Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production

PURPOSE: The goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies. MATERIALS AND METHODS: DNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone. CONCLUSION: DNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.

Syntenin Is Expressed in Human Follicular Dendritic Cells and Involved in the Activation of Focal Adhesion Kinase

Syntenin is an adaptor molecule containing 2 PDZ domains which mediate molecular interactions with diverse integral or cytoplasmic proteins. Most of the results on the biological function of syntenin were obtained from studies with malignant cells, necessitating exploration into the role of syntenin in normal cells. To understand its role in normal cells, we investigated expression and function of syntenin in human lymphoid tissue and cells in situ and in vitro. Syntenin expression was denser in the germinal center than in the extrafollicular area. Inside the germinal center, syntenin expression was obvious in follicular dendritic cells (FDCs). Flow cytometric analysis with isolated cells confirmed a weak expression of syntenin in T and B cells and a strong expression in FDCs. In FDC-like cells, HK cells, most syntenin proteins were found in the cytoplasm compared to weak expression in the nucleus. To study the function of syntenin in FDC, we examined its role in the focal adhesion of HK cells by depleting syntenin by siRNA technology. Knockdown of syntenin markedly impaired focal adhesion kinase phosphorylation in HK cells. These results suggest that syntenin may play an important role in normal physiology as well as in cancer pathology.

IL-4 and HDAC Inhibitors Suppress Cyclooxygenase-2 Expression in Human Follicular Dendritic Cells

Evidence for immunoregulatory roles of prostaglandins (PGs) is accumulating. Since our observation of PG production by human follicular dendritic cells (FDCs), we investigated the regulatory mechanism of PG production in FDC and attempted to understand the functions of released PGs in the responses of adjacent lymphocytes. Here, using FDC-like cells, HK cells, we analyzed protein expression alterations in cyclooxygenase-2 (COX-2) in the presence of IL-4 or histone deacetylase (HDAC) inhibitors. Both IL-4 and HDAC inhibitors suppressed COX-2 expression in dose-dependent manners. Their effect was specific to COX-2 and did not reach to COX-1 expression. Interestingly, HDAC inhibitors gave rise to an opposing effect on COX-2 expression in peripheral blood monocytes. Our results suggest that IL-4 may regulate COX-2 expression in FDCs by affecting chromatin remodeling and provide insight into the role of cellular interactions between T cells and FDC during the GC reaction. Given the growing interests in wide-spectrum HDAC inhibitors, the differential results on COX-2 expression in HK cells and monocytes raise cautions on their clinical use.

Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS-/- mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor NG-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca2+ levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca2+ levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol-treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca2+ chelator, calmodulin antagonist, and CaMKKbeta siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca2+-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca2+/CaMKKbeta-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization.

Production of Prostaglandin E2 and I2 Is Coupled with Cyclooxygenase-2 in Human Follicular Dendritic Cells

BACKGROUND: Prostaglandins (PGs) play pathogenic and protective roles in inflammatory diseases. The novel concept of PGs as immune modulators is being documented by several investigators. By establishing an in vitro experimental model containing human follicular dendritic cell-like cells, HK cells, we reported that HK cells produce prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) and that these PGs regulate biological functions of T and B cells. METHODS: To investigate the respective contribution of cyclooxygenase-1 (COX-1) and COX-2 to PGE2 and PGI2 production in HK cells, we performed siRNA technology to knock down COX enzymes and examined the effect on PG production. RESULTS: Both PGE2 and PGI2 productions were almost completely inhibited by the depletion of COX-2. In contrast, COX-1 knockdown did not significantly affect PG production induced by lipopolysaccharide (LPS). CONCLUSION: The current results suggest that mPGES-1 and PGIS are coupled with COX-2 but not with COX-1 in human follicular dendritic cell (FDC) and may help understand the potential effects of selective COX inhibitors on the humoral immunity.

Introduction to Autoimmune Disease

The immune system maintains the integrity of our bodies by warding off intruding microorganisms, but by sustaining tolerance to our own tissues. The immunologic tolerance is established by several layers of safeguards, including physical elimination of self-reactive lymphocytes during their development in the central lymphoid organs, anergy induction in autoreactive lymphocytes before their emigration to the periphery, or production regulatory T lymphocytes that suppress the activation, proliferation, and differentiation of various effector cells. The major regulatory T lymphocytes display their phenotype as CD4(+)CD25(+)Foxp3(+) and constitute about 10% of the peripheral T lymphocytes. Even with these safeguards, the immunologic tolerance sometimes fails and generates autoimmune diseases. Scientists studying the pathogenesis of autoimmune diseases pay particular attention to a CD4(+) T lymphocytes subset, Th17 lymphocytes, distinct from Th1 and Th2. Th17 produces diverse proinflammatory cytokines including IL-17 and TNF-alpha. Th17 and these cytokines are causatively associated with many episodes of autoimmune diseases. Accumulated data reveal the critical role of Th17 cells in the pathology of autoimmunity and portray them as an important target in the treatment of various autoimmune diseases. In this article, we will describe the main characteristics of regulatory T cells and Th17 cells and their cellular and molecular mechanisms of protective or destructive functions, respectively.

Introduction to Autoimmune Disease

The immune system maintains the integrity of our bodies by warding off intruding microorganisms, but by sustaining tolerance to our own tissues. The immunologic tolerance is established by several layers of safeguards, including physical elimination of self-reactive lymphocytes during their development in the central lymphoid organs, anergy induction in autoreactive lymphocytes before their emigration to the periphery, or production regulatory T lymphocytes that suppress the activation, proliferation, and differentiation of various effector cells. The major regulatory T lymphocytes display their phenotype as CD4(+)CD25(+)Foxp3(+) and constitute about 10% of the peripheral T lymphocytes. Even with these safeguards, the immunologic tolerance sometimes fails and generates autoimmune diseases. Scientists studying the pathogenesis of autoimmune diseases pay particular attention to a CD4(+) T lymphocytes subset, Th17 lymphocytes, distinct from Th1 and Th2. Th17 produces diverse proinflammatory cytokines including IL-17 and TNF-alpha. Th17 and these cytokines are causatively associated with many episodes of autoimmune diseases. Accumulated data reveal the critical role of Th17 cells in the pathology of autoimmunity and portray them as an important target in the treatment of various autoimmune diseases. In this article, we will describe the main characteristics of regulatory T cells and Th17 cells and their cellular and molecular mechanisms of protective or destructive functions, respectively.

Platelets Induce Proliferation of Human Umbilical Vein Endothelial Cells via CD154-CD40 Pathway Independently of VEGF

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.

Adhesion of CD40-stimulated Germinal Center B Cells to HK Cells Employs the CD11a/CD18-CD54 Interactions

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.