Koji Kitagori

Serum BAFF and APRIL levels in patients with IgG4-related disease and their clinical significance.

IntroductionB cell-activating factor of the tumor necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) play a crucial role in B cell development, survival, and antibody production. Here we analyzed the serum levels of BAFF and APRIL and their respective clinical associations in patients with an immunoglobulin (Ig) G4-related disease (IgG4-RD).MethodsWe measured serum levels of BAFF and APRIL in patients with IgG4-RD, primary Sjögrens syndrome (pSS), and healthy individuals. Serum BAFF and APRIL levels in IgG4-RD were assessed for correlations with serological parameters, including Ig, particularly IgG4, and the number of affected organs. Serum BAFF and APRIL levels in IgG4-RD were monitored during glucocorticoid (GC) therapy.ResultsSerum BAFF and APRIL levels in patients with IgG4-RD were significantly higher (P < 0.01) than in healthy individuals. The BAFF levels of patients with IgG4-RD were comparable to those of patients with pSS. Although clinical parameters, such as serum IgG4 and the number of affected organs, were not correlated with the levels of BAFF, serum APRIL levels were inversely correlated with serum IgG4 levels (r = -0.626, P < 0.05). While serum BAFF levels decreased following GC therapy, serum APRIL levels increased during follow-up.ConclusionThese results indicate that BAFF and APRIL might be useful markers for predicting disease activity in IgG4-RD. Further studies are needed to elucidate the role of BAFF and APRIL in the pathogenesis of IgG4-RD.

Cleaved Form of Osteopontin in Urine as a Clinical Marker of Lupus Nephritis

We assessed the utility of two forms of osteopontin (OPN), OPN full and its cleaved form (OPN N-half), in plasma and urine as markers of disease activity in lupus nephritis (LN). Samples were collected from patients with systemic lupus erythematosus (SLE) (LN: N = 29, non-LN: N = 27), IgA nephropathy (IgAN) (N = 14), minimal change nephrotic syndrome (MCNS) (N = 5), diabetic nephropathy (DN) (N = 14) and healthy volunteers (HC) (N = 17). While there was no significant difference in urine OPN full concentration between groups, urine OPN N-half concentration was significantly higher in patients with LN than HC (p < 0.05). Moreover, urine OPN N-half was higher in LN patients with overt proteinuria (urine protein/creatinine ratio: P/C > 0.5) than LN patients with minimal proteinuria (P/C < 0.5, p < 0.0001), and also higher than in DN patients with overt proteinuria (P/C > 0.5, p < 0.01). Urine thrombin activity correlated with urine OPN N-half concentration (p < 0.0001), but not with urine OPN full concentration. These results suggest that urine OPN N-half concentration reflects renal inflammation. Thus, urine OPN N-half may be a novel disease activity marker for LN.

A novel susceptibility locus in the IL12B region is associated with the pathophysiology of Takayasu arteritis through IL-12p40 and IL-12p70 production

BackgroundA previous study revealed the association between susceptibility to Takayasu arteritis (TAK) and a single nucleotide polymorphism (SNP) rs6871626 located in IL12B, which encodes interleukin (IL)-12p40, a common component of IL-12p70 and IL-23. We investigated the expression of these cytokines in patients with TAK, stratifying them into those with or without the risk allele at the rs6871626 SNP.MethodsPlasma levels of IL-12p40, IL-12p70, and IL-23 were quantified in 44 patients with TAK and 19 healthy controls (HCs) by enzyme-linked immunosorbent assays. Monocytes were obtained from 20 patients with TAK and 14 HCs, treated with interferon-γ (IFN-γ) and lipopolysaccharide, and then supernatant cytokines were quantified. In addition, the ratio of IFN-γ+ or IL-17A+ cells to CD4+ T cells was measured by flow cytometric analysis of peripheral blood mononuclear cells.ResultsThe levels of plasma IL-12p40, plasma IL-12p70, and supernatant IL-12p70 were significantly higher in patients with TAK than in HCs, whereas there were no significant differences in the levels of plasma IL-23, supernatant IL-23, or supernatant IL-12p40. The levels of plasma IL-12p70, supernatant IL-12p40, and supernatant IL-12p70 were significantly higher in patients with the risk allele than in those without. The ratio of CD4+IFN-γ+ cells was significantly higher in patients with the risk allele, whereas CD4+IL-17A+ cells showed no differences.ConclusionsThe rs6871626 SNP in IL12B may influence the increased expression of IL-12p40 and IL-12p70. These enhanced cytokines might play roles in the pathophysiology of TAK.

High Expression of Galectin-3 in Patients with IgG4-Related Disease: A Proteomic Approach

Objectives Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan condition manifesting itself in different forms. This study aimed to investigate protein expression profiles and to find the possible biomarker for IgG4-RD by liquid chromatography mass spectrometry (LC-MS) using tissue sections in IgG4-RD patients. Methods Protein expression profiles in five IgG4-related pancreatitis and three normal pancreatic samples were compared using LC-MS and were validated by quantitative real-time PCR (qRT-PCR), immunoblotting, and immunohistochemistry. ELISA was employed in the serum of 20 patients with systemic IgG4-RD before and during steroid treatment. Results LC-MS indicated that the levels of 17 proteins were significantly higher and 12 others were significantly lower in IgG4-related pancreatitis patients compared to controls. Among these proteins, galectin-3 levels were 13-fold higher in IgG4-related pancreatitis (P < 0.01). These results were confirmed by immunoblotting and qRT-PCR. The average number of galectin-3 + cells in various organs of IgG4-RD patients, including salivary glands, lungs, and lymph nodes, was higher than in controls. Galectin-3 was detectable in macrophages, dendritic cells, and stromal myofibroblast-like cells, but not in lymphocytes by immunofluorescence staining. Serum galectin-3 levels were higher in patients with IgG4-RD compared with healthy donors and remained high during steroid therapy. Conclusion Galectin-3 was overexpressed in IgG4-RD and the levels were indirectly related to clinical activity.

Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients

Transgelin-2 (TAGLN2) is an actin-binding protein that controls actin stability and promotes T cell activation. TAGLN2 is also expressed on B-cells but its function in B-cells is unknown. We found that TAGLN2-expressing B-cells were localized in the germinal center (GC) of secondary lymphoid tissues and TAGLN2 mRNA was significantly upregulated after IgM+IgG stimulation in primary human B-cells, suggesting that TAGLN2 was upregulated upon B-cell activation. In support of this, lymph nodes (LNs) from patients with systemic lupus erythematosus (SLE), in which the intense GC activity have been recognized, showed increased TAGLN2 expression in B-cells compared to control LNs. Moreover, TAGLN2+B-cells were distributed widely not only in the GC but also in the perifollicular areas in SLE LNs. In contrast, CD19+ B-cells and CD19+CD27+ memory-B cells in peripheral blood of SLE patients showed no increase in TAGLN2 mRNA. Two-photon excitation microscopy of Raji cells demonstrated that TAGLN2 colocalized with F-actin and moved together to the periphery upon stimulation. TAGLN2-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of TAGLN2-knockdown Raji cells showed decreased expression of the genes associated with immune function including CCR6 and as well as of those associated with regulation of the actin cytoskeleton including ABI2, compared to controls. These results suggest that TAGLN2 might regulate activation and migration of B-cells, in particular, the entry of activated B-cells into the follicle. We also suggest that TAGLN2 could be used as a marker for activated B-cells.

Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL‐6 and IL‐8

BEN domain‐containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3+ T cells). BEND3+ T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4+ and CD8+ T cells, and were also observed in cord blood. The stimulation of BEND3+ T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL‐6 and IL‐8 produced by BEND3+ T cells were higher than those by BEND3− T cells. The proportion of BEND3+ T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3+ T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3+ T cells may be of importance and useful in understanding human T cell immunology.

Osteopontin in systemic lupus erythematosus

In systemic lupus erythematosus (SLE), lupus nephritis (LN) is an important complication as an intractable condition and considered to be a prognostic factor. Based on the past reports that explain immunological functions of OPN and its relationship with autoimmune diseases such as LN or IgA nephropathy, we measured OPN full and OPN N-half in serum and urine of SLE patients and examined the possibility as a disease biomarker. OPN N-half is known as a more physiologically active form than OPN full. As a result, OPN N-half in urine was higher in LN than in healthy control, and also higher than in diabetic nephropathy that is a non-inflammatory nephropathy. In addition, OPN N-half in urine decreased after the treatment of LN, suggesting that OPN N-half in urine could be a biomarker for evaluating disease activity of LN.

OP0056 Association of IL-12P40 and IL-12P70 with Pathophysiology of Takayasu Arteritis

Background We preliminarily found a SNP (rs6871626, A vs. C) in IL-12B region as a susceptibility gene to Takayasu arteritis (TAK) by genome-wide association study. IL-12B encodes IL-12p40 that is a component of both IL-12p70 and IL-23. The significance of IL-12p40 in pathophysiology of TAK has not been fully investigated yet. Moreover, there has been no consistent evidence about whether IL-12p70 or IL-23 is important in TAK. Objectives 1) To investigate the expression of IL-12p40 in patients with TAK, 2) reveal which is more important to pathophysiology of TAK, IL-12p70 or IL-23, and 3) examine the influence of SNP to the cytokine production. Methods We collected sera from 43 TAK patients and 19 healthy controls (HCs), and measured IL-12p40, IL-12p70 and IL-23 with ELISA. Next, we collected whole blood from 18 TAK patients and 12 HCs, and isolated monocytes with RosetteSep™. The monocytes were incubated with IL-6, IL-10 and GM-CSF, and then stimulated with INF-γ and LPS. Then we measured the cytokines in culture supernatant with ELISA. Results IL-12p40 and IL-12p70 in sera of TAK patients were significantly higher than those of HCs (p=0.031 and p<0.001, respectively), whereas there were no differences in serum of IL-23 levels between TAK patients and HCs. IL-12p40 and IL-12p70 in sera of TAK patients whose allele at rs6871626 is AA or AC (defined as risk patients) were significantly higher than those of HCs (p=0.018 and p<0.001, respectively), whereas there were no differences in serum IL-23 levels among risk patients, TAK patients with CC allele (defined as non-risk patients) and HCs. Supernatant IL-12p40 and IL-12p70 levels from monocytes of risk patients were significantly higher than those of non-risk patients (p=0.044 and p=0.032, respectively) or those of HCs (p=0.032 and p=0.0015, respectively). Supernatant IL-23 from monocytes of risk patients were significantly higher than those of non-risk patients (p=0.04). Conclusions IL-12p40 and IL-12p70 but not IL-23 may play an important role in pathophysiology of TAK, and the risk allele of rs6871626 could contribute to production of these cytokines. Disclosure of Interest None declared