Cutoff Values of Serum IgG4 and Histopathological IgG4+ Plasma Cells for Diagnosis of Patients with IgG4-Related Disease

IgG4-related disease is a new disease classification established in Japan in the 21st century. Patients with IgG4-related disease display hyper-IgG4-gammaglobulinemia, massive infiltration of IgG4+ plasma cells into tissue, and good response to glucocorticoids. Since IgG4 overexpression is also observed in other disorders, it is necessary to diagnose IgG4-related disease carefully and correctly. We therefore sought to determine cutoff values for serum IgG4 and IgG4/IgG and for IgG4+/IgG+ plasma cells in tissue diagnostic of IgG4-related disease. Patients and Methods. We retrospectively analyzed serum IgG4 concentrations and IgG4/IgG ratio and IgG4+/IgG+ plasma cell ratio in tissues of 132 patients with IgG4-related disease and 48 patients with other disorders. Result. Serum IgG4 >135  mg/dl demonstrated a sensitivity of 97.0% and a specificity of 79.6% in diagnosing IgG4-related disease, and serum IgG4/IgG ratios >8% had a sensitivity and specificity of 95.5% and 87.5%, respectively. IgG4+cell/IgG+ cell ratio in tissues >40% had a sensitivity and specificity of 94.4% and 85.7%, respectively. However, the number of IgG4+ cells was reduced in severely fibrotic parts of tissues. Conclusion. Although a recent unanimous consensus of all relevant researchers in Japan recently established the diagnostic criteria for IgG4-related disease, findings such as ours indicate that further discussion is needed

Skewed production of IL-6 and TGFβ by cultured salivary gland epithelial cells from patients with Sjögren's syndrome.

OBJECTIVE: To determine the cytokine production profile of cultured salivary gland epithelial (SGE) cells obtained from patients with Sjögren's syndrome (SS). METHODS: SGE cells obtained from 9 SS patients and 6 normal controls were cultured in the presence of exogenous IFNγ. Cell proliferation and apoptosis in response to IFNγ were determined by WST1 assay and by FACS analysis. The concentrations of IL-6 and TGFβ secreted into culture supernatants were analyzed by ELISA. RESULTS: IFNγ did not significantly affect the proliferation or apoptosis of SGE cells. However, IL-6 concentrations were higher, and TGFβ concentrations were lower, in culture supernatants of SGE cells from SS patients than from normal controls. CONCLUSION: Cytokine production by SGE cells from SS patients showed a skewed balance compared with normal controls, with increased IL-6 and decreased TGFβ secretion. This imbalance may be critical in the regulation of Treg/Th17 cells and may foster a pathogenic milieu that may be causative and predictive in SS

Profile of patients included in the study.

<p>Nine patients (all women; mean age, 48±14 years) met both the 2002 American-European consensus group (AECG) criteria and the SICCA criteria for Sjögren's syndrome (SS), whereas the other six (all women; mean age, 57±8 years) did not (No).</p>*<p>Titers of anti-nuclear antibody (ANA).</p

Quantification of cytokines secreted into the culture supernatants of SGE cells.

<p>Confluent SGE cells obtained from 9 SS patients and 6 normal controls (non-SS) were incubated in the presence of IFNγ (1000 U/ml), and the culture supernatants were collected on days 0, 2, 4, and 6. Median concentrations (pg/ml) of IL-6 (A) and TGFβ (B) in the supernatants were determined by ELISA and compared by non-parametric Mann-Whitney tests (*, p<0.05).</p

(A) Expression of mRNA in salivary gland epithelial cells.

<p>Salivary gland epithelial cells were isolated from SS patients and cultured. Total RNA was isolated from these cells, and EGF-R, α amylase-1, and CD3δ mRNAs were assayed by RT-PCR, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045689#s2" target="_blank">Materials and Methods</a> section. Lane 1: salivary gland epithelial cells, Lane 2: labial salivary gland of the same patient, Lane 3: normal lymph node as a control for CD3δ. <b>(B and C) Effects of IFNγ on human SGE cells.</b> SGE cells were incubated with various concentration of IFNγ for 48 hours (B) or with 1000 U/ml of IFNγ for the indicated times (C), and the surface expression of CD40 was examined by FACS analysis.</p

Effects of IFNγ on the proliferation and apoptosis of SGE cells.

<p>(A) SGE cells, human airway epithelial cells (HBTEC) and human umbilical vein endothelial cells (HUVEC)were incubated with the indicated concentration of IFNγ, and proliferative responses were assessed at 48 h. Each bar shows mean + SD. IFNγ did not significantly affect the proliferation of any of these cells. The results shown are representative of three independent experiments. (B) SGE cells were incubated with the indicated concentration of IFNγ, and apoptosis was determined at 12 h by flow cytometry. Numbers in R1 and R2 indicate early and late apoptosis, respectively.</p

Decreased Expression of Innate Immunity-Related Genes in Peripheral Blood Mononuclear Cells from Patients with IgG4-Related Disease

<div><p>Background</p><p>IgG4-related disease (IgG4-RD) is a new clinical entity of unknown etiology characterized by elevated serum IgG4 and tissue infiltration by IgG4-positive plasma cells. Although aberrancies in acquired immune system functions, including increases in Th2 and Treg cytokines observed in patients with IgG4-RD, its true etiology remains unclear. To investigate the pathogenesis of IgG4-RD, this study compared the expression of genes related to innate immunity in patients with IgG4-RD and healthy controls.</p><p>Materials and Methods</p><p>Peripheral blood mononuclear cells (PBMCs) were obtained from patients with IgG4-RD before and after steroid therapy and from healthy controls. Total RNA was extracted and DNA microarray analysis was performed in two IgG4-RD patients to screen for genes showing changes in expression. Candidate genes were validated by real-time RT-PCR in 27 patients with IgG4-RD and 13 healthy controls.</p><p>Results</p><p>DNA microarray analysis identified 21 genes that showed a greater than 3-fold difference in expression between IgG4-RD patients and healthy controls and 30 genes that showed a greater than 3-fold change in IgG4-RD patients following steroid therapy. Candidate genes related to innate immunity, including those encoding Charcot–Leyden crystal protein (CLC), membrane-spanning 4-domain subfamily A member 3 (MS4A3), defensin alpha (DEFA) 3 and 4, and interleukin-8 receptors (IL8R), were validated by real-time RT-PCR. Expression of all genes was significantly lower in IgG4-RD patients than in healthy controls. Steroid therapy significantly increased the expression of DEFA3, DEFA4 and MS4A3, but had no effect on the expression of CLC, IL8RA and IL8RB.</p><p>Conclusions</p><p>The expression of genes related to allergy or innate immunity, including CLC, MS4A3, DEFA3, DEFA4, IL8RA and IL8RB, was lower in PBMCs from patients with IgG4-RD than from healthy controls. Although there is the limitation in the number of patients applied in DNA microarray, impaired expression of genes related to innate immunity may be involved in the pathogenesis of IgG4-RD as well as in abnormalities of acquired immunity.</p></div