spectrum of coexisting pANCA-associated diseases. In this respect, it might be of particular interest that PGRN-Abs were more frequently detected in UC (15/71; 20.13 %) than were pANCAs (4/65; 6.2 %; p = 0.012), cANCAs (7/65; 9.9 %; p = 0.101) and pANCAs and cANCAs together (9/65; 13.8 %, p = 0.266) (Fig. 1d). Surprisingly, in the present study cANCA/Pr3ANCA were slightly more frequent than pANCA/MPOANCA in patients with UC without any signs of vasculitis. The frequent presence of cANCA/Pr3-ANCA in UC was also reported recently [35]. In CD, PGRN-Abs were less frequent than ASCAs. Although the high percentage of missing data limits the validity of this analysis, ASCAs certainly have a higher
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specificity for CD than PGRN-Abs. However, it should be kept in mind that ASCAs also frequently occur in intestinal tuberculosis [36]. In conclusion, this study shows that IBD shares with several rheumatic autoimmune diseases the presence of PGRN-Abs, suggesting that these autoantibodies represent a common finding in autoimmune diseases affecting different organs. Paying attention to the obtained in-vitro data of the present study, PGRN-Abs presumably participate actively in the pathogenesis of the seropositive subgroup of patients with IBDs by their neutralizing effect on the plasma level of secreted anti-inflammatory PGRN. Nevertheless, the data on the frequency of PGRN-Abs in the course of IBD have to be regarded as preliminary, since multiple serum samples obtained at different points of time during the course of disease could only be tested for PGRN-Abs from 8.5 % of patients with CD (Table 2, supplementary material) and from 9.9 % of patients with UC (Table 1, supplementary material). Despite the low percentage of serial serum samples available for this study, the observed seroconversions suggest that PGRN-Abs can be found in a considerably higher percentage of patients at some time points during the course of their disease. Future studies could investigate possible family clustering of PGRN-Abs similar to those reported for anti-tropomyosin antibodies [37]. Furthermore, such studies should also investigate in vivo the suspected pathogenicity of PGRN-Abs. In vivo studies could combine either a passive transfer of murinized neutralizing anti-murine PGRN antibodies or an active immunization approach by the administration of N-terminal immunogenic fragments of PGRN with established mouse models for IBDs. Furthermore, future studies should focus on identifying the reason for the breakdown of self-tolerance against PGRN. Acknowledgments We are grateful to Jutta Conigliaro, Christian Schorpp, Ralph Hastenteufel, Gabi Carbon, the outpatient IBD clinic of Saarland University Medical Center in Homburg/Saar, and Diana Thurner and Bernhard Thurner, who helped with the collection of blood samples from IBD patients or gave helpful advice. The study was supported by a Saarland University Fellowship to L.T. and the HOMFOR program of the Saarland University Medical School to V.Z. Conflict of interest Saarland University, L.T., K.D.P., and M.P. filed 61/730,772, which covers the means and methods for detecting autoimmune disorders in which progranulin may be involved.
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