• BMB Reports
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• v.30 no.6
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• pp.431-437
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• 1997

Since the role of CD40 on the interleukin-4(IL-4) -induced B cell activation has been strongly implicated in the agumentation of IgE production and response, we have investigated the intracelluar signaling pathways utilized by IL-4 and CD40 for type II IgE receptor (CD23) expression. IL-4 and anti-CD40 antibody treatment of human B cells, independently caused a rapid induction of CD23 gene activation within 2 h. There was a noticeable synergism between the action of the two agents inducing CD23 expression: the addition of anti-CD40 to the IL-4-treated culture significantly agumented the IL-4-induced CD23 on both mRNA and surface protein levels, and the inclusion of IL-4 in the anti-CD40-treated cells caused a further increase of CD23 expression far above the maximal level induced by anti-CD40. Protein tyrosine kinase (PTK) inhibitors effectively suppressed the both IL-4- and anti -CD40-induced CD23 expression. whereas protein kinase C (PKC) inhibitors had no effects. Electrophoretic mobility shift assays (EMSA) have shown that IL-4 and anti-CD40 induce the activation of NF-IL-4 and $NF-_{K}B$, respectively, binding to the CD23 promoter, both in a PKC-independent and PTK-dependent manner. These data suggest that the synergistic activation of CD23 gene expression by IL-4 and anti-CD40 is mediated by co-operative action of distinct nuclear factors. each of which is rapidly activated via PKC-independent and PTK-dependent process.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.5
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• pp.255-259
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• 2003

Dendritic cells (DCs) play a critical role in various immune responses involving $CD4^+$ T cells and have been used to generate anti-tumor immunity. Chemotherapy induces severe side effects including immunosuppression in patients with cancer. Although immunosuppression has been studied, the effects of anticancer drugs on DCs are not fully determined. In this study, we demonstrated that CD40 activation strongly protected DCs from 5-fluorouracil (5-FU) or mitomycin C-induced apoptosis. DCspecific surface markers, including CD11c and major histocompatibility complex (MHC) class II, were used for identifying DCs. CD 40 activation with anti-CD40 mAb significantly enhanced the viability of DCs treated with 5-FU or mitomycin C, assayed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Fluorescence staining and analysis clearly confirmed the enhancing effect of anti-CD40 mAb on the viability of DCs, suggesting that CD40 activation may transduce critical signals for the viability of DCs. Annexin V staining assay showed that CD40 significantly protected DCs from 5-FU or mitomycin C-induced apoptosis. Taken together, this study shows that CD40 activation with anti-CD40 mAb has strong anti-apoptosis effect on DCs, suggesting that CD40 activation may overcome the immunosuppression, especially downregulation of number and function of DCs in chemotherapy-treated cancer patients.

• BMB Reports
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• v.31 no.1
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• pp.83-88
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• 1998

Recently a B cell surface molecule, CD40, has emerged as a receptor mediating a co-stimulatory signal for B cell proliferation and differentiation. To investigate the mechanism of synergy between interleukin-4 (IL-4) and CD40 ligation in B cell activation, we have examined the effect of CE40 cross-linking on the IL-4 receptor expression in human B cells using anti-CE40 antibody. We observed that IL-4 and anti-CD40 both induce IL-4 receptor gene expression with a rapid kinetics resulting in a noticeable accumulation of IL-4 receptor mRNA within 4 h. While IL-4 caused a dose-dependent induction of surface IL-4 receptor expression, the inclusion of anti-CD40 in the IL-4-treated culture, further up-regulated the IL-4-induced IL-4 receptor expression as analyzed by flow cytometry. Pretreatment of B cells with inhibitors of protein tyrosine kinase (PTK) resulted in a significant inhibition of both the IL-4- and anti-CD40-induced IL-4 receptor mRNA levels, while protein kinase C (PKC) inhibitors had no effects. These results suggest that IL-4 and CD40 ligation generate B cell signals, which via PTK-dependent pathways, lead to the synergistic induction of IL-4 receptor gene expression. The rapid induction of IL-4 receptor gene expression through the tyrosine kinase-mediated signal transduction by B cell activating stimuli, would provide cells capacity for an efficient response to IL-4 in the early phase of IL-4 action, and may in part constitute the molecular basis of the reported anti-CD40 co-stimulatory effect on the IL-4-induced response.

• IMMUNE NETWORK
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• v.8 no.3
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• pp.75-81
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• 2008

Background: Platelets take part in repairing the lesions of endothelial damage. To understand the molecular mechanism of this process, we tested the hypothesis that CD154 expressed on activated platelets stimulates proliferation of human endothelial cells. Methods: The expression levels of CD154 and CD40 on platelets and endothelial cells, respectively, were measured by flow cytometry and confocal microscopy. Function-blocking monoclonal antibody against CD154 was developed after immunization with CD154-transfected L cells. Results: An anti-CD40 agonist antibody and soluble CD154 both induced significant proliferation of endothelial cells. In addition, a function-blocking anti-CD154 antibody inhibited the platelet-induced proliferation of endothelial cells, indicating that the CD154-CD40 pathway is involved in these cellular interactions. An anti-VEGF antibody failed to inhibit the proliferation. This, in addition to the fact that very small amounts of VEGF are released from platelets or endothelial cells, suggests that VEGF does not play an important role in the platelet-stimulated proliferation of endothelial cells. Conclusion: Our results indicate that platelets induce proliferation of endothelial cells by CD154-CD40 interactions independently of VEGF.

• IMMUNE NETWORK
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• v.2 no.3
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• pp.142-149
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• 2002

Background: Our previous study showed that purified protein derivative (PPD)-stimulated pleural mononuclear cells (PMC) from tuberculous pleurisy (Tbp) produced significantly more $IFN-{\gamma}$ (10- to 70-fold) after in vitro PPD stimulation than freshly isolated pleural cells from malignant pleurisy. The present study was designed to determine whether blocking the CD40-CD40 ligand (CD40L) interaction decreases $IFN-{\gamma}$ production by altering IL-12 levels. Methods: IL-12 and $IFN-{\gamma}$ production after neutralizing anti-CD40L antibody treatment was compared to the efficacy of anti-CD80, anti-CD86, and a combination of anti-CD80 and CD86 (CD80+86) monoclonal antibodies (mAb). These activities were measured by enzyme-linked immunosorbent assays (ELISAs) and reverse transcription-polymerase chain reaction (RT-PCR), after in vitro stimulation with PPO antigen (Ag). Results: Neutralization of CD80, CD86 and CD80+86 did not decrease $IFN-{\gamma}$ and IL-12 production in Tbp-PMC, whereas neutralization of CD40L significantly depressed IL-12 p40 and $IFN-{\gamma}$. In addition, neutralization of CD40L completely inhibited IL-12 p40 and $IFN-{\gamma}$ mRNA expression. Conclusion: The CD40-CD40L interaction might play a major role in IL-12 and $IFN-{\gamma}$ production in Tbp-PMC, thus contributing to protective immunity in human tuberculosis.

• IMMUNE NETWORK
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• v.3 no.3
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• pp.176-181
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• 2003

Background: The molecular basis of follicular dendritic cells (FDC)-germinal center (GC) B cell interaction is largely unknown, although this cellular interaction is thought to be important for the whole process of GC B cell differentiation. Methods: Using FDC-like cells, HK, and highly purified GC B cells, we attempted to identify the molecules that play critical roles in the interactions between FDC and B cells. GC B cells were co-cultured with HK cells and soluble CD154 in the presence or absence of various function-blocking monoclonal antibodies to examine their effect on GC B cell binding to HK cells and B cell proliferation. Results: Anti-CD11a and anti-CD54 antibodies inhibited GC B cell binding to HK cells while anti-CD49d and anti-CD106 antibodies did not. GC B cell proliferation was not impaired by the disruption of GC B cell-HK cell adherence. Conclusion: Our results suggest that CD11a/CD18-CD54 interactions play an important roles in the initial binding of GC B cells to FDC and diffusible growth factors from FDC may be responsible the massive proliferation of GC B cells.

• Journal of Haehwa Medicine
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• v.13 no.2
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• pp.131-146
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• 2004

In the present study, we exarnined anti-allergic effect of SGBHB in cultured B cells. B cells were prepared from isolated murine splenocytes and activated by co-treatment of anti-CD40 monoclonal antibody and recombinant IL-4 allergens. Anti-allergic effects of SGBHB in activated B cells were determined by measuring B cell surface activated molecules (CD23+ and CD11a+), and expression levels of IL-$1{\beta}$, IL-6, IL-10, TNF-$\alpha$, IgE, and HRF. The major findings are summarized as follows. 1. SGBHB treatment did not produce significant cytotoxic effects on mouse lung fibroblast cells. 2. SGBHB produced significant inhibitory effect on the expression of B cell surface activated molecules (CD23+ and CD11a) in activated B cells. 3. SGBHB treatment significantly inhibited expression levels of IL-$1{\beta}$, IL-6, and TNF-$\alpha$ mRNAs in activated B cells.IL-6 protein levels were significantly decreased by $100{\mu}g/m{\ell}$ of SGBHB treatrrient, and TNF-$\alpha$ protein levels were decreased compared to the control group, but statistically insignificant. 4. SGBHB treatment significantly increased IL-10 at both mRNA and protein levels in activated B cells. 5. SGBHB treatment significantly inhibited levels of IgE production. Thus, the present data suggest that SGBHB has an anti-allergic effect on activated B cells by controlling irnmune responses, and further implicates the possibility on clinical application as a therapeutic agent.

• The Journal of Internal Korean Medicine
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• v.22 no.3
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• pp.405-413
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• 2001

Objective: Experimental studies were done to research the effects of Jahap-san(zige-san) on the allergic reaction Methods: For measuring the response about the antiallergic effect we investigated cytokines m-RNA expression of murine splenic B cell, production in anti-CD40 mAb-stimulated murine splenic B cells and the histamine release in IC-2 cells by anti-CD40 mAb-stimulated murine splenic B cells. Results: The extract of Jahap-san(zige-san) revealed significant decrease effect on cytokines m-RNA expression of murine splenic B cell, production in anti-CD40 mAb-stimulated murine splenic B cells and the histamine release in IC-2 cells by anti-CD40 mAb-stimulated murine splenic B cells. Conculusion : The extract of Jahap-san(zige-san) revealed significant effect on the antiallergy.

• IMMUNE NETWORK
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• v.7 no.2
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• pp.80-86
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• 2007

Background: CD40-activated B (CD40-B) cells might be an attractive source of autologous antigen-presenting cells (APCs) for immunotherapy due to the convenience to obtain from peripheral blood and expand in vitro. Moreover, CD40-B cells were found to be comparable with DCs in their capacity to raise antigen-specific CD8+ T cells. Here, we have established K562 cells expressing CD40L to expand CD40-activated B cells used for APCs. Methods: After activation of B cell by K562/CD40L, CD40-B cells were examined by counting B cell numbers. Surface expression of CD54, CD80, CD86 and HLA class II was measured by flow cytometry. The CD40-B cells were tested for its function as APC by mixed lymphocyte reactions (MLR) and by induction of T cell responses specific for pp65 peptide in vitro. Results: The expansion of B cells by K562/CD40L increased about 6-folds compared with anti-CD40 or K562. Furthermore, the expression of CD54, CD80, CD86 and HLA class II was up-regulated by K562/CD40L. B cells by K562/CD40L showed comparable antigen presentation activity with mature DCs as shown in MLR, INF-${\gamma}$ ELISPOT assay. Conclusion: These results suggest that K562/CD40L could be used to generate activated B cells as potent APCs which could be useful for cellular vaccination and adoptive immunotherapy.

• IMMUNE NETWORK
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• v.4 no.3
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• pp.143-154
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• 2004

Background: CD27 is recently known as a memory B cell marker and is mainly expressed in activated T cells, some B cell population and NK cells. CD27 is a member of tumor necrosis factor receptor family. Like CD40 molecule, CD27 has (P/S/T/A) X(Q/E)E motif for interacting with TNF receptor-associated factors (TRAFs), and TRAF2 and TRAF5 bindings to CD27 in 293T cells were reported. Methods: To investigate the CD27 signaling effect in B cells, human CD40 extracellular domain containing mouse CD27 cytoplamic domain construct (hCD40-mCD27) was transfected into mouse B cell line CH12.LX and M12.4.1. Results: Through the stimulation of hCD40-mCD27 molecule via anti-human CD40 antibody or CD154 ligation, expression of CD11a, CD23, CD54, CD70 and CD80 were increased and secretion of IgM was induced, which were comparable to the effect of CD40 stimulation. TRAF2 and TRAF3 were recruited into lipid-enriched membrane raft and were bound to CD27 in M12.4.1 cells. CD27 stimulation, however, did not increase TRAF2 or TRAF3 degradation. Conclusion: In contrast to CD40 signaling pathway, TRAF2 and TRAF3 degradation was not observed after CD27 stimulation and it might contribute to prolonged B cell activation through CD27 signaling.