Plasma cells within granulomatous inflammation display signs pointing to autoreactivity and destruction in granulomatosis with polyangiitis
- Antje Mueller1, 2Email author,
- Christoph Brieske1, 2,
- Susanne Schinke1, 2,
- Elena Csernok1, 2,
- Wolfgang L Gross1, 2,
- Katrin Hasselbacher3,
- Jan Voswinkel†4 and
- Konstanze Holl-Ulrich†5
© Mueller et al.; licensee BioMed Central Ltd. 2014
Received: 9 July 2013
Accepted: 11 February 2014
Published: 20 February 2014
Plasma cells residing in inflamed tissues produce antibodies in chronic inflammatory and systemic autoimmune diseases. This study examined if plasma cells, located within inflamed nasal tissue in granulomatosis with polyangiitis (GPA), express features potentially associated with the autoimmune and destructive character of this disease.
Ig gene mutation patterns of individual tissue-derived plasma cells from GPA (n = 5) were analyzed, by using laser-assisted microdissection followed by semi-nested polymerase chain reaction (PCR). Signs of B-lymphocyte maturation (ectopic lymphoid structures, ELS) and survival (a proliferation-inducing ligand, APRIL; B-cell maturation antigen, BCMA; transmembrane-activator and calcium modulator and cyclophilin interactor, TACI; receptor activator of nuclear factor κB ligand, RANKL) were examined in nasal tissues or serum, respectively, by using immunohistochemistry/fluorescence and enzyme-linked immunosorbent assay, ELISA.
Plasma-cell derived Ig genes (light- and heavy-chain pairs, n = 4; heavy chains, n = 33) resembled mutation patterns seen in other autoimmune diseases, predominantly displaying selection against replacement mutations within the framework region of Ig genes (10 of 15), which is responsible for structural integrity. Ectopic lymphoid structures were similar between GPA and a disease control (that is, unspecific chronic rhinosinusitis. However, histomorphologic features distinguishing GPA from rhinosinusitis (that is, neutrophilic microabscess and granuloma) expressed considerable amounts of membrane-associated and secreted APRIL, respectively. The latter was co-localized with CD138 and found in close proximity to cells expressing IgG, TACI, and BCMA. Interestingly, plasma cells strongly expressed receptor activator of nuclear factor κB ligand (RANKL), apart from fibroblast-like cells.
Plasma cells within granulomatous inflammation appear to display features that might be required for autoreactivity and, possibly, RANKL-mediated destruction in GPA.
Granulomatosis with polyangiitis (GPA/Wegener) is a multisystem disease of unknown etiology, characterized by granulomatous manifestations in the respiratory tract and systemic necrotizing vasculitis. Anti-neutrophilic cytoplasmic antibodies (ANCA) with specificity for proteinase 3 are a defining feature of this disease, but other autoantibodies are found as well[1, 2]. Clinical symptoms are often due to necrotizing granulomatous inflammation, predominantly in the respiratory tract, leading to fibroblast-mediated cartilage/bone destruction and to vasculitis, probably autoantibody mediated[1, 3, 4]. Inflamed tissue within nasal mucosa displays the pathognomonic triad consisting of ill-defined granuloma, geographic necrosis, and vasculitis, accompanied by prominent neutrophil infiltration (microabscess) and lymphoplasmocytic aggregates[3, 5, 6].
Recently, we detected mutated Ig variable (V) region genes in nasal tissue in GPA, and some of the CD20+ B cells produced autoantibodies. Thus, we assumed that autoreactivity develops in inflamed nasal tissue, probably via ectopic lymphoid structures (ELS). ELS are considered the morphologic basis of B-cell autoimmunity in rheumatoid arthritis (RA); however, this association was questioned. Further, B cells can be depleted via anti-CD20 therapy, inducing remission in GPA. Nonetheless, relapses occur, suggesting that plasma cells, surviving in niches and producing autoantibodies, could be responsible. Cells expressing B cell-activating factor, B cell-activating factor receptor, and a proliferation-inducing ligand (APRIL) were shown in GPA mucosa, promoting the niche concept. To search for alterations, plasma cells derived from inflamed nasal tissue in GPA were analyzed in terms of mutation pattern of their Ig genes and compared with controls, after laser-assisted microdissection and semi-nested PCR.
To investigate a relevance for B-cell autoimmunity in GPA, ELS were examined and compared with a non-autoimmune disease control, by using immunohistochemistry. Because plasma cell survival is mediated through APRIL signaling via B-cell maturation antigen (BCMA) or transmembrane-activator and calcium modulator and cyclophilin ligand interactor (TACI), their expressions were investigated, by using immunohistochemistry/-fluorescence and Elisa. Because APRIL binding to BCMA led to elevated receptor activator of nuclear factor κB ligand (RANKL) levels, its tissue expression was evaluated as well.
Our results indicate altered Ig V gene-mutation patterns in plasma cells residing in inflamed nasal tissue. The presence of ELS in GPA suggests the possibility of a role in developing autoreactive B cells; however, the phenotypical properties of ELS did not differ from a non-autoimmune inflammatory disorder (that is, chronic rhinosinusitis (CRS)). In contrast, plasma cell survival seems to be supported by distinct histomorphologic structures in GPA (that is, neutrophilic microabscess and granuloma), expressing the survival factor APRIL. Co-localization of APRIL and CD138 allows recognition by the receptor TACI. RANKL expression by cells with a plasma cell-like appearance might serve as an indication of binding between APRIL and the receptor BCMA.
Patients and tissues
Sinunasal biopsies were taken from 26 GPA patients, 20 patients with unspecific CRS, and one patient each with rheumatoid arthritis (RA) and sarcoidosis. Patients’ written consent according to the Declaration of Helsinki was obtained, and the study design was approved by the ethics committee of the University of Luebeck (07–058). Patient characteristics are summarized (Additional file1: Table S1). Formalin-fixed and paraffin-embedded nasal and lung biopsies of 22 GPA patients were selected for immunostaining (Additional file1: Table S2), and freshly frozen nasal biopsies of five GPA patients (proteinase 3-ANCA+) were chosen for Ig gene analysis. Markers of the histomorphologic triad of GPA were not present in these five biopsies, but lymphoplasmocytic infiltrates, and three patients had a history of GPA-related histology.
Isolation and characterization of plasma-cell-derived Ig V region genes
This was conducted as described before, with the exception of staining with anti-CD138 (MI15; Dako, Hamburg, Germany) followed by anti-mouse HRP-conjugate (Zytomed Systems, Berlin, Germany) and aminoethylcarbazole (Dako). PCR products were identified by using IMGT/V-Quest. All sequences have been submitted to GenBank (accession numbers: JN990775-JN990790, JN990797-JN990808, JQ240200-JQ240203, JQ693385-JQ693389, JQ715619-JQ715622).
The following primary mouse, rabbit, or rat antibodies were used: anti-CD3 (Dako), anti-CD4 (4B12; Biogenex, Fremont, CA, USA), anti-CD8 (BC/1A5; Biocare Medical, Concord, MA, USA), anti-CD20 (L26), anti-CD21 (1 F8), anti-CD35 (Ber-MAC-DRC), anti-CD57 (TB01), anti-CD68 (PGM1), (all Dako), anti-CD23 (SP23; Biomol, Hamburg, Germany), anti-CD38 (38C03; Labvision, Dreieich, Germany), anti-peripheral node addressin (PNAd: MECA-79; BD, Heidelberg, Germany), anti-APRIL (Stalk-1/ED; MyBioSource, San Diego, CA, USA; Ap2; Enzo Life Sciences, Lörrach, Germany), anti-RANKL (MIH24; Biolegend, San Diego, CA, USA), anti-TACI (R&D Systems, Wiesbaden, Germany), and anti-BCMA (Sigma, München, Germany). Neutrophils were detected enzymatically by using naphthol ASD chloroacetate. After deparaffinization and rehydration of sections (3 to 4 μm), antigens were retrieved by heating in pH 6.0 or pH 9.0 buffer. Nonspecific binding was minimized by using protein block (Dako). To provide for controls, sections were stained with appropriate isotype controls (Dako; AbD Serotec, Düsseldorf, Germany) instead of primary antibodies. Stainings for CD marker, PNAd, APRIL, TACI, and RANKL were carried out by applying a peroxidase-based protocol. CXCL13 and CCL21 were stained with goat anti-CXCL13 and goat anti-CCL21 (R&D Systems), respectively, by using an avidin/biotinylated enzyme complex-based protocol. Double stainings for secreted APRIL and goat anti-human IgG F(abʹ)2 fragment (Dianova, Hamburg, Germany), as well as for CD138 and BCMA, were performed by using the Envision Doublestain kit (Dako) according to the manufacturer’s instructions, with the exception of using donkey anti-goat-HRP-conjugated antibody (Dianova) for IgG detection. Images were acquired by using a slide scanner and corresponding software (Mirax Midi; Zeiss, Göttingen, Germany).
Double immunofluorescence staining
In brief, sections were stained for CD138 followed by donkey anti-mouse AF488 Fab fragment (Dianova). Thereafter, secreted APRIL was stained by using Ap2 antibody followed by goat anti-mouse AF568 IgG. Nuclear staining was done by using 4′,6-diamidin-2-phenylindol (DAPI) followed by mounting with ProLong Gold reagent (all Life Technologies, Karlsruhe, Germany). Sections were viewed on a laser-scanning microscope IX81 and confocal stacks modeled with the FV10-ASW v03 software (Olympus, Hamburg, Germany).
APRIL was measured in sera of GPA patients (n = 20), CRS patients (n = 10), and healthy controls (n = 13) by using an ELISA according to the manufacturer’s instructions (eBioscience, Frankfurt/M, Germany).
Altered mutation pattern of plasma cell-derived Ig genes in nasal tissue in GPA
Overview of selection ( P values) against or for replacement mutations within framework region (FR) and CDR of plasma cell-derived Ig genes in nasal tissue in GPA a
V region subfamily
P ≤ 0.002b
P ≤ 0.03b
P < 0.02
P ≤ 0.01(2),eP ≤ 0.02 (2),fP < -0.05
P < 0.01
P ≤ 0.02c
P ≤ 0.01c
P ≤ 0.001
P ≤ 0.02
P < 0.02
P ≤ 0.01d
P ≤ 0.03d
P < 0.03
P < 0.04
Ectopic lymphoid structures are similar between GPA and disease control
Neutrophils, macrophages, and giant cells forming granulomatous inflammation are major sources of APRIL
APRIL receptors (BCMA, TACI) and RANKL are expressed by plasma cells
BCMA was expressed by plasma cells (Figure 2E), lymphocytic and fibroblast-like cells. The latter also stained positive for RANKL, but unexpectedly, a substantial RANKL expression was found in cells exhibiting a plasma cell-like morphology (Figure 2F,G). The receptor TACI recognizes multi-/oligomerized APRIL cross-linked via heparan sulfate proteoglycans such as syndecans. Syndecan-1 (CD138) and secreted APRIL were colocalized on different cell types, including plasma cells (Figure 2H through J). Moreover, secreted APRIL was found in the direct vicinity of IgG+ cells (Figure 2K). Thus, clustered APRIL could interact with TACI, which was expressed by cells displaying a plasma-cell-like morphology (Figure 2L). Regarding CRS, cells expressing BCMA, RANKL, or TACI were present (Additional file1: Figure S5). Additional experiments are warranted to determine whether apparent differences (cell type, numbers) are relevant in comparison with GPA.
Based on previous findings in GPA[7, 12] and similarly to other chronic inflammatory diseases[8, 20, 21], we hypothesize that nonlymphoid tissue, such as the nasal mucosa in GPA, is a place where B/plasma cell-mediated autoreactivity could develop and/or be present. Overall, our results indicate alterations in the Ig V genes of tissue-derived plasma cells and histomorphologic support for plasma cell development and survival in nasal tissue in GPA. In more detail and regarding genetic features of plasma cells, we observed similarities between our current and previous results, a meta-analysis of other studies in various autoimmune diseases, and findings in rheumatoid synovium. Despite these similarities (showing, for instance, a prevalence of selection against mutations in the framework region of Ig V genes, which is not seen in controls), we are well aware that altered mutation patterns in the Ig V gene regions in themselves are not sufficient to indicate autoreactivity in the granulomatous inflammation. Moreover, aside from the mutation rate, physicochemical effects of mutations leading to amino acid exchanges have to be considered as well. In general, to demonstrate autoreactivity, this must be validated by cloning and expressing the antibodies from the isolated plasma cells and testing them for (auto)antigen specificity, which is part of ongoing work. Apart from a possible association with autoreactivity, mutations in Ig V gene regions could be induced by bacterial infections such as S. aureus, which has been linked to GPA pathogenesis. Superantigens from S. aureus-stimulating T cells have been demonstrated as a trigger for relapses. Nonetheless, it remains to be determined whether B-cell superantigens (for example, staphylococcal protein A, staphylococcal enterotoxin D) might play a role in GPA, which, for example, would lead to alterations in the Ig V gene repertoire of B cells.
Besides autoantibody-producing plasma cells, ELS have been implicated in B-cell autoimmunity; however, this is controversially discussed. Although our findings show that ELS are present within granulomatous inflammation and could thus be involved in selection and maturation of B lymphocytes, the results also support the concept that lymphoid structures are a result of chronic inflammation. To understand the role of ELS in GPA in terms of assisting in the development of (auto)immune reactions, more experimental work is needed. Of note, the presence of CD20+ cell aggregates and organized ELS in GPA contrasts with another study, reporting that B cells mostly are isolated cells. Further, a difference in CCL21 expression between rheumatoid synovium and nasal tissue in GPA with respect to perivascular stromal cells matches a polymorphism in the CCL21 gene, which is associated with RA but not GPA. Based on our results and corresponding to other studies[9, 22], we assume that highly organized ELS are not a specific prerequisite for maturation of (autoreactive) B lymphocytes in GPA. Regarding plasma cell survival and extending previous findings by discriminating between membrane-associated and secreted APRIL, GPA-characteristic histomorphology (neutrophilic microabscess, granuloma) seems to concentrate considerable amounts of APRIL, which is comparable with synovium, but differs from CRS tissue. The expression of RANKL by fibroblast-like and other cells could be due to an interaction between APRIL and its receptor BCMA, as has been demonstrated for synovial fibroblasts in RA. A contribution of RANKL+ plasma cells to destructive or other pathogenic mechanisms in GPA requires further investigation.
Although tissue-residing plasma cells in GPA display genetic features (indicated by their Ig V region gene-mutation pattern) that are similar to other autoimmune diseases, plasma cell (auto)reactivity in nasal mucosa remains to be demonstrated. A lack of difference between GPA and disease control in terms of ELS phenotype points to less relevance for autoimmune-specific processes. Neutrophils, macrophages, and giant cells forming GPA-characteristic granulomatous inflammation could support survival of plasma cells by providing considerable amounts of both membrane-associated and secreted APRIL. The latter might subsequently interact with the receptors TACI and/or BCMA, expressed by plasma cells. The strong expression of RANKL by plasma cell-like cells, which could be due to binding between APRIL and BCMA, might indicate an unrecognized role for these cells in destructive mechanisms in GPA.
Antineutrophil cytoplasmic antibody
a proliferation-inducing ligand
B-cell maturation antigen
ectopic lymphoid structures
follicular dendritic cells
granulomatosis with polyangiitis
peripheral node addressin
receptor activator of nuclear factor κB ligand
transmembrane-activator and calcium modulator and cyclophilin interactor
- V region:
variable Ig gene region.
The study was supported by grants from the German Research Society (DFG) to the Clinical Research Unit 170 (AM, KHU, WLG, EC), associated with the DFG-funded Cluster of Excellence “Inflammation at Interfaces” as junior research group (JRG I-h). The authors thank Dr. Martin Laudien (Department of Otorhinolaryngology, University of Kiel, Germany) for providing tissue samples and thank Drs. Nina Kesel and Sebastian Ullrich (Department of Anatomy & Experimental Morphology, University of Hamburg, Germany) for technical support. In addition, we thank Dr. Julia Holle (Department of Rheumatology & Clinical Immunology, University of Luebeck, & Medical Center, Bad Bramstedt, Germany), Dr. Stefan Wieczorek (Institute for Human Genetics, Ruhr University Bochum, Germany), Professor Dr. Rudolf Manz (Institute of Systemic Inflammation Research, University of Luebeck, Germany) and Professor Dr. Peter Lamprecht (Department of Rheumatology, University of Luebeck, Germany) for critical discussion. We appreciate the excellent technical assistance of Monika Backes.
- Jennette JC, Falk RJ, Hu P, Xiao H: Pathogenesis of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Annu Rev Pathol Mech Dis. 2013, 8: 139-160. 10.1146/annurev-pathol-011811-132453.View ArticleGoogle Scholar
- Thurner L, Preuss KD, Fadle N, Regitz E, Klemm P, Zaks M, Kemele M, Hasenfus A, Csernok E, Gross WL, Pasquali JL, Martin T, Bohle RM, Pfreundschuh M: Progranulin antibodies in autoimmune diseases. J Autoimmun. 2013, 42: 29-38.View ArticlePubMedGoogle Scholar
- Gadola SD, Gross WL: Vasculitis in 2011: the renaissance of granulomatous inflammation. Nat Rev Rheumatol. 2012, 8: 74-76.PubMedGoogle Scholar
- Kesel N, Köhler D, Herich L, Laudien M, Holl-Ulrich K, Jüngel A, Neidhart M, Gay S, Gay RE, Csernok E, Lamprecht P, Gross WL, Schumacher U, Ullrich S: Cartilage destruction in granulomatosis with polyangiitis (Wegener’s granulomatosis) is mediated by human fibroblasts after transplantation into immunodeficient mice. Am J Pathol. 2012, 180: 2144-2155. 10.1016/j.ajpath.2012.01.021.View ArticlePubMedGoogle Scholar
- Devaney KO, Travis WD, Hoffman G, Leavitt R, Lebovics R, Fauci AS: Interpretation of head and neck biopsies in Wegener’s granulomatosis. Am J Surg Pathol. 1990, 14: 555-564. 10.1097/00000478-199006000-00006.View ArticlePubMedGoogle Scholar
- Voswinkel J, Mueller A, Kraemer JA, Lamprecht P, Herlyn K, Holl-Ulrich K, Feller AC, Pitann S, Gause A, Gross WL: B lymphocyte maturation in Wegener’sgranulomatosis: a comparative analysis of VH genes from endonasal lesions. Ann Rheum Dis. 2006, 65: 859-864.View ArticlePubMedPubMed CentralGoogle Scholar
- Thurner L, Mueller A, Cerutti M, Martin T, Pasquali JL, Gross WL, Preuss KD, Pfreundschuh M, Voswinkel J: Wegener’s granuloma harbors B lymphocytes with specificities against a proinflammatory transmembrane protein and a tetraspanin. J Autoimmun. 2011, 36: 87-90. 10.1016/j.jaut.2010.09.002.View ArticlePubMedGoogle Scholar
- Humby F, Bombardieri M, Manzo A, Kelly S, Blades MC, Kirkham B, Spencer J, Pitzalis C: Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium. PLoS Med. 2009, 6: 59-75.View ArticleGoogle Scholar
- Cantaert T, Kolln J, Timmer T, van der Pouw Kraan TC, Vandooren B, Thurlings RM, Canete JD, Catrina AI, Out T, Verweij CL, Zhang Y, Tak PP, Baeten D: B lymphocyte autoimmunity in rheumatoid synovitis is independent of ectopic lymphoid neogenesis. J Immunol. 2008, 181: 785-794.View ArticlePubMedGoogle Scholar
- Cartin-Ceba R, Golbin JM, Keogh KA, Peikert T, Sanchez-Menendez M, Ytterberg SR, Fervenza FC, Specks U: Rituximab for remission induction and maintenance in refractory granulomatosis with polyangiitis (Wegener’s). Arthritis Rheum. 2012, 64: 3770-3778. 10.1002/art.34584.View ArticlePubMedGoogle Scholar
- Winter O, Dame C, Jundt F, Hiepe F: Pathogenic long-lived plasma cells and their survival niches in autoimmunity, malignancy and allergy. J Immunol. 2012, 189: 5105-5111. 10.4049/jimmunol.1202317.View ArticlePubMedGoogle Scholar
- Zhao Y, Odell E, Choong LM, Barone F, Fields P, Wilkins B, Tungekar FM, Patel P, Sanderson JD, Sangle S, D’Cruz D, Spencer J: Granulomatosis with polyangiitis involves sustained mucosal inflammation that is rich in B-cell survival factors and autoantigen. Rheumatology. 2012, 51: 1580-1586. 10.1093/rheumatology/kes123.View ArticlePubMedGoogle Scholar
- Zuckerman NS, Hazanov H, Barak M, Edelman H, Hess S, Shcolnik H, Dunn-Walters D, Mehr R: Somatic hypermutation and antigen-driven selection of B cells are altered in autoimmune diseases. J Autoimmun. 2010, 35: 325-335. 10.1016/j.jaut.2010.07.004.View ArticlePubMedGoogle Scholar
- Coquery CM, Erickson LD: Regulatory roles of the tumor necrosis factor receptor BCMA. Crit Rev Immunol. 2012, 32: 287-305. 10.1615/CritRevImmunol.v32.i4.10.View ArticlePubMedPubMed CentralGoogle Scholar
- Nagatani K, Itoh K, Nakajima K, Kuroki H, Katsuragawa Y, Mochizuki M, Aotsuka S, Mimori A: Rheumatoid arthritis fibroblast-like synoviocytes express BCMA and are stimulated by APRIL. Arthritis Rheum. 2007, 56: 3554-3563. 10.1002/art.22929.View ArticlePubMedGoogle Scholar
- Brochet X, Lefranc MP, Giudicelli V: IMGT/V-Quest: the highly customized and integrated system for Ig and TR-standardized V-J and V-D-J- sequence analysis. Nucleic Acid Res. 2008, 36: W503-W508. 10.1093/nar/gkn316.View ArticlePubMedPubMed CentralGoogle Scholar
- Hosokawa T, Kumon Y, Kobayashi T, Enzan H, Nishioka Y, Yuri K, Wakiguchi H, Sugiura T: Neutrophil infiltration and oxidant-production in human atherosclerotic carotid plaques. Histol Histopathol. 2011, 26: 1-11.PubMedGoogle Scholar
- Uduman M, Yaari G, Hershberg U, Stern JA, Shlomchik MJ, Kleinstein SH: Detecting selection in immunoglobulin sequences. Nucleic Acid Res. 2011, 39: W499-W504. 10.1093/nar/gkr413.View ArticlePubMedPubMed CentralGoogle Scholar
- Manzo A, Bugatti S, Caporali R, Prevo R, Jackson DG, Uguccioni M, Buckley CD, Montecucco C, Pitzalis C: CCL21 expression pattern of human secondary lymphoid organ stroma is conserved in inflammatory lesions with lymphoid neogenesis. Am J Pathol. 2007, 171: 1549-1562. 10.2353/ajpath.2007.061275.View ArticlePubMedPubMed CentralGoogle Scholar
- Gabay C, Krenn V, Bosshard C, Seemayer CA, Chizzolini C, Huard B: Synovial tissues concentrate secreted APRIL. Arthritis Res Ther. 2009, 11: R144-10.1186/ar2817.View ArticlePubMedPubMed CentralGoogle Scholar
- Szyszko E, Brokstad KA, Oijordsbakken G, Jonsson MV, Jonsson R, Skarstein K: Salivary glands of primary Sjögren’s syndrome patients express factors vital for plasma cell survival. Arthritis Res Ther. 2011, 13: R2-10.1186/ar3220.View ArticlePubMedPubMed CentralGoogle Scholar
- Scheel T, Gursche A, Zacher J, Häupl T, Berek C: V-Region gene analysis of locally defined synovial B and plasma cells reveals selected B cell expansion and accumulation of plasma cell clones in rheumatoid arthritis. Arthritis Rheum. 2011, 63: 63-72. 10.1002/art.27767.View ArticlePubMedGoogle Scholar
- Kallenberg CGM, Stegeman CA, Abdulahad WH, Heeringa P: Pathogenesis of ANCA-associated vasculitis: new possibilities for intervention. Am J Kidney Dis. 2013, doi.org/10.1053/j.ajkd.2013.05.009Google Scholar
- Silverman GJ, Goodyear CS: Confounding B-cell defences: lessons from a staphylococcal superantigen. Nat Rev Immunol. 2006, 6: 465-475. 10.1038/nri1853.View ArticlePubMedGoogle Scholar
- Kratz A, Campos-Neto A, Hanson MS, Ruddle NH: Chronic inflammation caused by lymphotoxin is lymphoid neogenesis. J Exp Med. 1996, 183: 1461-1472. 10.1084/jem.183.4.1461.View ArticlePubMedGoogle Scholar
- Wieczorek S, Holle JU, Müller S, Fricke H, Gross WL, Epplen JT: A functionally relevant IRF5 haplotype is associated with reduced risk to Wegener’s granulomatosis. J Mol Med. 2010, 88: 413-421. 10.1007/s00109-009-0580-y.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.