Search

ObjectivesTo explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren’s syndrome (SS) patients.MethodsSalivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production.ResultsTranscriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α).ConclusionsOverall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease resulting from the inflammatory infiltration of exocrine glands, mainly salivary and lacrimal glands, leading to secretory dysfunction and serious complications including debilitating fatigue, systemic autoimmunity, and lymphoma. Like other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, suggesting the involvement of innate immunity in pSS pathogenesis. NCR3/NKp30 is a natural killer (NK) cell-specific activating receptor regulating the cross talk between NK and dendritic cells including type II IFN secretion upon NK-cell activation. A genetic association between single-nucleotide polymorphisms (SNPs) in the NCR3/NKp30 promoter gene and a higher susceptibility for pSS has been previously described, with pSS patients most frequently carrying the major allele variant associated with a higher NKp30 transcript and IFN-γ release as a consequence of the receptor engagement. In the present study, we combined RNA-sequencing and histology from pSS salivary gland biopsies to better characterize NKp30 (NCR3) and its ligand B7/H6 (NCR3LG1) in pSS salivary gland tissues. Levels of NCR3/NKp30 were significantly increased both in salivary glands and in circulating NK cells of pSS patients compared with sicca controls, especially in salivary glands with organized ectopic lymphoid structures. In line with this observation, a strong correlation between NCR3/NKp30 levels and salivary gland infiltrating immune cells (CD3, CD20) was found. Furthermore, NCR3/NKp30 levels also correlated with higher IFN-γ, Perforin, and Granzyme-B expression in pSS SGs with organized ectopic lymphoid structures, suggesting an activation state of NK cells infiltrating SG tissue. Of note, NKp30+ NK cells accumulated at the border of the inflammatory foci, while the NKp30 ligand, B7/H6, is shown to be expressed mainly by ductal epithelial cells in pSS salivary glands. Finally, immunomodulatory treatment, such as the B-cell depleting agent rituximab, known to reduce the infiltration of immune cells in pSS SGs, prevented the upregulation of NCR3/NKp30 within the glands.

Salivary glands (SGs) represent a permissive site for several sialotropic viruses whose persistence is linked to the development of autoimmunity. Natural Killer (NK) cells play a key role in viral clearance but their involvement in viral infection control and in tertiary lymphoid structures (TLS) development within SGs is unknown. By using an inducible model of TLS in the SGs of wild-type C57BL/6 mice, induced by the local delivery of a replication-defective adenovirus (AdV), we demonstrated that circulating NK cells are rapidly recruited to SGs and highly enrich the early inflammatory infiltrate prior to TLS development. NK cells migrating to SGs in response to AdV infection up-regulate NKp46, undergo proliferation, acquire cytotoxic potential, produce Granzyme-B and IFN-γ, and reduce viral load in the acute phase of the infection. Nonetheless, the selective depletion of both circulating and infiltrating NK cells in AdV-infected mice neither affect the development and frequency of TLS nor the onset of autoimmunity. These data demonstrate that, upon local viral delivery of AdV, peripheral NK cells homing to SGs can exert an early control of the viral infection but are dispensable for the formation of TLS and breach of immunologic tolerance.

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, via the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.

Objective:To define the relationship of synovial B cells to clinical phenotypes at different stages of disease evolution and drug exposure in rheumatoid arthritis (RA). Methods:Synovial biopsy specimens and demographic and clinical data were collected from 2 RA cohorts (n = 329), one of patients with untreated early RA (n = 165) and one of patients with established RA with an inadequate response to tumor necrosis factor inhibitors (TNFi‐IR; n = 164). Synovial tissue was subjected to hematoxylin and eosin and immunohistochemical staining and semiquantitative assessment for the degree of synovitis (on a scale of 0–9) and of CD20+ B cell infiltrate (on a scale of 0–4). B cell scores were validated by digital image analysis and B cell lineage–specific transcript analysis (RNA‐Seq) in the early RA (n = 91) and TNFi‐IR (n = 127) cohorts. Semiquantitative CD20 scores were used to classify patients as B cell rich (≥2) or B cell poor ( Results:Semiquantitative B cell scores correlated with digital image analysis quantitative measurements and B cell lineage–specific transcripts. B cell–rich synovitis was present in 35% of patients in the early RA cohort and 47.7% of patients in the TNFi‐IR cohort (P= 0.025). B cell–rich patients showed higher levels of disease activity and seropositivity for rheumatoid factor and anti–citrullinated protein antibody in early RA but not in established RA, while significantly higher histologic synovitis scores in B cell–rich patients were demonstrated in both cohorts. Conclusion:We describe a robust semiquantitative histologic B cell score that closely replicates the quantification of B cells by digital or molecular analyses. Our findings indicate an ongoing B cell–rich synovitis, which does not seem to be captured by standard clinimetric assessment, in a larger proportion of patients with established RA than early RA.

Background:The pathogenic role of B-cells in primary Sjögren’s Syndrome (pSS) is well established and B cell abnormalities. Because of the substantial role of B-cells, rituximab (RTX), a chimeric anti-CD20 monoclonal antibody, has been considered as a potential biologic disease modifying drug to reduce disease activity in pSS. To date, the TRial for Anti-B-Cell Therapy In patients with pSS (TRACTISS) is the largest multi-centre, placebo-controlled trial with RTX. Despite the unmet primary endpoints (30% reduction in fatigue or oral dryness, measured by visual analogue scale), RTX treated patients showed an improvement in unstimulated whole salivary flow (Bowman et al. Arthritis Rheumatol 2017;69:1440–1450).Objectives:To provide the first longitudinal transcriptomic and histological analysis at 3 time points over 48 weeks of labial SGs of pSS patients treated with RTX, in comparison to placebo, from the TRACTISS cohort.Methods:26 pSS patients randomised to RTX or placebo arm consented for labial SG biopsies at baseline, weeks 16 and 48. Patients received two 1000mg cycles of RTX or placebo at baseline and week 24. SG focus score, inflammatory aggregate area fraction, B-cells (CD20+), T-cells (CD3+), follicular dendritic cells (FDCs) (CD21+) and plasma cells (CD138+) density were assessed by H&E and immunofluorescence staining. The histological analysis was performed by digital imaging using QuPath software. RNA was extracted from matched labial SG lobules and sequenced with Illumina platform. A Principal Component Analysis (PCA) and features driving the PCA were investigated along with the most influential gene loadings. The limma-voom R pipeline was used to extract Differential Expressed Genes (DEGs) between placebo and RTX group at week 48, and gene ontology (GO) enrichment analysis performed through EnrichR to derive GO terms and pathways associated with DEGs.Results:Placebo-treated labial SGs showed a worsening of inflammation highlighted by the increment of B-cell density, development of new FDC networks, and a higher ectopic GC prevalence at week 48, compared to RTX-treated patients. No difference in total T-cells and plasma cell infiltration was observed. RTX downregulated genes involved in immune cell recruitment and inflammatory aggregate organisation (e.g. CCR7, CCL19, CD52, and PDCD1) and gene signature-based analysis of 64 immune cell types highlighted how RTX preferentially blocked class-switched- and memory-B-cells infiltration in SGs at week 48. Pathway analyses confirmed the downregulation of leukocyte migration, MHC class II antigen presentation, and T-cell co-stimulation immunological pathways, such as the CD40 receptor complex pathway. The analysis of placebo SGs transcriptomic at week 48 showed a higher expression of genes linked to ectopic GC organisation, such as CXCL13, CCL19, LTβ, in female compared to male subjects. Gender was confirmed as a key co-variate responsible for most of the variation in the PCA, together with the SG focus score and the foci area fraction.Conclusion:Treatment with RTX showed beneficial effects on labial SG inflammatory infiltration in pSS, by downregulating genes involved in immune cell recruitment, activation and organisation in ectopic GCs. Class-switched-B-cells, memory-B-cells and FDC network development were primarily affected appearing to be responsible for the lack of progression in SG B cell infiltration in the RTX compared to the placebo arm in which clear worsening of SG immunopathology over 48 weeks was detected in female patients. Although a clear association with the clinical improvement in unstimulated salivary flow observed at week 48 in RTX-treated patients could not be established given the low number of patients consenting to 3 longitudinal biopsies it is conceivable that RTX is responsible for preserving exocrine function.Acknowledgements:SJB receives a salary contribution from the NIHR Birmingham Biomedical Research Centre.Disclosure of Interests:Elena Pontarini: None declared, Farzana Chowdhury: None declared, Elisabetta Sciacca: None declared, Sofia Grigoriadou: None declared, Felice Rivellese: None declared, Davide Lucchesi: None declared, Katriona Goldmann: None declared, Liliane Fossati-Jimack: None declared, Paul Emery: None declared, Wan Fai Ng: None declared, Nurhan Sutcliffe: None declared, Colin Everett: None declared, Catherine Fernandez: None declared, Anwar Tappuni: None declared, Myles Lewis: None declared, Costantino Pitzalis: None declared, Simon J. Bowman Consultant of: SJB In 2020 I have received consultancy fees from Novartis, Abbvie and Galapagos., Michele Bombardieri: None declared

The mechanisms underlying the transition of rheumatoid arthritis (RA) systemic autoimmunity to the joints remain largely unknown. Here, we demonstrate that macrophages in the secondary lymphoid organs (SLOs) and synovial ectopic lymphoid-like structures (ELSs) express peptidylarginine deiminase 4 (PAD4) in murine collagen induced arthritis (CIA) and synovial biopsies from RA patients. Moreover, peptidyl citrulline colocalized with macrophages in SLOs and ELSs, and depletion of macrophages in CIA decreased lymphoid tissue citrullination and serum anti-citrullinated protein/peptide antibody (ACPA) levels. Furthermore, PAD was released from activated murine and RA synovial tissue and fluid (SF) macrophages which functionally deiminated extracellular proteins/peptides in vitro. Additionally, activated murine and SF macrophages displayed macrophage extracellular trap formation (METosis) and release of intracellular citrullinated histones. Moreover, presentation of citrullinated proteins induced ACPA production in vitro. Thus, lymphoid tissue macrophages contribute to self-antigen citrullination and ACPA production, indicating that their selective targeting would potentially ameliorate citrullination-dependent autoimmune disorders.

ObjectivesMast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). However, their contribution remains controversial. To establish their role in RA, we analysed their presence in the synovium of treatment-naïve patients with early RA and their association and functional relationship with histological features of synovitis.MethodsSynovial tissue was obtained by ultrasound-guided biopsy from treatment-naïve patients with early RA (n=99). Immune cells (CD3/CD20/CD138/CD68) and their relationship with CD117+MCs in synovial tissue were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). The functional involvement of MCs in ectopic lymphoid structures (ELS) was investigated in vitro, by coculturing MCs with naïve B cells and anticitrullinated protein antibodies (ACPA)-producing B cell clones, and in vivo in interleukin-27 receptor alpha (IL27ra)-deficient and control mice during antigen-induced arthritis (AIA).ResultsHigh synovial MC counts are associated with local and systemic inflammation, autoantibody positivity and high disease activity. IHC/IF showed that MCs reside at the outer border of lymphoid aggregates. Furthermore, human MCs promote the activation and differentiation of naïve B cells and induce the production of ACPA, mainly via contact-dependent interactions. In AIA, synovial MC numbers increase in IL27ra deficient mice, in association with ELS and worse disease activity.ConclusionsSynovial MCs identify early RA patients with a severe clinical form of synovitis characterised by the presence of ELS.

Background:The study of synovial tissue in patients with Rheumatoid Arthritis (RA) has led to the identification of synovial patterns of immune cell infiltration and specific cellular subsets associated that have been disease activity and clinical outcomes(1–3). However, the relationship of circulating and synovial immune cell sub-sets with histopathological features and clinical outcomes remains to be defined.Objectives:To assess the relationship of peripheral blood and synovial immune cells with RA histopathology and clinical outcomes, by performing flow and digital cytometry in matched peripheral blood and synovial samples from patients with early RA.Methods:70 patients with early (1). Peripheral blood mononuclear cells (n=70) were analysed by flow cytometry. Matched synovial tissues (n=70) obtained by minimally invasive ultrasound-guided synovial biopsy underwent semi-quantitative scoring (0-4) of immune cell infiltration and classification into lympho-myeloid (LM), diffuse-myeloid (DM) and pauci-immune (PI) pathotypes, as previously described(1). 49 synovial and 36 matched peripheral blood samples underwent RNA-sequencing and were analysed by digital cytometry (Xcell) (4) and Singular Value Decomposition (SVD).Results:Circulating B cells and their subsets showed significant inverse correlations with inflammatory markers (ESR, CRP), disease activity (swollen joints, four components and two components(5) DAS28) and ultrasound scores (Fig 1A). Among T cell subsets, CXCR5-PD1hiICOS+CD4+ T cells (T peripheral helper cells, Tph) had strong positive correlations with inflammatory markers (ESR and CRP), disease activity (DAS28) and ultrasound scores (Fig 1B). Tph in the peripheral blood also correlated with immune cell infiltration in synovia (Fig 1C) and were significantly higher in patients with a LM pathotype (Fig 1D). Accordingly, circulating Tph were associated with synovial LM pathotype independently of clinical features such as DAS28, ACPA positivity, Body Mass Index (BMI) and age (AUC 0.821). By applying digital cytometry in matched synovial and peripheral blood samples, synovial B and T cells were significantly higher in patients with a LM pathotype, in line with the histological definition of the LM pathotype – rich in B and T cells. On the contrary, circulating B cells and total CD4 and CD8 T cells were significantly lower in patients with a synovial LM pathotype (Fig 1E). The Tph signature in synovia derived by Singular Value Decomposition (SVD) correlated with baseline ESR (R 0.38, pConclusion:By combining conventional flow cytometry in the peripheral blood and digital cytometry on matched synovial and peripheral blood samples, we highlight diverging associations of circulating immune cell subsets with synovial inflammation and pathotypes. Tph cells, in particular, emerge as predictors of lympho-myeloid synovial inflammation and disease progression.References:[1]F. Humby et al., Ann. Rheum. Dis. 78, 761–772 (2019), doi:10.1136/annrheumdis-2018-214539. [2]M. J. Lewis et al., Cell Rep. 28, 2455-2470.e5 (2019), doi:10.1016/j.celrep.2019.07.091.[3]D. a Rao et al., Nat. Publ. Gr. 542, 110–114 (2017), doi:10.1038/nature20810.[4]D. Aran et al., Genome Biol. 18, 220 (2017), doi:10.1186/s13059-017-1349-1.[5]E. M. A. Hensor et al., Rheumatology. 58, 1400–1409 (2019), doi:10.1093/rheumatology/kez049.Figure 1.Acknowledgements:The Pathobiology of Early Arthritis Cohort (PEAC) was supported by the MRC (grant 36661). Versus Arthritis provided funding infrastructure support (grant 20022). F. Rivellese is funded by an NIHR Transitional Research Fellowship (TRF-2018-11-ST2-002). We would like to thank the patients and the clinical and laboratory team (core team) at Queen Mary University of London.Disclosure of Interests:None declared

Background:The involvement of large joints at disease presentation in early Rheumatoid Arthritis (RA) has been associated with severe disease activity. At the same time, the clinical heterogeneity of RA is known to be mirrored by heterogeneity of synovial inflammation, with specific histological patterns (pathotypes) associated with treatment response and disease progression. However, it is not known whether joint size is associated with specific pathotypes.Objectives:To analyse histopathological features of synovial biopsies from joints of different sizes and establish the relationship with clinical outcomes in patients with early RA.Methods:167 patients with early (Results:The majority of synovial biopsies were performed on medium and small joints (60.6% and 19.4%) as compared to 21.3% in large joints (Table 1). At baseline, patients who underwent large joint biopsy showed significantly higher levels of inflammation (CRP 27.9±32.4 large, 20.7±26.9 medium, 10.4±9.8 small, p=0.007) and higher HAQ (1.8 ± 0.7 large, 1.4 ± 0.8 medium, 1.2 ±0.9 small, p=0.012), with no differences in DAS28. Significantly higher inflammatory scores and higher proportion of lympho-myeloid pathotype were observed in large joints (Table 1 and Figure 1). 6 months after treat-to-target treatment with csDMARDs, large joints patients had significantly higher HAQ and lower response (RR for low disease activity in large vs medium joints 0.5, 95%CI 0.2-0.9, p=0.03). Finally, differentially expressed genes by RNA-seq showed segregation according to joint size (Figure 2), with upregulation of genes of the Homeobox transcription factors family in large joints.Table 1.EULAR 2010 RA (n=167)Large joints#33 (19.4%)Medium joints#100 (60.6%)Small Joints#34 (20%)P*Clinical featuresESR mm/h,mean (SD)48.2 (31.5)39.6 (30.8)29.2 (17.3)nsCRP mg/L,mean (SD)27.9 (32.4)20.7 (26.9)10.4 (9.8)0.007DAS28, mean (SD)6 (1.2)5.7 (1.4)5.7 (1.5)nsHAQ, mean (SD)1.8 (0.7)1.4 (0.8)1.2 (0.9)0.012ACPA-positive, %70.9%77.3%83.9%nsRF-positive,%71.9%74.2%80.6%nsHistologyInflammatory score,median IQR)5 (3)4 (4)2 (2.75)Pathotype,%Ungraded6.1%7.8%2.9%0.014Fibroid6.1%24.3%32.3%Myeloid30.3%28.1%47.1%Lympho-myeloid57.6%39.8%17.6%Clinical outcomes at 6 monthsDAS28 6m,mean (SD)4.2 (1.8)3.4 (1.9)3.7 (1.5)nsHAQ 6m,mean (SD)1.2 (0.8)0.8 (0.8)0.8 (0.8)0.012DAS28 6m %23.3%48.8%37.9%0.04#Large joints: knees; Medium joints: wrists, ankle, elbows; Small joints: MCPs, MTPs, PIPs; * Chi-squared or Kruskal–Wallis as appropriate;Conclusion:Synovial biopsy of large joints as the most inflamed joints at disease presentation identified patients with early RA with specific histopathological features and clinical outcomes. Together with clustering of differentially expressed genes according to joint size, this suggests that the involvement of different joint compartments in early RA contributes to disease heterogeneity with potential physiopathological and clinical implications.References:[1]Humby et al Ann Rheum Dis. 2019 Jun;78(6):761-772[2]Lewis et al Cell Rep. 2019 Aug 27;28(9):2455-2470.e5[3]Linn-Rasker SP et al Ann Rheum Dis. 2007 May;66(5):646-50Acknowledgments:PEAChttp://www.peac-mrc.mds.qmul.ac.ukMRC grant 36661 & ARUK Grant 20022F. Rivellese NIHR Fellowship TRF-2018-11-ST2-002Disclosure of Interests:None declared

Introduction The role of citrullination in breach of tolerance in rheumatoid arthritis (RA) is well established, but the mechanisms linking protein citrullination and the generation of anti-citrullinated protein/p eptide antibodies (ACPA) are poorly understood. We have recently reported a critical role of peptidylarginine deiminase (PAD) 4 expression and extracellular trap formation by neutrophils in ACPA production in RA.1 In lymphoid tissues, neutrophils are scarce, whereas macrophages are abundant antigen presenting cells where they regulate T and B cell responses and play fundamental roles in the chronicity of RA. Objectives To test our hypothesis that antigen presenting cells (APCs) in lymphoid tissues express PADs and contribute to protein citrullination and ACPA production. Methods We tested our hypothesis in collagen induced arthritis (CIA) mice and synovial biopsies from RA patients. PAD4 and 2 expression was assessed in the spleens and lymph nodes of CIA mice and their distribution was revealed by immunohistochemistry (IHC). The impact of macrophage depletion on lymphoid tissues’ PAD activity, citrulline content, and serum ACPA levels was analysed; and the effect of macrophage stimulation on extracellular trap formation, PADs expression and release was investigated. Additionally, PAD4 expression in RA synovia was verified by IHC; and the correlation of PADI4 with macrophage-related genes was studied by RNA-Seq analysis. Results Our results indicated that serum ACPA were detectable in CIA sera before clinical arthritis; and PAD4 and 2 were expressed in the draining lymph nodes and spleens as shown by qPCR and Western blotting. In addition, PAD4 and 2 labelling specifically co-localised with macrophages but not other APCs. Moreover, PAD4 and −2-expressing tingible body macrophages (TBM) were associated with citrulline-retaining follicular dendritic cells (FDCs) in the germinal centres (GC). In vitro TLR activation of macrophages enhanced PAD4 and 2 expression; and stimulation of TBMs with TNF-α induced Macrophage Extracellular Trap formation (METosis), release of apoptotic bodies, and DNA-associated citrullinated proteins. Whereas retention of citrullinated proteins on FDCs induced ACPA production in in vitro GC reactions, macrophage depletion in CIA mice significantly reduced citrullinated proteins in lymphoid tissues and inhibited serum ACPA levels. In RA synovia, PAD4 colocalized with CD68 macrophages and was expressed in TBMs associated with citrulline-retaining FDCs in ectopic lymphoid structures. RNA-Seq analysis of PADI4 expression in RA synovial biopsies identified significant positive correlation with 279 genes associated with macrophage activation and function. Conclusions In conclusion, this work reports new mechanisms by which macrophages contribute to citrulline-induced autoimmunity. Therapeutic targeting of these mechanisms would inhibit citrullination, ACPA production and progression of autoimmune diseases. Reference . Corsiero E, Bombardieri M, Carlotti E, Pratesi F, Robinson W, Migliorini P, Pitzalis C. Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs. Ann.Rheum Dis2016;75:1866–1875. Disclosure of interest None declared

ObjectivesTo identify predictors of remission and disease activity patterns in patients with rheumatoid arthritis (RA) using individual participant data (IPD) from clinical trials.MethodsPhase II and III clinical trials completed between 2002 and 2012 were identified by systematic literature review and contact with UK market authorisation holders. Anonymised baseline and follow-up IPD from non-biological arms were amalgamated. Multiple imputation was used to handle missing outcome and covariate information. Random effects logistic regression was used to identify predictors of remission, measured by the Disease Activity Score 28 (DAS28) at 6 months. Novel latent class mixed models characterised DAS28 over time.ResultsIPD of 3290 participants from 18 trials were included. Of these participants, 92% received methotrexate (MTX). Remission rates were estimated at 8.4%(95%CI 7.4%to9.5%) overall, 17%(95%CI 14.8%to19.4%) for MTX-naïve patients with early RA and 3.2% (95% CI 2.4% to 4.3%) for those with prior MTX exposure at entry. In prior MTX-exposed patients, lower baseline DAS28 and MTX reinitiation were associated with remission. In MTX-naïve patients, being young, white, male, with better functional and mental health, lower baseline DAS28 and receiving concomitant glucocorticoids were associated with remission. Three DAS28 trajectory subpopulations were identified in MTX-naïve and MTX-exposed patients. A number of variables were associated with subpopulation membership and DAS28 levels within subpopulations.Conclusions Predictors of remission differed between MTX-naïve and prior MTX-exposed patients at entry. Latent class mixed models supported differential non-biological therapy response, with three distinct trajectories observed in both MTX-naïve and MTX-exposed patients. Findings should be useful when designing future RA trials and interpreting results of biomarker studies.

Regulatory B cells (Breg) have attracted increasing attention for their roles in maintaining peripheral tolerance. Interleukin 33 (IL-33) is a recently identified IL-1 family member, which leads a double-life with both pro- and anti-inflammatory properties. We report here that peritoneal injection of IL-33 exacerbated inflammatory bowel disease in IL-10-deficient (IL-10−/−) mice, whereas IL-33-treated IL-10-sufficient (wild type) mice were protected from the disease induction. A phenotypically unconventional subset(s) (CD19+CD25+CD1dhiIgMhiCD5-CD23-Tim-1-) of IL-10 producing Breg-like cells (BregIL-33) was identified responsible for the protection. We demonstrated further that BregIL-33 isolated from these mice could suppress immune effector cell expansion and functions and, upon adoptive transfer, effectively blocked the development of spontaneous colitis in IL-10−/− mice. Our findings indicate an essential protective role, hence therapeutic potential, of BregIL-33 against mucosal inflammatory disorders in the gut., Highlights • IL-33, a branded ‘Th2’ cytokine, could accelerate the Th1-mediated colitis under an IL-10 deficient condition. • IL-10 sufficient mice were protected from the IL-33-mediated mucosal inflammation. • IL-33 induced a phenotypically unique subset of IL-10-producing regulatory B cells (BregIL-33). • Adoptive transfer of BregIL-33 blocked the spontaneous colitis in IL-10-deficient mice (therapeutic potential). • Our findings offer new insights into the immunological mechanisms underlying mucosal inflammation, and its regulation.

The association between systemic lupus erythematosus (SLE) and cardiovascular events is well established in epidemiologic studies. In particular, results have shown a substantial increase in angina and myocardial infarction in patients with SLE (1). Imaging studies have shown an increased prevalence of carotid plaque and coronary calcification in patients with SLE, suggesting that there is an accelerated burden of atherosclerosis in these patients (2, 3). Given the inflammatory nature of atherosclerosis, the acceleration of disease in the presence of SLE is perhaps not surprising. However, the exact underlying mechanisms involved are still poorly defined. Studies utilizing murine models of atherosclerosis in combination with a lupus-like disease have been reported (4–8). Accelerated atherosclerosis has been described in apolipoprotein E (ApoE)–deficient mice with either the gld or lpr mutation (4, 6, 8), and in bone marrow chimeras of gld mice transplanted into low-density lipoprotein (LDL) receptor–deficient (Ldlr−/−) mice (gld→Ldlr−/−) (7). Radiation chimeras using the triple-congenic lupus mouse strain B6.Sle1.2.3 transplanted into Ldlr−/− mice (Sle1.2.3→Ldlr−/−) also showed increased atherosclerosis, which was thought to be due to systemic activation of B and T cells (5). As immune complex (IC) formation is a feature of SLE, it seems logical to suppose that accelerated atherosclerosis in SLE could be attributed to increased IC deposition within the arterial walls, with subsequent activation of complement. However, there is no experimental evidence to support this hypothesis. Hypocomplementemia has been linked to defective clearance of apoptotic cells (9), which is considered to be a key pathogenetic mechanism in the development of both lupus and atherosclerosis (4). Acceleration of murine atherosclerosis attributed to accumulation of apoptotic cells within atherosclerotic lesions has been described in the context of deficiency of complement C1q (10), Mer receptor tyrosine kinase (11, 12), and lactadherin (13). A lupus-like phenotype associated with defective uptake of apoptotic cells has also been demonstrated in these mice (9, 14, 15). Even though all these findings indicate that synergistic mechanisms may operate in both conditions, whether the same pathways lead to an accelerated development of atherosclerosis and increased renal damage remains to be elucidated. Herein we describe a novel mouse model of lupus and atherosclerosis (Sle16.Ldlr−/−) generated by crossing Ldlr−/− mice with the congenic line Sle16 (16). The Sle16 line carries, on chromosome 1, an interval originating in the 129 mouse strain, and these mice develop autoantibodies and mild renal inflammation with IC deposition without proteinuria (16). We observed that both nephritis and atherosclerosis were accelerated in the Sle16.Ldlr−/− mice, suggesting that synergistic interactions take place between systemic immune dysregulation and dyslipidemia. Importantly, our data indicate that distinct complement-related mechanisms operate in the kidney when compared to the arterial walls.

Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.

The 129-derived Sle16 is a susceptibility locus for systemic autoimmunity when present on the C57BL/6 (B6) background. Genetic analysis of a (129×B6)F2 cross identified a region from the B6 chromosome 3 (Sle18) with positive linkage to antinuclear Abs. In this study, we have generated a B6 congenic strain harboring the 129 allele of Sle18 and intercrossed this line with the lupus-prone B6.129-Sle16 strain. The presence of the 129-Sle18 allele in the B6.129-Sle16Sle18 double congenic mice suppressed the development of Sle16-mediated autoantibody production and ameliorated the renal pathology. The 129-Sle18 locus rectified the B cell abnormalities detected in the B6.129-Sle16 mice, such as the reduction in the percentage of marginal zone B and B1a cells and the increased number of germinal centers. The B6.129-Sle16Sle18 spleens still displayed an increased percentage of activated T and B cells. However, in the B6.129-Sle16Sle18 strain the percentage of naive T cells was equivalent to that in B6.129-Sle18 and B6 mice and these cells showed a reduced proliferative response to anti-CD3 stimulation compared with B6.129-Sle16 T cells. There was a significant increase in the percentage of CD4+FoxP3+regulatory T cells in all congenic strains. These cells had normal regulatory function when tested in vitro. Thus, 129-Sle18 represents a novel, non-MHC lupus-suppressor locus probably operating as a functional modifier of B cells that, in combination with other factors, leads to lupus resistance. Further characterization of this locus will help to uncover the immune mechanism(s) conferring protection against lupus.

Epistatic interactions between the non-autoimmune strains 129 and C57BL/6 (B6), used for generating gene-targeted animals, can induce a lupus-like disease. Genome-wide scan analyses of testcross progeny between these two strains have identified several lupus susceptibility loci, with the strongest linkage to the production of autoantibodies (auto-Abs) displayed by an interval on chromosome 1 of 129 origin (Sle16). However, the contribution of B6 loci to the lupus phenotype remained unknown. We used a congenic approach to deduce the contribution to the autoimmune traits of the B6 genomic interval on chromosome 3 (Sle18), previously shown to be linked to antinuclear Ab production. This interval, when transferred on a 129 background (a strain termed 129.B6-Sle18), promoted auto-Ab production targeting a broad spectrum of autoantigens, expansion of activated CD4(+)T and B cells and mild glomerulonephritis. Surprisingly, these immunological and serological defects were accompanied by a significant increase in the percentage of regulatory T cells (Tregs; CD4(+) Foxp3(+)). However, these cells, that expressed lower levels of Foxp3, had no impaired regulatory function when tested in vitro. These findings illustrate further the efficacy of congenic dissection for functional characterisation of individual lupus susceptibility loci and highlight the contribution of loci derived from non-autoimmune strains to the disease pathogenesis.

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine lupus nephritis is secreted by hepatocytes as an acute phase protein, and it has been thought to be a product of an endogenous xenotropic virus, NZB-X1. However, since endogenous polytropic (PT) and modified polytropic (mPT) viruses encode gp70s that are closely related to xenotropic gp70, these viruses can be additional sources of serum gp70. To better understand the genetic basis of the expression of serum gp70, we analyzed the abundance of xenotropic, PT, or mPT gp70 RNAs in livers and the genomic composition of corresponding proviruses in various strains of mice, including two different Sgp (serum gp70 production) congenic mice. Our results demonstrated that the expression of different viral gp70 RNAs was remarkably heterogeneous among various mouse strains and that the level of serum gp70 production was regulated by multiple structural and regulatory genes. Additionally, a significant contribution of PT and mPT gp70s to serum gp70 was revealed by the detection of PT and mPT, but not xenotropic transcripts in 129 mice, and by a closer correlation of serum levels of gp70 with the abundance of PT and mPT gp70 RNAs than with that of xenotropic gp70 RNA in Sgp3 congenic mice. Furthermore, the injection of lipopolysaccharides selectively up-regulated the expression of xenotropic and mPT gp70 RNAs, but not PT gp70 RNA. Our data indicate that the genetic origin of serum gp70 is more heterogeneous than previously thought, and that distinct retroviral gp70s are differentially regulated in physiological vs inflammatory conditions.

Systemic lupus erythematosus (SLE) is an autoimmune disease that displays highly variable clinical features. The serologic hallmark of SLE is the production of autoantibodies directed against a wide spectrum of self antigens, especially nuclear components, such as DNA. Several lines of evidence indicate that both in humans and in animal models, SLE susceptibility is inherited as a multifactorial genetic trait (1). To identify the genetic components of SLE, linkage analyses have been performed in spontaneous lupus–prone mice, and these studies have led to the identification of several genomic intervals (2). Interestingly, a great proportion of the intervals detected are strain-specific, confirming the genetic complexity of the disease and indicating the presence of extensive heterogeneity in the genes that contribute to the pathogenesis of SLE. However, 1 interval, located on the distal region of chromosome 1 in the mouse and on its orthologous region in humans, has shown strong linkage in several human and murine studies (3,4). One of the best characterized loci in this region is Sle1b, which was identified via linkage analysis of the lupus-prone NZM2410 mouse model (5). This locus, when expressed on the C57BL/6 genetic background (B6.Sle1b), can induce a relatively benign autoimmune disease characterized by loss of tolerance to nuclear antigens, an increase in the proportion of activated T cells and B cells, and mild splenomegaly. These features seem to be linked to a specific haplotype of the signaling lymphocytic activation molecule (SLAM)/CD2 family of genes, indicating that these genes might mediate the autoimmune phenotype associated with the Sle1b locus (6,7). However, in the same chromosome 1 region, there are several other candidate genes, such as Apcs, Fcgr2, Cd55, Pdcd1, and Ro antigen. All of these genes were inactivated in 129-derived embryonic stem cells, and the knockout models displayed a variable degree of autoimmunity (8–12). However, the interpretation of the autoimmune phenotype (13) observed in these gene-targeted models has recently been questioned (14). Studies by our group and others (8,15–17) have shown that hybrid strains between 129 and C57BL/6 (B6) mice, commonly used in the generation of gene-targeted mice, are spontaneously predisposed to the development of humoral autoimmunity, with low levels of renal disease. Genome-wide linkage studies conducted on the 129 × B6 hybrid mice identified a strong association between a 129-derived segment on chromosome 1, now named Sle16, and the expression of autoantibodies (14,18). The autoimmune effect of this locus has recently been defined further by the study of B6 subcongenic strains carrying 129 fragments of different lengths (14,19). The analysis of these lines revealed that a 129 interval between 87.9 cM and 100 cM (D1Mit15 and D1Mit115) was sufficient to induce the autoimmune phenotype, and the congenic line carrying this fragment, named B6.129chr1b, was selected for the present study. It is noteworthy that the B6.129chr1b congenic mice mirror the combination of genes created when a gene in this region is targeted on 129 embryonic stem cells and then backcrossed onto the B6 genetic background. Immunoglobulin-transgenic models have been instrumental in understanding B cell regulation, revealing several key mechanisms including receptor editing, deletion, anergy, and ignorance (20–24). Chen et al (25) generated an anti-DNA–knockin model in which the rearranged variable heavy chain (VH) gene of an anti-DNA antibody (VH3H9) was inserted at the Igh locus. This allowed the transgenic locus to undergo normal editing, isotype switching, and somatic mutation. A variety of light chains can combine with the VH3H9 to yield anti-DNA antibodies (26), but only a few light chains are able to “silence” VH3H9 so that it no longer binds to DNA. By virtue of this characteristic, the mice expressing only the VH3H9 chain (VH3H9R mice) can generate anti-DNA specificities. When the VH3H9R mice were crossed with the knockin transgenic light chain Vκ8 mice (27), the antibody generated in the double-transgenic mice (VH3H9R/Vκ8R mice) (28) bound only single-stranded DNA (ssDNA) and not double-stranded DNA (dsDNA) (27). Previous studies with the VH3H9R mice showed that autoreactive transgenic B cells accumulated in the splenic marginal zone and were regulated by anergy in the presence of a nonautoimmune background, such as BALB/c (25,29) and B6 (30,31), but were activated in a model of chronic graft-versus-host disease (30). Similarly, the VH3H9R/Vκ8R-knockin transgenic B cells were regulated by anergy in nonautoimmune disease–prone BALB/c mice, while in autoimmune disease–prone MRL/Mp.lpr/lpr animals, transgenic B cells escaped tolerance induction and underwent class switching and affinity maturation (32). To determine whether the Sle16 locus could influence the selection of self-reactive B cells, we bred the B6.129chr1b mice with the VH3H9R.B6 mice and the VH3H9R/Vκ8R.B6 mice and monitored the regulation and activation of anti-DNA–transgenic B cells over a period of 10 months. A similar analysis was also performed with mice deficient in serum amyloid P component (B6. Apcs−/−), chosen as an example of an autoimmune mouse strain with a targeted deletion of 1 of the candidate genes located within the 129chr1b region. The analysis of these mice showed that hemizygosity of the Sle16 locus was sufficient to impair B cell anergy and to induce class switching and epitope spreading. The immunologic alterations were more pronounced in mice that were homozygous for the Sle16 locus and were similar in the B6. Apcs−/− mice, indicating that the lack of serum amyloid P component had no additional effect on the loss of B cell tolerance.

C1q-deficient mice have been shown to develop a lupus-like disease and to display an impaired clearance of apoptotic cells that are enriched in lupus autoantigens. However, the role of C1q in the regulation of autoreactive B cells remains debatable. To explore this we crossed MRL/Mp C1q-deficient mice with knock-in transgenic (Tg) mice expressing an anti-ssDNA antibody (VH3H9R and VH3H9R/VLkappa8R). Analysis of the VH3H9R mice showed that in the absence of C1q higher titres of Tg-derived IgM and IgG3 anti-ssDNA antibodies were detectable. In contrast, in the VH3H9R/VLkappa8R C1q-deficient animals no increase in Tg antibody levels was observed. In both models the lack of C1q induced a marked reduction of marginal zone B cells and this was paralleled by a significant increase in the percentage of plasmocytes. Thus, one could postulate that in the absence of C1q the failure to clear efficiently dying cells provides an additional stimulus to the autoreactive Tg B cells resulting in their emigration from the marginal zone B cell compartment with subsequent increase in plasmocytes. However, the lack of C1q led to an increased production of Tg IgM and IgG3 antibodies only in VH3H9R mice indicating that additional genetic susceptibility factors are required to break self-tolerance.

Extensive evidence indicates that genetic predisposition is a central element in susceptibility to systemic lupus erythematosus both in humans and animals. We have previously shown that a congenic line carrying a 129-derived chromosome 1 interval on the C57BL/6 background developed humoral autoimmunity. To further dissect the contribution to autoimmunity of this 129 interval, we have created six subcongenic strains carrying fractions of the original 129 region and analyzed their serological and cellular phenotypes. At 1 year of age the congenic strain carrying a 129 interval between the microsatellites D1Mit15 (87.9 cM) and D1Mit115 (99.7 cM) (B6.129chr1b) had high levels of autoantibodies, while all the other congenic lines were not significantly different from the C57BL/6 controls. The B6.129chr1b strain displayed only mild proliferative glomerulonephritis despite high levels of IgG and C3 deposited in the kidneys. FACS analysis of the spleens revealed that the B6.129chr1b mice had a marked increase in the percentage of activated T cells associated with a significant reduction in the proportion of CD4+CD25high regulatory T cells. Moreover, this analysis showed a significantly reduced percentage of marginal zone B cells that preceded autoantibody production. Interestingly the 129chr1b-expressing bone marrow-derived macrophages displayed an impaired uptake of apoptotic cells in vitro. Collectively, our data indicate that the 129chr1b segment when recombined on the C57BL/6 genomic background is sufficient to induce loss of tolerance to nuclear Ags. These findings have important implication for the interpretation of the autoimmune phenotype associated with gene-targeted models.

C1q deficiency in both humans and mice is strongly associated with autoimmunity. We have previously shown that bone marrow transplantation (BMT) restored C1q levels in C1q-deficient (C1qa(-/-)) mice. Here, we studied the effect of BMT on autoimmunity in C1qa(-/-) mice. Following irradiation, young C1qa(-/-) or wild-type MRL/Mp mice received bone marrow cells (BMC) from strain-matched wild-type or C1qa(-/-) animals. C1q levels increased rapidly when C1qa(-/-) mice received BMC from wild-type mice. Conversely, they decreased slowly in wild-type mice transplanted with C1qa(-/-) BMC. C1qa(-/-) animals transplanted with C1qa(-/-) BMC demonstrated accelerated disease when compared with wild-type mice given wild-type BMC. In contrast, a significant delay in the development of autoantibodies and glomerulonephritis was observed in C1qa(-/-) mice reconstituted with wild-type BMC, and the impaired clearance of apoptotic cells, previously described in C1qa(-/-) mice, was rectified. Moreover, the autoimmune disease was accelerated in wild-type mice given C1qa(-/-) BMC compared to animals transplanted with wild-type cells. These results provide supporting evidence that BMT may be a therapeutic option in the treatment of autoimmunity associated with human C1q deficiency.

C1q deficiency has been shown to accelerate spontaneous autoimmunity in mice. We studied the time course of activation of monocytes and lymphocytes in autoimmune and non-autoimmune mice in the presence or absence of C1q as a disease accelerator. Autoimmune MRL\Mp.C1qa−/− and non-autoimmune C57BL\6.C1qa−/− mice were analysed at various time points between 6 and 33 weeks of age and compared to strain- and age-matched C1q-sufficient controls. Splenic and peritoneal leucocytes were analysed by flow cytometry and plasma levels of immunoglobulin M (IgM), total IgG, IgG subclasses and IgM autoantibodies were measured. Both C1q-deficient strains had significantly more splenic monocytes than their controls at all time points analysed. In addition, MRL\Mp.C1qa−/− but not C57BL/6.C1qa−/− mice developed splenic hypercellularity starting at about 12–17 weeks old, had signs of accelerated CD4+ T-cell activation and showed a marked increase in splenic plasma cells and total serum IgM levels from about 22 weeks of age. The accelerated CD4+ T-cell activation was not due to a direct inhibitory effect of C1q on T cells. These data show that C1q deficiency causes splenic monocytosis together with accelerated T-cell activation in an autoimmune-prone mouse strain.

Human CD93 (known as C1qRp) has been shown to be a phagocytic receptor involved in the in vitro C1q-dependent enhancement of phagocytosis. However, binding of CD93 to C1q and its function remain controversial. In this study, we have generated CD93-deficient mice (CD93−/−) to investigate its biological role(s). The CD93−/− mice were viable and showed no gross abnormalities in their development. Thioglycolate-elicited peritoneal macrophages deficient in CD93 showed a similar enhancement in complement- and FcγR-dependent uptake of RBC to the wild-type macrophages when plated on C1q-coated surfaces suggesting that the lack of this receptor had no effect on these C1q-mediated events. There was no impairment in either complement- or FcγR-dependent phagocytic assays in vivo. By contrast, the CD93−/− mice had a significant phagocytic defect in the clearance of apoptotic cells in vivo (human Jurkat T cells and murine thymocytes: p = 0.0006 and p = 0.0079, respectively) compared with strain-matched controls. However, in vitro, the CD93−/− macrophages showed similar engulfment of apoptotic cells to wild-type macrophages. Furthermore, no supporting evidence for a role of CD93 as an adhesion molecule was found using intravital microscopy or analyzing peritoneal cell recruitment in response to three different inflammatory stimuli (thioglycolate, zymosan A, and IL-1β). Thus, our findings indicate that murine CD93 is expressed on the peritoneal macrophage, especially on thioglycolate-elicited cells, but does not appear to play a key role in C1q-mediated enhancement of phagocytosis or in the intercellular adhesion events tested. However, our results suggest that it may contribute to the in vivo clearance of dying cells.

Objective CD22, a B cell–restricted transmembrane glycoprotein, regulates B cell antigen receptor signaling upon interaction with α2,6-linked sialic acid–bearing glycans, which act as ligands and are expressed on B and T cells. In this study, we investigated how the expression of CD22 ligand (CD22L) is modulated following lymphocyte activation or during the course of systemic lupus erythematosus (SLE). Methods The expression levels of CD22L on B and T cells in nonautoimmune mice were assessed by flow cytometric analysis using a soluble recombinant form of CD22, following stimulation with antigen or mitogen in vitro. In addition, the expression levels of CD22L on circulating lymphocytes were correlated with the progression of SLE in lupus-prone mice. Results We observed a constitutive expression of CD22L on mature B cells, but not T cells, in nonautoimmune mice. However, CD22L levels were up-regulated selectively on T cells (but not B cells) stimulated with antigens in vitro, while their expression levels on B cells was up-modulated following polyclonal activation with lipopolysaccharide. Furthermore, expression of CD22L was increased on circulating B cells (and to a lesser extent on T cells) in parallel with progression of SLE in several different lupus-prone mice and in a cohort of (C57BL/6 × [NZB × C57BL/6.Yaa]F1) backcross mice. Conclusion The expression of CD22L is differentially regulated in B and T cells, and high expression of CD22L on circulating B cells is a marker for development of severe SLE, suggesting a role for CD22–CD22L interactions in SLE as well as in the regulation of humoral immunity.

Nephrotoxic nephritis is mediated by Fcγ receptors on circulating leukocytes and not intrinsic renal cells. Background There is evidence that mesangial cells express Fcγ receptors in vitro, but the in vivo relevance of this is not known. FcRγ-/- mice lack the gamma chain signaling subunit and therefore do not express the activator Fcγ receptors (FcγRI and FcγRIII) or the high affinity IgE receptor, FcγRI. FcRγ-/- mice were protected from renal inflammation following the induction of accelerated nephrotoxic nephritis using sheep anti-mouse glomerular basement membrane anti-serum in mice sensitized to sheep IgG. Methods In order to test whether Fcγ receptors had a role on intrinsic renal cells during nephritis, bone marrow cells were transplanted between wild-type (WT) mice and mice with a gene-targeted deletion of FcRγ. Donor marrow reconstitution was confirmed by flow cytometric analysis of peripheral blood for FcγRIII, and the susceptibility of the transplanted mice to accelerated nephrotoxic nephritis was tested. Results Following the induction of nephrotoxic nephritis, FcRγ-/- mice transplanted with WT bone marrow developed as much renal disease as WT mice transplanted with WT bone marrow. In contrast, WT mice transplanted with FcRγ-/- marrow were completely protected from glomerular crescents, thrombosis, albuminuria and renal impairment, as were FcRγ-/- mice transplanted with FcRγ-/- marrow. Mice with FcRγ-/- marrow had prolonged survival, but by day 28 after nephrotoxic serum injection they had developed mesangial hypercellularity and a macrophage influx caused by non-FcRγ dependent mechanisms. Conclusion Despite previous evidence that mesangial cells express Fcγ receptors in vitro, they have no role in an FcRγ-dependent model of glomerulonephritis in vivo.

SummaryAn increased number of apoptotic bodies have been detected in glomeruli of non-nephritic kidneys of C1q-deficient mice. In these mice an in vivo impaired uptake of apoptotic cells by peritoneal macrophages was also demonstrated. Here we investigated whether C1q plays a role in the in vitro clearance of apoptotic cells by glomerular mesangial cells. Phagocytosis was assessed using a novel flow cytometric assay that was validated by immunofluorescence studies. The uptake of apoptotic cells by mesangial cells, measured as percentage of mesangial cells ingesting apoptotic cells, was ∼25%, 10% and 10% for a T cell lymphoma line (RMA), thymocytes and neutrophils, respectively. The uptake reached a plateau phase after 3 h, was specific for apoptotic cells and was mediated by serum but not by complement components C1q or C3. The phagocytosis of apoptotic cells was significantly inhibited by Arg-Gly-Asp-Ser (RGDS), a peptide capable of blocking the interaction of thrombospondin with CD36 or the vitronectin receptor. Pretreatment of the mesangial cells with dexamethasone (200 nm) but not with LPS increased the uptake markedly. These findings indicate that murine mesangial cells are capable of taking up syngeneic apoptotic cells, although much less efficiently than professional phagocytic cells. They also show that serum proteins other than complement components mediate the removal of apoptotic cells by murine mesangial cells in vitro.

By generating four IgG isotype-switch variants of the high affinity 34–3C anti-erythrocyte autoantibody, and comparing them to the IgG variants of the low affinity 4C8 anti-erythrocyte autoantibody that we have previously studied, we evaluated in this study how high affinity binding to erythrocytes influences the pathogenicity of each IgG isotype in relation to the respective contributions of Fcγ receptor (FcγR) and complement. The 34–3C autoantibody opsonizing extensively circulating erythrocytes efficiently activated complement in vivo (IgG2a = IgG2b > IgG3), except for the IgG1 isotype, while the 4C8 IgG autoantibody failed to activate complement. The pathogenicity of the 34–3C autoantibody of IgG2b and IgG3 isotypes was dramatically higher (>200-fold) than that of the corresponding isotypes of the 4C8 antibody. This enhanced activity was highly (IgG2b) or totally (IgG3) dependent on complement. In contrast, erythrocyte-binding affinities only played a minor role in in vivo hemolytic activities of the IgG1 and IgG2a isotypes of 34–3C and 4C8 antibodies, where complement was not or only partially involved, respectively. The remarkably different capacities of four different IgG isotypes of low and high affinity anti-erythrocyte autoantibodies to activate FcγR-bearing effector cells and complement in vivo demonstrate the role of autoantibody affinity maturation and of IgG isotype switching in autoantibody-mediated pathology.

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 × C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp+/+ strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp+/+ animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp+/+ mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp+/+ strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.

Background The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. Methods 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. Results In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. Conclusions There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations. Electronic supplementary material The online version of this article (doi:10.1186/s12989-014-0064-2) contains supplementary material, which is available to authorized users.

Cryoglobulin activity associated with murine immunoglobulin G3 (IgG3) has been shown to play a significant role in the development of murine lupuslike glomerulonephritis. A fraction, but not all, IgG3 monoclonal antibodies are capable of inducing a severe acute lupuslike glomerulonephritis as a result of direct localization of IgG3 cryoglobulins, suggesting the importance of qualitative features of cryoglobulins in their nephritogenic activities. Here a remarkable difference is shown in the renal pathogenicity of 2 murine IgG3 monoclonal cryoglobulins, identical in the amino acid sequences of their heavy and light chains but different in galactosylation patterns of oligosaccharide side chains because of their synthesis in different myeloma cells. The antibody lacking the capacity to induce severe glomerulonephritis displayed an increased proportion of galactosylated heavy chains. Changes in conformation, as revealed by gel filtration analysis, reduced cryoglobulin activity, and accelerated clearance could account for the lack of the renal pathogenicity of the more galactosylated variant. This observation provides a direct demonstration for the role of IgG galactosylation in the pathogenic potential of cryoglobulins. (Blood. 2001;97:3537-3543)

Murine acquired immunodeficiency syndrome (MAIDS) is characterized by lymphoproliferation, polyclonal B cell activation resulting in the production of autoantibodies, and a progressive immunodeficiency. These are all hallmarks of some autoimmune diseases. Yaa is a Y-chromosome-linked gene that accelerates autoimmune diseases in some autoimmune-prone strains of mice. To further elucidate a possible relationship with autoimmunity, the effect of the Yaa gene on MAIDS was investigated. Analysis of phenotypic and functional disease parameters revealed that Yaa does not accelerate MAIDS disease. This is probably due to the generalized activation of most or all lymphoid cells in MAIDS, which cannot be enhanced by the Yaa gene. This result is in accordance with the selective enhancing effect of the Yaa gene on the immune response against self and foreign antigens in a specific genetic background. It suggests that the autoimmune response associated with MAIDS is a secondary phenomenon. Interestingly, even in wild-type C57BL/6 mice, autoantibody production may contribute overproportionally to the hypergammaglobulinemia associated with MAIDS.

To assess the potency of low-affinity anti–red blood cell (RBC) autoantibodies in the induction of anemia, we generated an immunoglobulin (Ig)G2a class-switch variant of a 4C8 IgM anti–mouse RBC autoantibody, and compared its pathogenic potential with that of its IgM isotype and a high-affinity 34-3C IgG2a autoantibody. The RBC-binding activity of the 4C8 IgG2a variant was barely detectable, at least 1,000 times lower than that of its IgM isotype, having a high-binding avidity, and that of the 34-3C IgG2a monoclonal antibody (mAb). This low-affinity feature of the 4C8 mAb was consistent with the lack of detection of opsonized RBCs in the circulating blood from the 4C8 IgG2a–injected mice. However, the 4C8 IgG2a variant was highly pathogenic, as potent as its IgM isotype and the 34-3C IgG2a mAb, due to its capacity to interact with Fc receptors involved in erythrophagocytosis. In addition, our results indicated that the pentameric form of the low-affinity IgM isotype, by promoting the binding and agglutination of RBCs, is critical for its pathogenic activity. Demonstration of the remarkably high pathogenic potency of low-affinity autoantibodies, if combined with appropriate heavy chain effector functions, highlights the critical role of the Ig heavy chain constant regions, but the relatively minor role of autoantigen-binding affinities, in autoimmune hemolytic anemia.

Fas ligand is a well-known inducer of apoptosis in cells expressing its receptor Fas; it also prevents autoimmunity by inducing apoptosis of activated T cells. However, Fas ligand also mediates non-apoptotic functions involving inflammatory cell migration and cytokine responses. We sought here to study the role of Fas ligand in nephrotoxic nephritis, a model of crescentic glomerulonephritis, using generalized lymphoproliferative disorder (GLD) mice on a C57BL/6 background, which have defective Fas ligand and display only mild autoimmunity. These mice were significantly protected from glomerular crescent formation, glomerular thrombosis, renal impairment, and albuminuria 15 days after the induction of glomerulonephritis in comparison with wild-type mice. There were a reduced number of apoptotic cells in the glomeruli of nephritic GLD mice but no defect in their antibody responses or splenocyte proliferation at 15 days following the induction of glomerulonephritis. Bone marrow transplantation from wild-type mice restored disease susceptibility to GLD mice; however, wild-type mice were not protected when transplanted with bone marrow from GLD mice. Mesangial cells express Fas ligand in vitro, and these cells isolated from GLD mice produced lower amounts of monocyte chemoattractive protein-1 following interleukin-1 stimulation compared with cells from wild-type mice. Thus, Fas ligand-defective mice are protected from nephrotoxic nephritis, a disease in which both circulating and intrinsic renal cells appear to have a role.

The development of systemic lupus is accelerated by the Yaa (Y-linked autoimmune acceleration) mutation, which is the consequence of a translocation of the telomeric end containing the Tlr7 gene from the X chromosome onto the Y chromosome. However, the loss of marginal zone (MZ) B cells, one of the Yaa-linked cellular abnormalities, has previously been shown to be unrelated to the Tlr7 gene duplication, and the present study therefore aimed to investigate the mechanism responsible for MZ B-cell loss. Analyses of Yaa and non-Yaa C57BL/6 male mice expressing an MD4 anti-HEL IgM transgene or those deficient in fms-like tyrosine kinase 3 ligand (FL) revealed that the proportion of MZ B cells in these Yaa mice was comparable to that of the respective non-Yaa control mice. Notably, the activation of MZ B cells was compromised in both of these transgenic model systems, due to the absence of cognate antigens or the impaired development of dendritic cells, respectively. These results contrasted with the loss of MZ B cells in non-Yaa mice treated with FL and the lack of accumulation of MZ B cells in Yaa mice treated with a B-cell survival factor, BAFF. Taken together, our results suggest that the persistent and enhanced activation of Yaa-bearing hyperactive MZ B cells by dendritic cells is responsible for the loss of this B-cell subset in Yaa mice.

By generating IgM and IgA switch variants of the 34-3C IgG2a anti–red blood cell (RBC) autoantibody, we evaluated the pathogenic activity of these 2 isotypes in view of the Fc-associated effector functions (ie, complement activation and polyvalency-dependent agglutination). We found that polymeric forms of 34-3C IgM and IgA anti-RBC autoantibody were as pathogenic as IgG2a, which was the most pathogenic among 4 different IgG subclasses, whereas their monomeric variants completely lacked pathogenic effects. Histological examination showed that 34-3C IgM and IgA autoantibodies caused anemia as a result of multivalency-dependent hemaggultination and subsequent sequestration of RBC in the spleen, in contrast to Fc receptor– and complement receptor–mediated erythrophagocytosis by Kupffer cells with IgG isotypes. In addition, the development of anemia induced by IgM and IgA isotypes of 34-3C antibody and by 2 additional IgM anti-RBC monoclonal autoantibodies was not inhibited at all in C3-deficient mice, indicating the lack of involvement of complement activation in the pathogenesis of IgM- and IgA-induced anemia. Our data demonstrate a remarkably high pathogenic potential of polymeric forms of IgM and IgA anti-RBC autoantibodies due to their ability to induce hemagglutination but completely independent of complement activation.

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of the Y-linked autoimmune acceleration (Yaa) mutation, which induces an age-dependent monocytosis. Using a cohort of C57BL/6 (B6) × (NZB × B6)F1 backcross male mice bearing the Yaa mutation, we defined the pathogenic role and genetic basis for Yaa-associated monocytosis. We observed a remarkable correlation of monocytosis with autoantibody production and subsequent development of lethal lupus nephritis, indicating that monocytosis is an additional useful indicator for severe SLE. In addition, we identified an NZB-derived locus on chromosome 1 predisposing to the development of monocytosis, which peaked at Fcgr2b encoding FcγRIIB and directly overlapped with the previously identified NZB autoimmunity 2 (Nba2) locus. The contribution of Nba2 to monocytosis was confirmed by the analysis of Yaa-bearing B6 mice congenic for the NZB-Nba2 locus. Finally, we observed a very low-level expression of FcγRIIB on macrophages bearing the NZB-type Fcgr2b allele, compared with those bearing the B6-type allele, and the development of monocytosis in FcγRIIB haploinsufficient B6 mice carrying the Yaa mutation. These data suggest that the Nba2 locus may play a supplementary role in the pathogenesis of SLE by promoting the development of monocytosis and the activation of effector cells bearing stimulatory FcγR, in addition to its implication in the dysregulated activation of autoreactive B cells.

Background: It has been suggested that defective handling of apoptotic cells by macrophages plays a key role in the development of systemic lupus erythematosus (SLE). The relative contribution of intrinsic defects and serum factors remains controversial. Objective: To compare monocytes from SLE patients, patients with rheumatoid arthritis, and healthy controls for their ability to differentiate in vitro into macrophages and to bind/engulf apoptotic cells. Methods: Peripheral blood derived monocytes from healthy donors or from patients with SLE or rheumatoid arthritis were allowed to differentiate into macrophages. The in vitro uptake of apoptotic cells by macrophages was evaluated by a flow cytometry assay that allowed discrimination between binding and internalisation. Results: Monocytes from SLE and rheumatoid patients showed a striking defect in adherence to plastic compared with healthy donors. Absence or heat inactivation of serum resulted in a reduction in the binding and engulfment of apoptotic cells by macrophages. Macrophages from rheumatoid and SLE patients had similar percentages of apoptotic cells bound to their surface compared with normal controls. However, macrophages from SLE patients showed a significant defect in the internalisation of apoptotic cells compared with those from healthy controls, even in the presence of normal human serum. Conclusions: Monocytes from patients with SLE and rheumatoid arthritis have a similar defect in their capacity to adhere to plastic. However, only macrophages from SLE patients showed an impaired ability to engulf apoptotic cells, which indicates that an intrinsic cellular defect may be responsible for this phenomenon.

OBJECTIVE: Monocytosis is a unique cellular abnormality associated with the Yaa (Y-linked autoimmune acceleration) mutation. The present study was designed to define the cellular mechanism responsible for the development of monocytosis and to characterize the effect of the Yaa mutation on the development of monocyte subsets. METHODS: We produced bone marrow chimeras reconstituted with a mixture of Yaa and non-Yaa bone marrow cells bearing distinct Ly-17 alloantigens, and determined whether monocytes of Yaa origin became dominant. Moreover, we defined the 2 major inflammatory (Gr-1+,CD62 ligand [CD62L]+) and resident (Gr-1-,CD62L-) subsets of blood monocytes in aged BXSB Yaa male mice, as compared with BXSB male mice lacking the Yaa mutation. RESULTS: Analysis of the Ly17 allotype of blood monocytes in chimeric mice revealed that monocytes of both Yaa and non-Yaa origin were similarly involved in monocytosis. Significantly, the development of monocytosis paralleled a selective expansion of the resident monocyte subset compared with the inflammatory subset, and the former expressed CD11c, a marker of dendritic cells. Neither monocytosis nor the change in monocyte subpopulations, including CD11c expression, was observed in Yaa-bearing C57BL/6 mice, in which systemic lupus erythematosus (SLE) fails to develop. CONCLUSION: Our results suggest that Yaa-associated monocytosis is not attributable to an intrinsic abnormality in the growth potential of monocyte lineage cells bearing the Yaa mutation and that the Yaa mutation could lead to the expansion of dendritic cells, thereby contributing to the accelerated development of SLE.

Using a cohort of C57BL/6 (B6) × (NZB × B6)F1 backcross male mice bearing the Yaa (Y-linked autoimmune acceleration) mutation, we mapped and characterized the NZB-derived susceptibility loci predisposing to the development of autoimmune hemolytic anemia (AHA). Our analysis identified 2 major loci on NZB chromosome 7 and chromosome 1 linked with Coombs antierythrocyte autoantibody production, and their contributions were confirmed by the analysis of B6.Yaa mice (B6 mice bearing the Yaa mutation) congenic for each NZB-derived susceptibility interval. A newly identified Aia3 (autoimmune anemia 3) locus present on NZB chromosome 7 selectively regulated Coombs antibody responses, while the second locus, directly overlapping with Nba2 (NZB autoimmunity 2) on chromosome 1, promoted the development of AHA, likely as part of its effect on overall production of lupus autoantibodies. A higher incidence of Coombs antibody production in B6.Aia3 congenic mice (B6 mice bearing the NZB-Aia3 locus) than B6.Nba2 mice (B6 mice bearing the NZB-Nba2 locus) indicated a major role for Aia3 in AHA. Notably, lack of expansion of B1 cells in B6.Aia3 congenic mice argued against the involvement of this subset in AHA. Finally, our analysis of BC mice also demonstrated the presence of a B6-derived H2-linked locus on chromosome 17 that apparently regulated the production of Coombs antibodies as a result of its overall autoimmune promoting effect.

Many forms of glomerulonephritis are triggered by Ab localization in the glomerulus, but the mechanisms by which this induces glomerular inflammation are not fully understood. In this study we investigated the role of complement in a mouse model of cryoglobulin-induced immune complex glomerulonephritis. Several complement-deficient mice on a C57BL/6 and BALB/c genetic background were used and compared with strain-matched, wild-type controls. Cryoglobulinemia was induced by i.p. injection of 6-19 hybridoma cells producing an IgG3 cryoglobulin with rheumatoid factor activity against IgG2a of allotype a present in BALB/c, but not C57BL/6, mice. Thus, the cryoprecipitate in C57BL/6 mice consisted of the IgG3 cryoglobulin only (type I cryoglobulinemia) compared with IgG3-IgG2a complexes in BALB/c (type II cryoglobulinemia). The survival of mice was not affected by complement deficiency. Glomerular influx of neutrophils was significantly less in C3-, factor B-, and C5-deficient mice compared with wild-type and C1q-deficient mice. It did not correlate with C3 deposition, but did correlate with the amount of C6 deposited. Deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration, suggesting that the generation of C5a accounts for the effects observed. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Our results suggest that in this model of cryoglobulin-induced glomerulonephritis the neutrophil influx was mediated by C5 activation with the alternative pathway playing a prominent role in its cleavage. Thus, blocking C5 is a potential therapeutic strategy for preventing renal injury in cryoglobulinemia.

By assessing the development of Y-linked autoimmune acceleration (Yaa) gene-induced systemic lupus erythematosus in C57BL/6 (B6) × (New Zealand Black (NZB) × B6.Yaa)F1 backcross male mice, we mapped three major susceptibility loci derived from the NZB strain. These three quantitative trait loci (QTL) on NZB chromosomes 1, 7, and 13 differentially regulated three different autoimmune traits: anti-nuclear autoantibody production, gp70-anti-gp70 immune complex (gp70 IC) formation, and glomerulonephritis. Contributions to the disease traits were further confirmed by generating and analyzing three different B6.Yaa congenic mice, each carrying one individual NZB QTL. The chromosome 1 locus that overlapped with the previously identified Nba2 (NZB autoimmunity 2) locus regulated all three traits. A newly identified chromosome 7 locus, designated Nba5, selectively promoted anti-gp70 autoantibody production, hence the formation of gp70 IC and glomerulonephritis. B6.Yaa mice bearing the NZB chromosome 13 locus displayed increased serum gp70 production, but not gp70 IC formation and glomerulonephritis. This locus, called Sgp3 (serum gp70 production 3), selectively regulated the production of serum gp70, thereby contributing to the formation of nephritogenic gp70 IC and glomerulonephritis, in combination with Nba2 and Nba5 in NZB mice. Among these three loci, a major role of Nba2 was demonstrated, because B6.Yaa Nba2 congenic male mice developed the most severe disease. Finally, our analysis revealed the presence in B6 mice of an H2-linked QTL, which regulated autoantibody production. This locus had no apparent individual effect, but most likely modulated disease severity through interaction with NZB-derived susceptibility loci.

SUMMARYThe New Zealand Black (NZB) mouse strain is a model of autoimmune haemolytic anaemia (AHA) and systemic lupus erythematosus (SLE), characterized by the production of anti-red blood cell (RBC) antibodies and anti-nuclear antibodies (ANA), respectively. A linkage analysis was carried out in an (NZB × BALB/c) F2 cross in order to identify loci involved in the production of both anti-RBC IgM and IgG antibodies. These regions of linkage were compared with linkage data to ANA from the same cohort and other linkage analyses involving New Zealand mice. Four previously described NZB loci linked to anti-RBC antibodies were confirmed, and eight novel loci linked to this trait were also mapped: five of which were of NZB origin, and three derived from the non-autoimmune BALB/c background. A comparison between loci linked with anti-RBC antibodies and ANA demonstrated many that co-localize, suggesting the presence of genes that result in the general breaking of tolerance to self-antigen. Furthermore, the observation that some loci were associated only with the anti-RBC response suggests an antigen specific mechanism in addition to a general breaking of tolerance. A locus linked with anti-RBC antibodies and ANA on distal chromosome 7 in this cohort is orthologous to one on the q arm of human chromosome 11, a region linked to AHA and ANA in human SLE.

To dissect the individual effects of the four non-MHC, autosomal loci (Bxs1 to Bxs4) that contribute to SLE susceptibility in BXSB mice, we generated congenic lines from chromosome 1 on a C57BL/10.YBXSB (B10.Yaa) background for the intervals (values in megabases (Mb)) Bxs1 (46.3-89.2 Mb), Bxs1/4 (20.0-65.9 Mb), Bxs1/2 (64.4-159.0 Mb), and Bxs2/3 (105.4-189.0 Mb). Glomerulonephritis, qualitatively similar to that seen in the parental BXSB strain, developed in three of these congenic strains. Early onset, severe disease was observed in B10.Yaa.BXSB-Bxs2/3 congenic mice and caused 50% mortality by 12 mo. In B10.Yaa.BXSB-Bxs1/4 mice disease progressed more slowly, resulting in 13% mortality at 12 mo. The progression of renal disease in both of these strains was correlated with the level of anti-dsDNA Abs. B10.Yaa.BXSB-Bxs1 mice, despite their genetic similarity to B10.Yaa.BXSB-Bxs1/4 mice, developed a low-grade glomerulonephritis in the absence of anti-dsDNA Abs. Thus, Bxs4 directed an increase in titer and spectrum of autoantibodies, whereas Bxs1 promoted the development of nephritis. The Bxs2 interval was linked to the production of anti-dsDNA Abs without concomitant glomerulonephritis. In contrast, the Bxs3 interval was sufficient to generate classic lupus nephritis in a nonautoimmune–prone strain. Immune phenotype differed between controls and congenics with a significant increase in B220+ cells in BXSB and B10.Yaa.BXSB-Bxs2/3, and an increase in CD4 to CD8 ratio in both BXSB and B10.Yaa.BXSB-Bxs1/4. Disease in the Bxs3 mice was delayed in comparison to the BXSB parental strain, emphasizing the necessity for multiple interactions in the production of the full BXSB phenotype.

The presence of rheumatoid factors (RF) is a characteristic feature of patients with rheumatoid arthritis, but not systemic lupus erythematosus. In this study, we have explored the role of the anti-apoptotic Bcl-2 protein and the Y-linked autoimmune acceleration (Yaa) mutation in the production of IgG RF in comparison with IgG anti-DNA autoimmune responses. Analysis in C57BL/6 mice, in their F1 hybrids with lupus-prone NZW mice, and in bone marrow chimeras containing mixtures of C57BL/6 bcl-2-transgenic and BXSB non-transgenic cells demonstrated that an enforced Bcl-2 expression in B cells promoted the induction of IgG anti-DNA production in these mice, while significant IgG RF responses were observed only in mice developing high levels of gp70-anti-gp70 immune complexes and lethal glomerulonephritis. Moreover, in contrast to a synergistic interaction between the Yaa mutation and Bcl-2 overexpression on IgG anti-DNA production, the Yaa mutation failed to enhance the production of IgG RF induced in bcl-2-transgenic mice. Our results reveal that defects in the regulation of B cell apoptosis play a critical role in the production of IgG RF, and that the Yaa mutation differentially modulates RF and anti-DNA autoimmune responses, likely related to the nature of autoantigens involved in each autoimmune response.

Autoantibodies of the cryoprecipitating IgG3 isotype have been shown to play a significant role in the development of murine lupus–like autoimmune syndrome. At present, the structural basis of IgG3 cryoprecipitation and its role in autoantibody pathogenicity remain to be defined. Using molecular variants of an IgG3 monoclonal rheumatoid factor, 6-19, derived from an autoimmune MRL-Faslpr mouse, we have investigated the implication of charged residues in the heavy-chain variable (VH) region, potential CH3-linked oligosaccharides, and galactosylation of CH2-linked oligosaccharides in its cryoglobulin activity. The cryoglobulin activity of the IgG3 6-19 mutant bearing more negatively charged residues at VH 6 and 23 was found to be reduced but still highly significant, whereas that of the mutant lacking a potential CH3 glycosylation site remained unchanged. In marked contrast, IgG3 6-19 variants obtained from 6-19 heavy-chain transgenic mice displayed barely detectable cryoglobulin activity associated with an increased level of galactosylation in the CH2 oligosaccharide side chains. Thus, our data strongly suggest that the cryoglobulin activity of IgG3 6-19 autoantibody is critically determined by levels of galactosylation in the CH2 oligosaccharide side chains, whereas VH residues play a secondary role in 6-19 IgG3 cryoglobulin activity.

Mice implanted with hybridoma secreting 6-19 IgG3 anti-IgG2a rheumatoid factor (RF) with cryoglobulin activity develop acute glomerulonephritis and cutaneous leukocytoclastic vasculitis. As the RF activity is implicated in the skin, but not glomerular lesions, it is still unclear whether the renal pathogenicity is determined by 6-19 H chains alone or their combination with L chains. To address this question, we have generated transgenic mice expressing only the H chain gene or both H and L chain genes of the 6-19 IgG3 anti-IgG2a RF and determined the development of glomerular and vascular lesions. H-single and H/L-double transgenic mice displayed comparable high amounts of IgG3 cryoglobulins, but only H/L-double transgenic mice having 10-fold higher levels of IgG3 anti-IgG2a RF progressively developed chronic, lethal glomerulonephritis. The severe glomerular lesions observed at 8–10 mo of age were very heterogeneous (membranoproliferative changes, crescents, and sclerosis); in addition, one-third of them had necrotizing arteritis in the kidneys and skeletal muscles. These renal and vascular changes were very different from those observed in the acute cryoglobulinemia, characterized by mainly “wire-loop” glomerular lesions and a cutaneous leukocytoclastic form of vasculitis. Thus, our data demonstrate the importance of a unique combination of the H and L chains for the expression of the pathogenic activity of IgG3 cryoglobulins and that a single autoantibody is able to induce different types of glomerular and vascular complications, depending on its production levels and kinetics.

We investigated the impact of changes occurring during red blood cell (RBC) ageing on the RBC-binding activity of pathogenic anti-erythrocyte monoclonal antibodies derived from autoimmune-prone New Zealand black (NZB) mice. As assessed by flow cytometric analysis on in vivo biotinylated RBCs, all five NZB-derived anti-RBC mAb exhibited more efficient binding to aged RBCs than to young RBCs, and resulted in a selective elimination of more aged RBCs from the circulating blood. In addition, treatment of RBCs with proteases markedly enhanced the binding of all five anti-RBC mAb, raising the possibility that increased exposure of autoimmune epitopes on aged RBCs may be in part, a result of contacts with proteolytic enzymes during the lifetime of circulating RBCs. In marked contrast, the binding activity of mAb raised in non-autoimmune animals against antigens expressed on RBCs, such as CD44, CD47, CD147 and TER-119, was either decreased or unchanged with RBC ageing, and these epitopes, except for that recognized by anti-CD47 mAb, were highly sensitive to mild treatment with proteases. Our data unravel the unique molecular feature of RBC epitopes involved in autoimmune haemolytic anaemia, suggesting that membrane alterations in aged RBCs might play a significant role in the development of the autoantibody response to RBCs.

Using three different Fcgamma receptor (FcgammaR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcgammaR, FcgammaRI, and FcgammaRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcgammaRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcgammaRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcgammaRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, approximately 20-100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcgammaRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcgammaRIII was revealed by the use of two different IgG2a anti-red blood cell autoantibodies, which displayed a striking preferential utilization of FcgammaRIII, compared with the high-affinity FcgammaRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcgammaRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity.