Immunoglobulin D (IgD) and IgD receptor expression in diffuse large B-cell lymphoma.

Objective: Immunoglobulin D (IgD) levels are often elevated in patients with autoimmune diseases. However, the oncogenic activities of IgD and IgD receptor (IgDR) in diffuse large B-cell lymphoma (DLBCL) have not been reported in detail. Therefore, we aimed to investigate the expression of IgD and IgDR in patients with DLBCL. Methods: Membrane IgD (mIgD) and IgDR expression in tissue samples was analyzed using IHC, mIgD and IgDR expression on peripheral blood mononuclear cells (PBMCs) was analyzed by FCM, and secreted IgD (sIgD) level was analyzed by ELISA. Fisher's exact test and Spearman correlation analysis were used to evaluate the relationship between IgD, IgDR, and clinical parameters. Results: The pathological lymph nodes of 34 patients with DLBCL were studied, and mIgD and IgDR expression was found in 16 and 19 patients. mIgD and IgDR expression was upregulated in patients with DLBCL and mIgD expression was significantly associated with IgDR expression. Further correlation analysis showed that mIgD expression was correlated with serum ß2-MG level and Hans algorithm as germinal center B (GCB), whereas IgDR expression correlated with serum LDH level, IPI score and GCB. ELISA showed that sIgD level was significantly increased in DLBCL patients and it correlated with serum ß2-MG and LDH levels. FCM showed that mIgD and IgDR expression in PBMCs of patients with DLBCL was significantly higher than that in healthy controls. Conclusion: Our findings suggest that overexpression of IgD and IgDR is an abnormal activation state in DLBCL.

Regulatory effects of paeoniflorin-6'-O-benzene sulfonate (CP-25) on dendritic cells maturation and activation via PGE2-EP4 signaling in adjuvant-induced arthritic rats.

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease. Dendritic cells (DCs) are one of the most powerful antigen-presenting cells, and they play an important role in RA pathogenesis. Prostaglandin E2 (PGE2) is a potent lipid mediator that can regulate the maturation and activation of DCs, but the molecular mechanisms have not been elucidated. In this study, both in vitro and in an RA rat model, we investigated the mechanisms involved by focusing on PGE2-mediated signaling and using a novel anti-inflammatory compound, paeoniflorin-6'-O-benzene sulfonate (CP-25). PGE2 combined with tumor necrosis factor-α promoted DC maturation and activation through EP4-cAMP signaling. Treatment with CP-25 increased the endocytic capacity of DCs induced by PGE2. CP-25 inhibited the potency of DCs induced by the EP4 receptor agonist, CAY10598, to stimulate allogeneic T cells. Consistent with these findings, the CAY10598-induced upregulation of DC surface activation markers and production of IL-23 was significantly inhibited by CP-25 in a concentration-dependent manner. In vivo administration of CP-25 alleviated adjuvant arthritis (AA) in rats through inhibition of DC maturation and activation. Our results indicate that PGE2-EP4-cAMP signal hyperfunction can lead to abnormal activation of DC functions, which correlates with the course of disease in AA rats and provides a possible treatment target. The inhibition of DC maturation and activation by CP-25 interference of the PGE2-EP4 pathway may significantly contribute to the immunoregulatory profile of CP-25 when used to treat RA and other immune cell-mediated disorders.

Angiotensin II and tumor necrosis factor-α stimulate the growth, migration and invasion of BEL-7402 cells via down-regulation of GRK2 expression.

PURPOSE: To investigate the effects of angiotensin II (Ang II) and tumor necrosis factor-α (TNF-α) on the biological characteristics of hepatocellular carcinoma (HCC) cells and the associated changes in G protein-coupled receptor kinase 2 (GRK2) expression. METHODS: The mean serum levels of Ang II and TNF-α in normal subjects and patients with benign liver tumors (BLTs) and HCC were evaluated by enzyme-linked immunosorbent assay (ELISA), and liver samples from the patients with HCC and HCC mice were used to assess the protein levels of both cytokines, their major receptors and GRK2. In addition, the dynamics of Bel-7402 cells were determined with cell counting kit-8 (CCK-8) and Transwell experiments, while the levels of the primary cytokine receptors Ang II type-1 receptor (AT1R) and type-2 receptor (AT2R) as well as TNF receptor 1 (TNFR1) were detected by flow cytometry (FCM). The effects of Ang II and TNF-α on the GRK2 levels in Bel-7402 cells and on the dynamics of GRK2-knockdown HCC cells were also investigated. RESULTS: Both cytokines independently enhanced Bel-7402 cell growth, migration and invasion by decreasing the GRK2 level. In contrast, down-regulating the GRK2 level in Bel-7402 cells suppressed these effects. No synergistic effects were discovered when Ang II and TNF-α were administered together. Furthermore, increased AT1R and TNFR1 levels stimulated HCC initiation and progression, whereas AT2R overexpression produced the opposite effect. CONCLUSIONS: The present results suggested that Ang II and TNF-α promote Bel-7402 cell growth, migration and invasion by down-regulating GRK2 expression, and that the associated receptors AT1R, AT2R and TNFR1 participate in HCC initiation and progression.

Paeoniflorin-6'-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a new ester derivative of paeoniflorin with improved lipid solubility and oral bioavailability, as well as better anti-inflammatory activity than its parent compound. In this study we explored whether CP-25 exerted therapeutic effects in collagen-induced arthritis (CIA) mice through regulating B-cell activating factor (BAFF)-BAFF receptors-mediated signaling pathways. CIA mice were given CP-25 or injected with biological agents rituximab or etanercept for 40 days. In CIA mice, we found that T cells and B cells exhibited abnormal proliferation; the percentages of CD19+ total B cells, CD19+CD27+-activated B cells, CD19+BAFFR+ and CD19+TACI+ cells were significantly increased in PBMCs and spleen lymphocytes. CP-25 suppressed the indicators of arthritis, alleviated histopathology, accompanied by reduced BAFF and BAFF receptors expressions, inhibited serum immunoglobulin levels, decreased the B-cell subsets percentages, and prevented the expressions of key molecules in NF-κB signaling. Furthermore, we showed that treatment with CP-25 reduced CD19+TRAF2+ cell expressions stimulated by BAFF and decreased TRAF2 overexpression in HEK293 cells in vitro. Thus, CP-25 restored the abnormal T cells proliferation and B-cell percentages to the normal levels, and normalized the elevated levels of IgA, IgG2a and key proteins in NF-κB signaling. In comparison, rituximab and etanercept displayed stronger anti-inflammatory activities than CP-25; they suppressed the elevated inflammatory indexes to below the normal levels in CIA mice. In summary, our results provide evidence that CP-25 alleviates CIA and regulates the functions of B cells through BAFF-TRAF2-NF-κB signaling. CP-25 would be a soft immunomodulatory drug with anti-inflammatory effect.

The influence of TNF-α and Ang II on the proliferation, migration and invasion of HepG2 cells by regulating the expression of GRK2.

PURPOSE: Hepatocellular carcinoma (HCC) is a common digestive system malignancy that is associated with a poor prognosis. This study researched the interaction of tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) in HCC cells proliferation, migration and invasion and examined their influence on the expression of G protein-coupled receptor kinase 2 (GRK2) and relevant receptors. METHODS: Cell Counting Kit-8 and Transwell assays were performed to evaluate the effects of TNF-α and Ang II on HepG2 cells proliferation, migration and invasion. Flow cytometry was used to investigate the expression of tumor necrosis factor receptor 1 (TNFR1), angiotensin II type 1 (AT1R) and type 2 receptors (AT2R) on the surface of HepG2 cells. Additionally, Western blot was performed to assess the modulation of GRK2 expression by TNF-α and Ang II in HepG2 cells. Meanwhile, GRK2 siRNA-transfected HepG2 cells were used to confirm the effects of GRK2, TNF-α and Ang II on the proliferation, migration and invasion of GRK2-knockdown HCC cells. Finally, the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues were tested by immunohistochemistry. RESULTS: The data demonstrated that TNF-α and Ang II can enhance the proliferation, migration and invasion of HepG2 cells through suppressing GRK2 expression but that the two reagents combined did not have synergistic effects. Moreover,overexpression of TNFR1 and AT1R perhaps promoted the formation and progression of HCC, while high AT2R expression had the opposite effect. CONCLUSIONS: This study provides new ideas for the prevention and treatment of HCC by researching the interaction and probable mechanism of different bioactive factors associated with HCC.

CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

Paeoniflorin-6'-O-benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19+ B cells, CD19+CD20+ B cells, CD19+CD27+ B cells and CD19+CD20+CD27+ B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

BAFF upregulates CD28/B7 and CD40/CD154 expression and promotes mouse T and B cell interaction in vitro via BAFF receptor.

AIM: B cell-activating factor belonging to the TNF family (BAFF) is a member of TNF family and required for peripheral B cell survival and homeostasis. BAFF has been shown to promote the proliferation of T and B cells. In this study we examined whether and how BAFF mediated the interaction between mouse T and B cells in vitro. METHODS: BAFF-stimulated B or T cells were co-cultured with T or B cells. The interactions between T and B cells were analyzed by measuring the expression of co-stimulatory molecules (CD28/CD80 or CD40/CD154), the proliferation and secretion of T and B cells and other factors. Two siRNAs against the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) were used to identify the receptors responsible for the actions of BAFF. RESULTS: BAFF-stimulated B cells significantly promoted the proliferation and activity of co-cultured T cells, and increased the percentages of CD4(+)CD28(+) and CD4(+)CD154(+) T cells. Similarly, BAFF-stimulated T cells significantly promoted the proliferation and activity of co-cultured B cells, and increased CD19(+)CD80(+) and CD19(+)CD40(+)B cell subpopulations. BAFF-R siRNA-silenced B cells showed significantly lower expression of CD40 and CD80 than the control B cells. When the BAFF-R siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was not increased. TACI siRNA-silenced B cells exhibited higher expression of CD40 and CD80 than the control B cells. When the TACI siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was significantly increased. CONCLUSION: BAFF upregulates CD28/B7 and CD40/CD154 expression, and promotes the interactions between T and B cells in a BAFF-R-dependent manner.

Ginsenoside compound K suppresses the abnormal activation of T lymphocytes in mice with collagen-induced arthritis.

AIM: To investigate the anti-arthritis and immunomodulatory activities of ginsenoside compound K (C-K) in mice with collagen-induced arthritis (CIA). METHODS: DBA/1 mice with CIA were treated with C-K (28, 56 or 112 mg·kg(-1)·d(-1), ig) or the positive control methotrexate (2 mg/kg, ig, every 3 d) for 34 d. Splenic T and B lymphocytes were positively isolated using anti-CD3-coated magnetic beads or a pan B cell isolation kit. T lymphocyte subsets, and CD28, T cell receptor (TCR), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) expression in purified splenic T lymphocytes were analyzed using flow cytometry, Western blotting and laser confocal microscopy. RESULTS: C-K treatment significantly ameliorated the pathologic manifestations of CIA mice, remarkably inhibited T lymphocyte proliferation, and marginally inhibited the proliferation of B lymphocytes. C-K treatment significantly suppressed TNF-α and anti-CII antibody levels, and increased IFN-γ level in the joints of CIA mice, but did not alter IL-4 production. Treatment of CIA mice with C-K significantly decreased the percentages of activated T cells, co-stimulatory molecule-expressing T cells and effector memory T cells, and increased the frequencies of naive T cells and regulatory T cells. Furthermore, C-K treatment significantly decreased the expression of CD28 and TCR, whereas it increased the expression of CTLA-4 and PD-1 on T lymphocytes of CIA mice. Methotrexate treatment exerted comparable effects in all these experiments. CONCLUSION: C-K suppresses the progression of CIA through regulating TCR, CD28, CTLA-4 and PD-1 expression, thus inhibiting the abnormal activation and differentiation of T lymphocytes.

Total glucosides of paeony inhibit the proliferation of fibroblast-like synoviocytes through the regulation of G proteins in rats with collagen-induced arthritis.

The aim of this study was to investigate the expression of G proteins in fibroblast-like synoviocytes (FLSs) from rats with collagen-induced arthritis (CIA) and to determine the effect of total glucosides of paeony (TGP). CIA rats were induced with chicken type II collagen (CCII) in Freund's complete adjuvant. The rats with experimental arthritis were randomly separated into five groups and then treated with TGP (25, 50, and 100mg/kg) from days 14 to 35 after immunization. The secondary inflammatory reactions were evaluated through the polyarthritis index and histopathological changes. The level of cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The FLS proliferation response was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The toxin-catalyzed ADP-ribosylation of G proteins was performed through autoradiography. The results show that TGP (25, 50, and 100mg/kg) significantly decreased the arthritis scores of CIA rats and improved the histopathological changes. TGP inhibited the proliferation of FLSs and increased the level of cAMP. Moreover, the FLS proliferation and the level of Gαi expression were significantly increased, but the level of Gαs expression was decreased after stimulation with IL-1ß (10ng/ml) in vitro. TGP (12.5 and 62.5µg/ml) significantly inhibited the FLS proliferation and regulated the balance between Gαi and Gαs. These results demonstrate that TGP may exert its anti-inflammatory effects through the suppression of FLS proliferation, which may be associated with its ability to regulate the balance of G proteins. Thus, TGP may have potential as a therapeutic agent for the treatment of rheumatoid arthritis.

BF02, a recombinant TNFR2 fusion protein, alleviates adjuvant arthritis by regulating T lymphocytes in rats.

AIM: To investigate the therapeutic effects of BF02 on adjuvant arthritis (AA) in rats and the regulatory effects of BF02 on T lymphocyte function. METHODS: SD rats received a single intradermal injection of Freund's complete adjuvant emulsion into the right hind metatarsal footpad. After the onset of AA, the rats were injected BF02 (1, 3, or 9 mg/kg, sc) every 3 d for a total of 15 d. Intragastric administration of methotrexate (MTX, 0.5 mg/kg, every 3 d for a total of 15 d) was taken as the positive control drug. Arthritis index, swollen joint count, ankle joint histopathology, spleen histopathology and the paw radiography were used for evaluating the drug effects on AA rats. T lymphocyte function was assessed by measuring T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression levels, and percentage of T lymphocyte subsets. RESULTS: In the AA rats, remarkable secondary inflammatory responses exhibited, accompanied by significantly higher levels of IL-1, IL-6, TNF-α, IL-17, LTα, RANKL, and MMP-13. The expression of IL17 and TNF-α mRNAs was also substantially higher than in normal rats. The percentages of CD3(+)CD4(+) and CD4(+)CD25(+) T lymphocytes were increased, whereas the percentages of CD4(+)CD62L(+) and CD4(+)CD25(+)FoxP3(+) T lymphocytes were decreased. Treatment of the AA rats with BF02 (9 mg/kg) or MTX significantly decreased the arthritis index, swollen joint count and arthritis global assessment. Moreover, both BF02 (9 mg/kg) and MTX significantly inhibited T lymphocyte proliferation, and blocked the above mentioned aberrance in T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression, and percentages of T lymphocyte subsets. CONCLUSION: BF02 exerts therapeutic effects on AA rats via the regulation of T lymphocytes.

Depletion of ß-arrestin2 in hepatic stellate cells reduces cell proliferation via ERK pathway.

ß-Arrestins are multifunctional adaptor proteins. Recently, some new roles of ß-arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of ß-arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of ß-arrestins with the experimental hepatic fibrosis and the proliferation of hepatic stellate cells (HSCs) were investigated. Porcine serum induced liver fibrosis was established in this study. At five time points, the dynamic expression of ß-arrestin1, ß-arrestin2, and α-smooth muscle actin (α-SMA) in rat liver tissues, was measured by immunohistochemical staining, double immunofluorescent staining, and Western blotting. This study showed that aggravation of hepatic fibrosis with gradually increasing expression of ß-arrestin2 in the hepatic tissues, but not ß-arrestin1. Further, as hepatic fibrosis worsens, ß-arrestin2-expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of ß-arrestin2 had a significant positive correlation with the expression of α-SMA, an activated HSCs marker. In vitro studies, the dynamic expression of ß-arrestin1 and ß-arrestin2 in platelet derived growth factor-BB (PDGF-BB) stimulated HSCs was assessed by Western blotting. The expression of ß-arrestin2 was remarkably increased in PDGF-BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of ß-arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting ß-arrestin2 mRNA (siß-arrestin2) into HSCs led to a 68% and 70% reduction of ß-arrestin2 mRNA and protein expression, respectively. siß-arrestin2 abolished the effect of PDGF-BB on the proliferation of HSCs. In addition, siß-arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The present study provided strong evidence for the participation of the ß-arrestin2 in the pathogenesis of hepatic fibrosis. The ß-arrestin2 depletion diminishes HSCs ERK1/2 signaling and proliferation stimulated by PDGF-BB. Selective targeting of ß-arrestin2 inhibitors to HSCs might present as a novel strategy for the treatment of hepatic fibrosis.

Etanercept attenuates collagen-induced arthritis by modulating the association between BAFFR expression and the production of splenic memory B cells.

Anti-tumour necrosis factor-α (TNF-α) drugs are approved for the treatment of rheumatoid arthritis (RA). Many studies have investigated the effect of these drugs on the T cell response; however, some clues have indicated that it may also target B cells. This study was carried out to explore the potential effects and mechanisms of etanercept, a soluble TNF-α receptor, on the function of B cells and their development into memory B cells in type II collagen (CII)-induced arthritis (CIA). Beginning on day 24 after CII immunisation, the mice were evaluated every 2-3 days to determine two clinical parameters: their arthritis global assessment and swollen joint count (SJC). The serum concentrations of IgG1, IgG2a and anti-CII antibodies and the splenic pathology and proliferation of B cells were measured. The percentage of total memory B cells in the spleen was analysed with flow cytometry. BAFFR was detected by immunohistochemistry. In CIA mice, etanercept markedly suppressed the arthritis global assessment and the SJC, reduced the production of anti-CII, IgG1 and IgG2a antibodies, and prevented spleen histopathology to varying degrees; however, it had no obvious effect on splenic B cell proliferation. Etanercept also decreased the percentage of total CD27(+) memory B cells in the spleen. Treatment with etanercept was associated with a further increase in BAFFR expression, a significant reduction in CD27 expression, and a negative correlation between the levels of BAFFR and the percentage of memory B cells. Our findings showed that increased BAFFR expression has a regulatory effect on the activation of B cells and the generation of memory B cells, which may be one of the mechanisms of the therapeutic effects of etanercept.

Expression and function of ß-arrestin 2 stimulated by IL-1ß in human fibroblast-like synoviocytes and the effect of paeoniflorin.

The aim of this study was to explore the expression and function of ß-arrestin 2 in human fibroblast-like synoviocytes (FLS) stimulated by IL-1ß and the effect of paeoniflorin (Pae). We isolated and cultured human FLS, which were stimulated by IL-1ß. The FLS proliferations were detected by [3H] thymidine incorporation. The level of cAMP stimulated by IL-1ß on different times was investigated by radioimmunoassay, and the activity of PKA was measured by luminescent kinase assay. The expression of ß-arrestin 2 was measured by western blot. We found that the human FLS proliferation increased apparently in 24 h, and the activities of PKA and cAMP accumulation increased significantly in 6 h after stimulated by IL-1ß, while cAMP accumulation and the activities of PKA decreased especially in 24 h when the expression of ß-arrestin 2 increased significantly. Decreased cAMP accumulation and the increased expression of ß-arrestin 2 may reveal a positive correlation with the FLS proliferation. Pae (10(-5), 10(-6), 10-7 mol•L(-1)) in vitro could suppress the FLS proliferation and the high expression of ß-arrestin 2. The expression of ß-arrestin 2 may have a positive correlation with the human FLS proliferation, while the activities of PKA and cAMP accumulation have a negative correlation with the proliferation. The increased ß-arrestin 2 may down-regulate cAMP-PKA signaling pathway and promote FLS proliferation. Pae may suppress the expression of ß-arrestin 2 and up-regulate cAMP-PKA signaling, which may be one of the mechanisms for the effects of Pae on inhibiting human FLS proliferation.