Autoantibody Immune Complexes Research Articles

B-360 Usefulness of the Ratios of CYFRA21-1 and p53 Autoantibody Immune Complexes to Their Free Antigens in Lung Cancer

Abstract Background Autoantibodies against specific cancer-associated antigens are produced in the early stages of tumorigenesis. In lung cancer, the possibility of autoantibodies as diagnostic biomarkers has been proposed. This study was to evaluate the usefulness of novel DNA-guided sandwich immunoassay detecting CYFRA21-1 and p53 autoantibody immune complexes and their free antigens in diagnosis of lung cancer. Methods In total, 100 patients with lung cancer and 119 healthy controls were analyzed with 9G testTM Lung/Cancer (Biometrix Technology, South Korea) measuring plasma autoantibody immune complexes (CYFRA21-1 with anti-CYFRA21-1 autoantibody immune complex (CIC), p53 with anti-p53 autoantibody immune complex (PIC)) and their free antigens (CYFRA 21-1 and p53). The ratios of CIC to CYFRA21-1 (CIC/CYFRA21-1) and PIC to p53 (PIC/p53), and the multiplication of the two ratios, named as LC index were calculated. Results The levels of CIC and PIC were significantly increased in lung cancer compared with those in healthy control (P = 0.0062 and P = 0.0026, respectively), while free antigens didn’t distinguish between lung cancer and healthy control. The ratios CIC/CYFRA21-1 and PIC/p53 were significantly higher in lung cancer than those in healthy control (all, P < 0.0001). Diagnostic performances expressed as area under the curve (AUC) were higher in the ratios of autoantibody immune complexes to each free antigen (PIC/p53 and CIC/CYFRA21-1) than autoantibody immune complexes (PIC and CIC) (AUC; 0.887, 0.847 vs 0.618, 0.608, respectively). The most excellent diagnostic performance was proven with LC index at diagnostic cut-off 3.60, which showed 81% sensitivity and 95% specificity with 0.945 AUC (P < 0.001). It was more sensitive to early lung cancer than to advanced lung cancer (sensitivity 87.5% vs 72.7%). Conclusion The ratios of CIC to CYFRA21-1 and PIC to p53 are useful biomarkers in lung cancer diagnosis and the combination of the ratios, LC index could be complementary tool for lung cancer screening with radiological tests.

Ras family signaling pathway in immunopathogenesis of inflammatory rheumatic diseases.

The Ras (rat sarcoma virus) is a GTP-binding protein that is considered one of the important members of the Ras-GTPase superfamily. The Ras involves several pathways in the cell that include proliferation, migration, survival, differentiation, and fibrosis. Abnormalities in the expression level and activation of the Ras family signaling pathway and its downstream kinases such as Raf/MEK/ERK1-2 contribute to the pathogenic mechanisms of rheumatic diseases including immune system dysregulation, inflammation, and fibrosis in systemic sclerosis (SSc); destruction and inflammation of synovial tissue in rheumatoid arthritis (RA); and autoantibody production and immune complexes formation in systemic lupus erythematosus (SLE); and enhance osteoblast differentiation and ossification during skeletal formation in ankylosing spondylitis (AS). In this review, the basic biology, signaling of Ras, and abnormalities in this pathway in rheumatic diseases including SSc, RA, AS, and SLE will be discussed.

21746 Antigen discovery in membranous glomerulopathy using laser capture microdissection and mass spectrometry

ABSTRACT IMPACT: Identifying the causative antigen in membranous glomerulopathy cohorts enables the development of serum assays to detect and monitor disease progression without the need for invasive kidney biopsies. OBJECTIVES/GOALS: Primary membranous glomerulopathy is caused by the formation of autoantibody immune complexes which deposit in the glomerulus and obstruct kidney function. Causative antigens remain to be identified in roughly 20% of cases. Our goal is to identify the antigen in these cohorts, so that non-invasive assays can be developed for disease monitoring. METHODS/STUDY POPULATION: Renal biopsy tissue from known antigen cases (PLA2R, THSD7A), and unknown cases were included in the analysis. Renal biopsy tissue from formalin fixed paraffin embedded tissue was cut at a thickness of 10 µm onto Leica PET-membrane frame slides. These slides were then stained with hematoxylin. The glomeruli were microdissected into microcentrifuge tubes using a Leica DM6000B microscope. The microdissected glomeruli were lysed in 2% SDS and 0.1M DTT at 99 degrees Celsius for 1 hour and processed by filter assisted sample preparation (FASP). Digested peptides were analyzed by liquid chromatography-mass spectrometry using an Orbitrap Fusion Lumos using data-dependent acquisition. RESULTS/ANTICIPATED RESULTS: Mass spectrometry data collected from the laser captured glomeruli was searched against the human proteome fasta database from Uniprot using MaxQuant. IBAQ values were used for quantitation and statistical analysis. Null hypothesis significance testing was performed for each protein by comparing each sample group to the rest of the samples in the data set. In the control groups, the causative antigens PLA2R and THSD7A were detected and quantified with the largest magnitude fold change in their respective category, validating the experimental design. Using this approach, the proteins SAP, NELL1, and NCAM1 were identified and subsequently validated as causative antigens in distinct patient cohorts. DISCUSSION/SIGNIFICANCE OF FINDINGS: Here, we share the results of our efforts to comprehensively identify the spectrum of causative antigens in membranous glomerulopathy. In this context, antigen discovery is an essential first step for the development of non-invasive assays to inform prognosis, monitor response to treatment, and better understand disease etiology.

Limited Effect of Indolamine 2,3-Dioxygenase Expression and Enzymatic Activity on Lupus-Like Disease in B6.Nba2 Mice.

B6.Nba2 mice spontaneously develop a lupus-like disease characterized by elevated levels of serum anti-nuclear autoantibody (ANA) immune complexes and constitutive type I interferon (IFNα) production. During disease progression, both plasmacytoid dendritic cells (pDCs) and antibody secreting plasma cells accumulate in spleens of B6.Nba2 mice. Indoleamine 2,3-dioxygenase (IDO) has been suggested to play a role in several autoimmune diseases including in the MRL/lpr model of mouse lupus-like disease; however, it remains unknown if IDO is involved in disease development and/or progression in other spontaneous models. We show here that IDO1 protein and total IDO enzymatic activity are significantly elevated in lupus-prone B6.Nba2 mice relative to B6 controls. IDO1 expression was restricted to PCs and SignR1+ macrophages in both strains, while significantly increased in B6.Nba2-derived SiglecH+ (SigH+) pDCs. Despite this unique expression pattern, neither pharmacologic inhibition of total IDO nor IDO1 gene ablation altered serum autoantibody levels, splenic immune cell activation pattern, or renal inflammation in B6.Nba2 mice. Interestingly, IDO pharmacologic inhibition, but not IDO1 deficiency, resulted in diminished complement factor C'3 fixation to kidney glomeruli, suggesting a possible therapeutic benefit of IDO inhibition in SLE patients with renal involvement.

Autoreactive helper T cells alleviate the need for intrinsic TLR signaling in autoreactive B cell activation.

T cells play a significant role in the pathogenesis of systemic autoimmune diseases, including systemic lupus erythematosus; however, there is relatively little information on the nature and specificity of autoreactive T cells. Identifying such cells has been technically difficult because they are likely to be rare and low affinity. Here, we report a method for identifying autoreactive T cell clones that recognize proteins contained in autoantibody immune complexes, providing direct evidence that functional autoreactive helper T cells exist in the periphery of normal mice. These T cells significantly enhanced autoreactive B cell proliferation and altered B cell differentiation in vivo. Most importantly, these autoreactive T cells were able to rescue many aspects of the TLR-deficient AM14 (anti-IgG2a rheumatoid factor) B cell response, suggesting that TLR requirements can be bypassed. This result has implications for the efficacy of TLR-targeted therapy in the treatment of ongoing disease.

Fc-gamma receptors: Attractive targets for autoimmune drug discovery searching for intelligent therapeutic designs.

Autoantibody immune complexes (ICs) mediate pathogenesis in multiple autoimmune diseases via direct interference with target function, complement fixation, and interaction with Fc-gamma receptors (FcγRs). Through high avidity interactions, ICs are able to crosslink low affinity FcγRs expressed on a wide variety of effector cells, leading to secretion of pro-inflammatory mediators and inducing cytotoxicity, ultimately resulting in tissue injury. Given their relevance in numerous autoimmune diseases, FcγRs have been considered as attractive therapeutic targets for the last three decades. However, a limited number of investigational drug candidates have been developed targeting FcγRs and only a few approved therapeutics have been associated with impacting FcγRs. This review provides a historical overview of the different therapeutic approaches used to target FcγRs for the treatment of autoimmune and inflammatory diseases.

C‐Reactive Protein Inhibits Plasmacytoid Dendritic Cell Interferon Responses to Autoantibody Immune Complexes

C-reactive protein (CRP) is a serum pattern recognition molecule that binds to apoptotic cells and nucleoprotein autoantigens and Fcγ receptors (FcγR). In systemic lupus erythematosus (SLE), immune complexes (ICs) containing nucleoprotein autoantigens activate plasmacytoid dendritic cells (PDCs) to produce type I interferon (IFN), which contributes to disease pathogenesis. Autoantibody ICs are taken up by PDCs through FcγR type IIa into endosomes, where the nucleic acid components activate Toll-like receptor 7 (TLR-7) or TLR-9. The objective of this study was to investigate the effect of CRP on PDC and monocyte responses to nucleoprotein autoantigens and ICs. Peripheral blood mononuclear cells (PBMCs), purified monocytes, and PDCs were isolated from healthy volunteers and stimulated with autoantibody ICs containing apoptotic cells, small nuclear RNPs (snRNPs), or DNA, or directly with TLR-7 and TLR-9 agonists. Supernatants were analyzed for IFNα and cytokine levels by enzyme-linked immunosorbent assay and multiplex assay. Small nuclear RNPs were fluorescence-labeled, and the effect of CRP on binding, uptake, and intracellular localization of autoantibody snRNP complexes was measured by flow cytometry and confocal microscopy. CRP bound to autoantigen did not induce IFNα in PBMCs or PDCs, whereas complexes formed with autoantibody did. Significantly, CRP inhibited the IFNα response to both anti-U1 RNP-snRNP complexes and anti-DNA-DNA complexes, but not to other TLR-7 and TLR-9 agonists. CRP directly inhibited PDC IFNα release, promoted PDC differentiation, and increased late endosome localization of autoantigen in PDCs and monocytes. CRP is a regulator of the type I IFN response to SLE ICs. CRP increased the intracellular processing of ICs in late endosomes, which is associated with decreased synthesis of type I IFN after intracellular TLR activation.

Inflammatory monocytes and Fcγ receptor IV on osteoclasts are critical for bone destruction during inflammatory arthritis in mice

Destruction of bone tissue by osteoclasts represents a severe pathological phenotype during inflammatory arthritis and results in joint pain and bone malformations. Previous studies have established the essential role of cytokines including TNFα and receptor-ligand interactions, such as the receptor activator of nuclear factor-kappa B-receptor activator of nuclear factor-kappa B ligand interaction for osteoclast formation during joint inflammation. Moreover, autoantibodies contribute to joint inflammation in inflammatory arthritis by triggering cellular fragment crystallizable (Fc)γ receptors (FcγR), resulting in the release of proinflammatory cytokines and chemokines essential for recruitment and activation of innate immune effector cells. In contrast, little is known about the expression pattern and function of different FcγRs during osteoclast differentiation. This would allow osteoclasts to directly interact with autoantibody immune complexes, rather than being influenced indirectly via proinflammatory cytokines released upon immune complex binding to other FcγR-expressing innate immune cells. To address this question, we studied FcγR expression and function on osteoclasts during the steady state and during acute joint inflammation in a model of inflammatory arthritis. Our results suggest that osteoclastogenesis is directly influenced by IgG autoantibody binding to select activating FcγRs on immature osteoclasts, resulting in enhanced osteoclast generation and, ultimately, bone destruction.

Natural antibody to apoptotic cell membranes inhibits the proinflammatory properties of lupus autoantibody immune complexes

Naturally arising IgM antibodies (NAb) to apoptotic cell (AC) determinants are present from birth and can be further induced by AC challenge. In systemic lupus erythematosus, lower anti-AC NAb levels have been associated with higher disease activity. We have recently shown that a prototypical AC-specific IgM NAb can suppress proinflammatory responses to purified agonists of Toll-like receptors and block the in vivo induction of IgG immune complex (IC)-induced arthritis. Nuclear antigens, which activate dendritic cells (DCs), form complexes with IgG autoantibody, and these have been implicated in the pathogenesis of autoimmune disease. In this study, we sought to investigate potential roles of such NAb for regulating IC-mediated activation of DCs, which is believed to be involved in disease initiation and perpetuation. Bone marrow-derived myeloid DCs were stimulated with ICs composed of IgG autoantibody and chromatin or IgG autoantibody and RNA. Outcome was evaluated according to the production of inflammatory cytokines, as determined by enzyme-linked immunosorbent assay, and the expression of costimulatory molecules (markers of DC activation), as determined by flow cytometry. MAPK activation was evaluated by phospho-flow analysis and immunofluorescence microscopy. IgM anti-AC NAb dose-dependently suppressed the production of DNA IC- and RNA IC-induced interleukin-6 and DNA IC-induced tumor necrosis factor α, as well as the RNA IC-induced up-regulation of CD86 and CD40 on DCs. IgM NAb-mediated inhibition was associated with suppression of IC-mediated p38 MAPK activation and nuclear localization. We demonstrated a direct in vitro inhibitory effect of IgM NAb on inflammatory responses induced by IgG-nucleic acid ICs. These findings contribute to emerging evidence that regulatory NAb to AC determinants may oppose the influence of pathogenic lupus autoantibody ICs and thereby play roles in the maintenance of immune homeostasis.

CXCR3 promotes the production of IgG1 autoantibodies but is not essential for the development of lupus nephritis in NZB/NZW mice

Autoantibody immune complexes and cellular infiltrates drive nephritis in patients with systemic lupus erythematosus (SLE) and in murine lupus. The chemokine receptor CXCR3 is assumed to promote cellular infiltration of inflamed tissues. Moreover, CXCR3 deficiency ameliorates lupus nephritis in the MRL/MpJ-Fas(lpr) (MRL/lpr) mouse model of SLE. Hence, CXCR3 blockade has been suggested as a novel therapeutic strategy for the treatment of lupus nephritis. We undertook this study to test the effect of CXCR3 in the (NZB × NZW)F(1) (NZB/NZW) mouse model of SLE. CXCR3(-/-) NZB/NZW mice were generated and monitored for survival, proteinuria, and kidney infiltration. Anti-double-stranded DNA (anti-dsDNA) and total IgG1, IgG2a, and IgG2b antibody levels were determined by enzyme-linked immunosorbent assay. T cell and plasma cell infiltrates in the kidneys and interferon-γ production were determined by flow cytometry. Plasma cell infiltrates were measured using enzyme-linked immunospot assay. Kidney tissue was evaluated for pathologic changes. CXCR3(-/-) NZB/NZW mice exhibited reduced production of total and anti-dsDNA antibodies of the IgG1 subclass, but had normal titers of IgG2a and IgG2b antibodies compared to CXCR3(+/+) NZB/NZW mice. Cellular infiltrates and glomerulonephritis were not reduced in CXCR3(-/-) mice. CXCR3 has an effect on (auto)antibody production but is not essential for lupus pathogenesis in NZB/NZW mice, indicating that the effect of CXCR3 on the development of kidney disease varies between MRL/lpr and NZB/NZW mice. These results suggest that CXCR3-dependent and -independent mechanisms can mediate lupus nephritis. Hence, therapeutic CXCR3 blockade could be beneficial for only a subgroup of patients with SLE.

Foxp3+ Regulatory T Cells Control Humoral Autoimmunity by Suppressing the Development of Long-Lived Plasma Cells

Foxp3(+) regulatory T cells (Tregs) are crucial for maintaining T cell tolerance, but their role in humoral autoimmunity remains unclear. To address this, we combined a model of autoantibody-dependent arthritis (K/BxN) with Foxp3 mutant scurfy mice to generate Treg-deficient K/BxN mice, referred to as K/BxNsf mice. The disease symptoms of K/BxNsf mice were exacerbated, and this coincided with increases in extrafollicular Th cells, follicular Th cells, and germinal centers. Surprisingly, the K/BxNsf mice exhibited an abnormal accumulation of mature plasma cells in their spleens and a corresponding loss of bone marrow plasma cells. The plasma cells were unresponsive to the bone marrow homing chemokine CXCL12, despite normal expression of the chemokine receptor CXCR4. Importantly, they were long-lived and less susceptible to the cytotoxic action of cyclophosphamide. They also expressed less FcγRIIb and were less apoptotic in response to autoantigen-autoantibody immune complexes. This suggests that Tregs control plasma cell susceptibility to cell death induced by engagement of FcγRIIb with immune complexes. Direct cytotoxic effects of Tregs also contribute to the death of plasma cells. Thus, our results reveal that Tregs suppress the emergence of long-lived splenic plasma cells by affecting plasma cell-autonomous mechanisms as well as T cell help, thereby avoiding the persistence of humoral autoimmunity.

Neutrophil surface-bound immunoglobulin-a feature of Felty's syndrome?

In a study of eight patients with Felty's syndrome, surface-bound immunoglobulin (IgG +/- IgM and complement) was demonstrated in all cases by a fluorescein-labelled antihuman globulin technique using paraformaldehyde-fixed neutrophils to prevent non-specific surface adsorption of immunoglobulin. The test was negative in control patients with rheumatoid arthritis alone. The neutrophil binding of immunoglobulin occurred in vivo and could not be reproduced with the patient's serum and normal neutrophils. The presence of alloantibodies due to previous pregnancy or transfusion can obscure this picture. A neutrophil autoantibody or cell-bound immune complexes may be the cause of this phenomenon, which may be a useful marker of Felty's syndrome in patients with rheumatoid arthritis.

Neutrophil surface-bound immunoglobulin-a feature of Felty's syndrome?

Summary In a study of eight patients with Felty's syndrome, surface-bound immunoglobulin (IgG ± IgM and complement) was demonstrated in all cases by a fluorescein-labelled antihuman globulin technique using paraformaldehyde-fixed neutrophils to prevent non-specific surface adsorption of immunoglobulin. The test was negative in control patients with rheumatoid arthritis alone. The neutrophil binding of immunoglobulin occurred in vivo and could not be reproduced with the patient's serum and normal neutrophils. The presence of alloantibodies due to previous pregnancy or transfusion can obscure this picture. A neutrophil autoantibody or cell-bound immune complexes may be the cause of this phenomenon, which may be a useful marker of Felty's syndrome in patients with rheumatoid arthritis.

Autoimmunity and Inflammation Due to a Gain-of-Function Mutation in Phospholipase Cγ2 that Specifically Increases External Ca 2+ Entry

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.