Lorenz Thurner

Progranulin antibodies in autoimmune diseases

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasus arteritis (4/13), classical panarteritis nodosa (4/10), Behcets disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis.

Progranulin antibodies entertain a proinflammatory environment in a subgroup of patients with psoriatic arthritis

IntroductionPsoriatic arthritis (PsA) is a distinctive inflammatory arthritis which may typically develop in a subgroup of individuals suffering from psoriasis. We recently described progranulin autoantibodies (PGRN-Abs) in the sera of patients with different autoimmune diseases including seronegative polyarthritis. In the present study we investigated the occurrence of PGRN-Abs in PsA.MethodsPGRN-Abs were determined in 260 patients with PsA, 100 patients with psoriasis without arthritic manifestations (PsC) and 97 healthy controls using a recently described ELISA. PGRN plasma levels were determined from subgroups by a commercially available ELISA-kit. Possible functional effects of PGRN-antibodies were analysed in vitro by tumour necrosis factor (TNF)-α mediated cytotoxicity assays using WEHI-S and HT1080 cells.ResultsPGRN-Abs were detected with relevant titres in 50/260 (19.23%) patients with PsA, but in 0/100 patients with psoriasis without arthritic manifestations (P = 0.0001). All PGRN-Abs belonged to immunoglobulin G (IgG). PGRN-Abs were significantly more frequent in PsA patients with enthesitis or dactylitis. PGRN-Abs were also more frequent in PsA patients receiving treatment with TNF-α-blockers than in patients treated without TNF-α-blockers (20.8% versus 17.4%; P = 0.016). PGRN plasma levels were significantly lower in PGRN-Ab-positive patients with PsA than in healthy controls and patients with psoriasis without arthritic manifestations (P < 0.001), indicating a neutralizing effect of PGRN-Abs. Moreover cytotoxicity assays comparing PGRN-antibody positive with negative sera from matched patients with PsA, clearly showed a proinflammatory effect of PGRN antibodies.ConclusionNeutralizing PGRN-Abs occur with relevant titres in a subgroup of patients with PsA, but not in patients without arthritic manifestations (PsC). PGRN-Ab-positive patients had more frequent enthesitis or dactylitis. TNF-α-induced cytotoxicity assays demonstrated that the protective effects of progranulin were inhibited by serum containing PGRN-Abs. This suggests that PGRN-Ab might not only be useful as a diagnostic and prognostic marker, but may provide a proinflammatory environment in a subgroup of patients with PsA.

Wegener’s granuloma harbors B lymphocytes with specificities against a proinflammatory transmembrane protein and a tetraspanin

Wegeners granulomatosis (WG) is a severe autoimmune disorder ranging from localized granulomatous disease to generalised anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. A previous analysis of immunoglobulin heavy chain genes derived from tissue, i.e. Wegeners granuloma indicated selection and affinity maturation towards local antigen(s). The current study focused on determining the specificity of immunoglobulins from distinct B lymphocytes out of Wegeners granuloma. Four pairs of variable region immunoglobulin light and heavy chain genes, isolated before, were recombinantly expressed using the baculovirus/insect cell system. These immunoglobulins were then analysed for their antigenic target employing a protein macroarray based upon a human fetal brain tissue cDNA expression library. The lysosomal transmembrane protein 9B, a key regulator for TNFα activation, was identified as the putative antigenic target of two immunoglobulins and a tetraspanin, which might play a role in leukocyte activation and motility, was identified as the putative antigenic target of another one. Recombinant monoclonal antibodies out of Wegeners granuloma represent a new tool aiding in elucidation of its and WG immunopathogenesis.

Progranulin and Amyloid-β Levels: Relationship to Neuropsychology in Frontotemporal and Alzheimer’s Disease

BACKGROUND Analysis of cerebrospinal fluid (CSF) has improved over the last few years; thus specific markers for different diseases have emerged, e.g., amyloid-β (Aβ) for Alzheimers disease (AD) and progranulin for frontotemporal dementia (FTD). OBJECTIVE Evaluation of correlation between biomarkers in CSF and cognitive performance in populations with AD and FTD. METHODS 27 patients with AD and 16 with FTD were included. CSF tau, P-tau(181P), Aβ₄₂, and progranulin (PGRN) were measured and a standardized neuropsychological test battery applied. Olfactory testing was additionally included where available. RESULTS For all patients across both groups, an association between PGRN and categoric (p = 0.016) and letter fluency (p = 0.029), naming (p = 0.003), and overall cognition (Mini-Mental State Examination: p = 0.04) was observed. Aβ42 was strongly associated with memory function (learning: p = 0.001; recall: p = 0.002). A correlation between Aβ₄₂ and memory performance was moreover found for each group separately, while PGRN also showed a correlation with recognition memory (p = 0.04) in AD. Furthermore, an association between reduced PGRN and olfactory dysfunction was revealed (p = 0.01). CONCLUSIONS CSF-levels of PGRN and Aβ₄₂ levels express deficits in cognition differentially, with PGRN being predominantly associated with frontal and Aβ₄₂ with temporal dysfunction. This mirrors the cerebral occurrence of these proteins. These associations appear to be consistent across both disease groups. The relationship between PGRN and olfaction further underpins the association between PRGN and frontal dysfunction.

Review: Novel Insights Into Tumor Necrosis Factor Receptor, Death Receptor 3, and Progranulin Pathways in Arthritis and Bone Remodeling

Approximately 30 members of the tumor necrosis factor receptor superfamily (TNFRSF) have been identified. They are transmembrane proteins with cysteine‐rich motifs in their extracellular domains that bind to their cognate ligands 1. They are categorized into 3 groups: death domain–containing receptors, decoy receptors, and TNFR‐associated factor–binding receptors. Only 8 TNFRSF members contain a death domain (TNFR type I [TNFRI], death receptor 3 [DR‐3], DR‐4, DR‐5, DR‐6, Fas, nerve growth factor receptor, and ectodysplasin A receptor [EDAR]), of which TNFRI and DR‐3 constitute the principal focus of this article. Interactions between TNF superfamily (TNFSF) ligands and TNFRSF receptors help maintain tissue homeostasis by controlling survival, proliferation, differentiation, and effector function of immune cells. We limit our review to recent advances and novel insights into the roles of TNFRI and DR‐3 in bone and joint biology. Bone cells (osteoblasts, osteoclasts, and osteocytes), fibroblast‐like synoviocytes, chondrocytes, and immune cells that infiltrate the arthritic joint will at different times express a wide range of TNFRSF members and TNFSF ligands. An overview of the current status of our knowledge in this regard is provided in Table 1. The impact of TNFRI activation on bone and inflammatory joint diseases has been researched in great depth 2, 3, but little or no data in the field have been reported on other more recently discovered TNFRSF members such as TROY (TNFRSF expressed on the mouse embryo; TNFRSF19), EDAR, and XEDAR (X‐linked ectodysplasin receptor; TNFRSF27). The unexpected interaction between progranulin (PGRN) and both TNFRI and TNFRII is particularly interesting in the context of arthritis‐associated bone pathology. PGRN levels are elevated in the synovial fluid of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and other arthropathies 4, 5, 6, and PGRN has been shown to inhibit TNF‐induced osteoclastogenesis and promote osteoblast differentiation in mice 7. However, PGRN has a higher binding affinity for TNFRII (antiinflammatory with osteoprotective function) than for TNFRI (predominantly proinflammatory with degenerative function), which suggests conflicting actions. The potential overall impact of these divergent PGRN signaling pathways on the architecture of the arthritic joint has been evaluated 8. Table 1 Cellular expression of death domain–containing TNFRSF members and their association with arthritis*

Novel insights into TNF receptor, DR3 and progranulin pathways in arthritis and bone remodeling

Approximately 30 members of the tumor necrosis factor receptor superfamily (TNFRSF) have been identified. They are transmembrane proteins with cysteine‐rich motifs in their extracellular domains that bind to their cognate ligands 1. They are categorized into 3 groups: death domain–containing receptors, decoy receptors, and TNFR‐associated factor–binding receptors. Only 8 TNFRSF members contain a death domain (TNFR type I [TNFRI], death receptor 3 [DR‐3], DR‐4, DR‐5, DR‐6, Fas, nerve growth factor receptor, and ectodysplasin A receptor [EDAR]), of which TNFRI and DR‐3 constitute the principal focus of this article. Interactions between TNF superfamily (TNFSF) ligands and TNFRSF receptors help maintain tissue homeostasis by controlling survival, proliferation, differentiation, and effector function of immune cells. We limit our review to recent advances and novel insights into the roles of TNFRI and DR‐3 in bone and joint biology. Bone cells (osteoblasts, osteoclasts, and osteocytes), fibroblast‐like synoviocytes, chondrocytes, and immune cells that infiltrate the arthritic joint will at different times express a wide range of TNFRSF members and TNFSF ligands. An overview of the current status of our knowledge in this regard is provided in Table 1. The impact of TNFRI activation on bone and inflammatory joint diseases has been researched in great depth 2, 3, but little or no data in the field have been reported on other more recently discovered TNFRSF members such as TROY (TNFRSF expressed on the mouse embryo; TNFRSF19), EDAR, and XEDAR (X‐linked ectodysplasin receptor; TNFRSF27). The unexpected interaction between progranulin (PGRN) and both TNFRI and TNFRII is particularly interesting in the context of arthritis‐associated bone pathology. PGRN levels are elevated in the synovial fluid of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and other arthropathies 4, 5, 6, and PGRN has been shown to inhibit TNF‐induced osteoclastogenesis and promote osteoblast differentiation in mice 7. However, PGRN has a higher binding affinity for TNFRII (antiinflammatory with osteoprotective function) than for TNFRI (predominantly proinflammatory with degenerative function), which suggests conflicting actions. The potential overall impact of these divergent PGRN signaling pathways on the architecture of the arthritic joint has been evaluated 8. Table 1 Cellular expression of death domain–containing TNFRSF members and their association with arthritis*

The molecular basis for development of proinflammatory autoantibodies to progranulin.

Recently we identified in a wide spectrum of autoimmune diseases frequently occurring proinflammatory autoantibodies directed against progranulin, a direct inhibitor of TNFR1 & 2 and of DR3. In the present study we investigated the mechanisms for the breakdown of self-tolerance against progranulin. Isoelectric focusing identified a second, differentially electrically charged progranulin isoform exclusively present in progranulin-antibody-positive patients. Alkaline phosphatase treatment revealed this additional progranulin isoform to be hyperphosphorylated. Subsequently Ser81, which is located within the epitope region of progranulin-antibodies, was identified as hyperphosphorylated serine residue by site directed mutagenesis of candidate phosphorylation sites. Hyperphosphorylated progranulin was detected exclusively in progranulin-antibody-positive patients during the courses of their diseases. The occurrence of hyperphosphorylated progranulin preceded seroconversions of progranulin-antibodies, indicating adaptive immune response. Utilizing panels of kinase and phosphatase inhibitors, PKCβ1 was identified as the relevant kinase and PP1 as the relevant phosphatase for phosphorylation and dephosphorylation of Ser81. In contrast to normal progranulin, hyperphosphorylated progranulin interacted exclusively with inactivated (pThr320) PP1, suggesting inactivated PP1 to cause the detectable occurrence of phosphorylated Ser81 PGRN. Investigation of possible functional alterations of PGRN due to Ser81 phosphorylation revealed, that hyperphosphorylation prevents the interaction and thus direct inhibition of TNFR1, TNFR2 and DR3, representing an additional direct proinflammatory effect. Finally phosphorylation of Ser81 PGRN alters the conversion pattern of PGRN. In conclusion, inactivated PP1 induces hyperphosphorylation of progranulin in a wide spectrum of autoimmune diseases. This hyperphosphorylation prevents direct inhibition of TNFR1, TNFR2 and DR3 by PGRN, alters the conversion of PGRN, and is strongly associated with the occurrence of neutralizing, proinflammatory PGRN-antibodies, indicating immunogenicity of this alternative secondary modification.

A high number of IgG4-positive plasma cells rules out nodular lymphocyte predominant Hodgkin lymphoma

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a subtype of Hodgkin lymphoma that frequently shows a nodal growth pattern with abundant reactive B cells in the microenvironment. Early NLPHL cases can be particularly difficult to differentiate from progressively transformed germinal centers (PTGC). Since PTGC have been described to be IgG4 associated in a relatively high proportion of cases, the aim of the present study was to determine if IgG4 immunostaining can be helpful in the differential diagnosis between NLPHL and PTGC. We furthermore aimed to learn if LP cells can express IgG4. For this purpose, 58 cases of PTGC and 56 cases of NLPHL were assessed using IgG4 immunostaining. We could confirm that a significant number of PTGC cases showed high numbers of IgG4-positive plasma cells (22/58, 38%), whereas hot spot areas of IgG4-positive plasma cells were not found in any of the NLPHL cases. In lymph node areas with the differential diagnosis of NLPHL and PTGC, IgG4 immunostaining can therefore provide a helpful diagnostic tool to rule out NLPHL when a high number of IgG4-positive plasma cells are encountered. We also assessed 13 cases with a combination of NLPHL and PTGC in the same lymph node. Five of these cases presented hot spot areas of IgG4-positive plasma cells in the PTGC regions, while no significant numbers of IgG4-positive plasma cells were observed in the NLPHL part of the lymph node. LP cells were never IgG4 positive. Furthermore, immunoglobulin heavy chain rearrangements of single IgG4-positive plasma cells were analyzed, revealing a polyclonal plasma cell population. In summary, our data suggest that IgG4 immunostaining can provide additional information in the diagnostic workup of cases with the differential diagnosis of NLPHL and PTGC. IgG4’s inefficiency in clearing antigens may explain why lymph nodes with PTGC are usually strongly enlarged and develop a high number of hyperplastic germinal centers. Polyclonal immunoglobulin heavy chain rearrangements in IgG4-positive plasma cells further support the hypothesis that PTGC represent a misled immune reaction.

LRPAP1 is a frequent proliferation-inducing antigen of BCRs of mantle cell lymphomas and can be used for specific therapeutic targeting

The predominant usage of VH4-34 and V3-21 and reports of stereotyped CDR3s suggest a shared antigenic target of B-cell receptors (BCR) from mantle cell lymphomas (MCL). To identify the target antigens of MCL–BCRs, BCRs from 21 patients and seven MCL cell lines were recombinantly expressed and used for antigen screening. The BCRs from 8/21 patients and 2/7 MCL cell lines reacted specifically with the autoantigen low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1). High-titered and light chain-restricted anti-LRPAP1 serum antibodies were found in MCL patients, but not in controls. LRPAP1 induced proliferation by BCR pathway activation, while an LRPAP1–ETA′ toxin-conjugate specifically killed MCL cells with LRPAP1-specific BCRs. Our results suggest a role of LRPAP1 in lymphomagenesis and maintenance of a considerable proportion of MCL cases by chronic autoantigenic stimulation, likely evolving from a chronic autoreactive B-cell response. Importantly, LRPAP1 can be used for a novel therapeutic approach that targets MCL with LRPAP1-reactive BCRs with high specificity.

JUNB, DUSP2, SGK1, SOCS1 and CREBBP are frequently mutated in T-cell/histiocyte rich large B-cell lymphoma

T-cell/histiocyte-rich large B-cell lymphoma is a rare aggressive lymphoma showing histopathological overlap with nodular lymphocyte-predominant Hodgkin lymphoma. Despite differences in tumor microenvironment and clinical behavior, the tumor cells of both entities show remarkable similarities, suggesting that both lymphomas might represent a spectrum of the same disease. To address this issue, we investigated whether these entities share mutations. Ultra-deep targeted resequencing of six typical and 11 histopathological variants of nodular lymphocyte-predominant Hodgkin lymphoma, and nine cases of T-cell/histiocyte-rich large B-cell lymphoma revealed that genes recurrently mutated in nodular lymphocyte-predominant Hodgkin lymphoma are affected by mutations at similar frequencies in T-cell/histiocyte-rich large B-cell lymphoma. The most recurrently mutated genes were JUNB, DUSP2, SGK1, SOCS1 and CREBBP, which harbored mutations more frequently in T-cell/histiocyte-rich large B-cell lymphoma and the histopathological variants of nodular lymphocyte-predominant Hodgkin lymphoma than in its typical form. Mutations in JUNB, DUSP2, SGK1 and SOCS1 were highly enriched for somatic hypermutation hotspot sites, suggesting an important role of aberrant somatic hypermutation in the generation of these somatic mutations and thus in the pathogenesis of both lymphoma entities. Mutations in JUNB are generally rarely observed in malignant lymphomas and thus are relatively specific for nodular lymphocyte-predominant Hodgkin lymphoma and T-cell/histiocyte-rich large B-cell lymphoma at such high frequencies (5/17 and 5/9 cases with JUNB mutations, respectively). Taken together, the findings of the present study further support a close relationship between T-cell/histiocyte-rich large B-cell lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma by showing that they share highly recurrent genetic lesions.