Human Follicular Dendritic Cell Research Articles

Akt, IL-4, and STAT Proteins Play Distinct Roles in Prostaglandin Production in Human Follicular Dendritic Cell-like Cells.

This study aimed to explore the role of Akt protein in the induction and inhibition of prostaglandin (PG) in human follicular dendritic cell (FDC)-like cells. FDC-like cells and B cells were isolated from human tonsils. PG production was assessed using enzyme immunoassay, while the upstream cyclooxygenase-2 (COX-2) protein levels were measured using immunoblotting with FDC-like cells transfected with Akt siRNA to analyze the impact of Akt knockdown. The COX-2 expression and PG production induced with IL-1β were significantly increased by Akt knockdown. However, IL-1β did not significantly alter either total or phosphorylated Akt protein levels. Akt knockdown resulted in the augmentation of COX-2 expression induced by B cells, although the addition of B cells did not significantly modulate both total and phosphorylated Akt proteins. In contrast, IL-4 specifically exhibited a potent inhibitory effect on COX-2 protein induction and PG production via STAT6. The inhibitory activity of IL-4 was not hampered by Akt knockdown. Interestingly, COX-2 expression levels induced with IL-1β were markedly modulated with STAT1 and STAT3 knockdown. STAT1 silencing resulted in further augmentation of COX-2, whereas STAT3 silencing prohibited IL-1β from stimulating COX-2 expression. The current results suggest that Akt, IL-4, and STAT1 play inhibitory roles in PG production in FDC-like cells and expand our knowledge of the immune inflammatory milieu.

The Migration of Human Follicular Dendritic Cell-Like Cell Is Facilitated by Matrix Metalloproteinase 3 Expression That Is Mediated through TNFα-ERK1/2-AP1 Signaling.

Follicular dendritic cells are important stromal components of the germinal center (GC) and have pivotal roles in maintaining the GC microenvironment for high-affinity antibody production. Tumor necrosis factor-α (TNFα) is essential for the development and functions of follicular dendritic cells. Despite the importance of follicular dendritic cells in humoral immunity, their molecular control mechanisms have yet to be fully elucidated due to the lack of an adequate investigation system. Here, we have used a unique human primary follicular dendritic cell-like cell (FDCLC) to demonstrate that the migration of these cells is enhanced by TNFα-mediated metalloproteinase 3 (MMP3) expression. MMP3 was found to be highly expressed in normal human GCs and markedly upregulated in human primary FDCLCs by TNFα. TNFα induced ERK1/2 phosphorylation and the transcription of MMP3 through AP1. TNFα treatment increased FDCLC migration, and a knockdown of MMP3 significantly reduced the TNFα-induced migration of FDCLCs. Overall, we have newly identified a control mechanism for the expression of MMP3 in FDCLCs that modulates their migration and may indicate an important role in GC biology. Since GCs are observed in the lesions of autoimmune diseases and lymphomas, targeting the MMP3/TNFα-mediated migration of stromal cells in the B cell follicle may have great potential as a future therapeutic modality against aberrant GC-associated disorders.

15(S)-HETE plays a regulatory role in the immune inflammatory responses

Abstract Arachidonic acids are subjected to the enzymatic transformation by PGH2 synthase or lipoxygenase upon the liberation from the membrane phospholipid. Using in vitro and in vivo experimental models, we have investigated the cellular and molecular mechanisms in the production and function of arachidonic acid metabolites. Here we report that a downstream product of 15-lipoxygenase, 15-S-hydroxyeicosatetraenoic acid (15-S-HETE) is not produced by human follicular dendritic cell (FDC)-like cells but exerts a regulatory effect on prostaglandin production by this type of cells. 15-S-HETE appears to derive from endothelial cells. FDC-like cells, which were isolated from human tonsil, do not express 15-lipoxyenase or 5-lipoxygenase but metabolize 15-S-HETE into downstream products. Immunoblotting analysis and results with chemical inhibitors imply that 15-S-HETE acts on FDC-like cells via PPAR-γ and leukotriene B4 receptor 2. 15-S-HETE enhances or inhibits the expression of PGH2 synthase and resultant prostaglandin production in FDC-like cells, which depends on the types of co-stimulation. Based on these data, we suggest a novel cellular and molecular pathway of immune regulation mediated by arachidonic acid metabolites.

Opposing roles of TGF-β in prostaglandin production by human follicular dendritic cell-like cells.

Prostaglandins (PGs) are recognized as important immune regulators. Using human follicular dendritic cell (FDC)-like HK cells, we have investigated the immunoregulatory role of PGs and their production mechanisms. The present study was aimed at determining the role of TGF-β in IL-1β-induced cyclooxygenase-2 (COX-2) expression by immunoblotting. COX-2 is the key enzyme responsible for PG production in HK cells. TGF-β, when added simultaneously with IL-1β, gave rise to an additive effect on COX-2 expression in a dose-dependent manner. However, TGF-β inhibited IL-1β-stimulated COX-2 expression when it was added at least 12h before IL-1β addition. The inhibitory effect of TGF-β was specific to IL-1β-induced COX-2 expression in HK cells. The stimulating and inhibitory effects of TGF-β were reproduced in IL-1β-stimulated PG production. Based on our previous results of the essential requirement of ERK and p38 MAPKs in TGF-β-induced COX-2 expression, we examined whether the differential activation of these MAPKs would underlie the opposing activities of TGF-β. The phosphorylation of ERK and p38 MAPKs was indeed enhanced or suppressed by the simultaneous treatment or pre-treatment, respectively. These results suggest that TGF-β exerts opposing effects on IL-1β-induced COX-2 expression in HK cells by differentially regulating activation of ERK and p38 MAPKs.

Suppressor of cytokine signaling 1 is a positive regulator of TGF-β-induced prostaglandin production in human follicular dendritic cell-like cells.

PGs are emerging as important immune modulators. Since our report on the expression of PG synthases in human follicular dendritic cells, we investigated the potential immunoregulatory function of PGs and their production mechanisms. In this study, we explored the intracellular signaling molecules mediating TGF-β-induced cyclooxygenase (COX)-2 augmentation in follicular dendritic cell-like cells. TGF-β triggered phosphorylation of Smad3 and ERK, which were essential for the increase in COX-2 protein. Interestingly, depletion of suppressor of cytokine signaling 1 (SOCS1) resulted in an almost complete inhibition of Smad3 phosphorylation and COX-2 induction. Nuclear translocation of Smad3 was inhibited in SOCS1-depleted cells. SOCS1 knockdown also downregulated TGF-β-stimulated Snail expression and its binding to the Cox-2 promoter. In contrast, overexpression of SOCS1 gave rise to a significant increase in Snail and COX-2 proteins. SOCS1 was reported to be a negative regulator of cytokine signaling by various investigators. However, our current data suggest that SOCS1 promotes TGF-β-induced COX-2 expression and PG production by facilitating Smad3 phosphorylation and Snail binding to the Cox-2 promoter. The complete understanding of the biological function of SOCS1 might be obtained via extensive studies with diverse cell types.

Human Tonsil-Derived Follicular Dendritic-Like Cells are Refractory to Human Prion Infection in Vitro and Traffic Disease-Associated Prion Protein to Lysosomes

The molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined. This is due, in part, to the absence of any well characterized and relevant cultured human cells susceptible to infection with human prions, such as those involved in Creutzfeldt-Jakob disease. In variant Creutzfeldt-Jakob disease, prion replication is thought to occur first in the lymphoreticular system and then spread into the brain. We have, therefore, examined the susceptibility of a human tonsil-derived follicular dendritic cell-like cell line (HK) to prion infection. HK cells were found to display a readily detectable, time-dependent increase in cell-associated abnormal prion protein (PrP(TSE)) when exposed to medium spiked with Creutzfeldt-Jakob disease brain homogenate, resulting in a coarse granular perinuclear PrP(TSE) staining pattern. Despite their high level of cellular prion protein expression, HK cells failed to support infection, as judged by longer term maintenance of PrP(TSE) accumulation. Colocalization studies revealed that exposure of HK cells to brain homogenate resulted in increased numbers of detectable lysosomes and that these structures immunostained intensely for PrP(TSE) after exposure to Creutzfeldt-Jakob disease brain homogenate. Our data suggest that human follicular dendritic-like cells and perhaps other human cell types are able to avoid prion infection by efficient lysosomal degradation of PrP(TSE).

STAT6 and JAK1 are essential for IL-4-mediated suppression of prostaglandin production in human follicular dendritic cells: Opposing roles of phosphorylated and unphosphorylated STAT6

Prostaglandins (PGs) are emerging as important immune mediators. Since our first report on the expression of prostacyclin synthase in the germinal centers, we have investigated production mechanisms and biological functions of PG using human follicular dendritic cell (FDC)-like cells. In the previous report, we observed that TGF-β enhances PG production, and IL-4 prevents this upregulation. To elucidate the inhibitory mechanism of IL-4, its effects on the key enzyme leading to PG production were analyzed in this study. IL-4 but not IL-10 inhibited TGF-β-induced COX-2 expression at both mRNA and protein levels. Next the early signaling molecules of IL-4 were identified by siRNA technology. IL-4 induced tyrosine phosphorylation of STAT1, 3, and 6, but only JAK1-STAT6 pathway was responsible for the prevention of COX-2 augmentation and PG production. Phosphorylated STAT6 accumulated in the nucleus rapidly upon IL-4 addition, and the complete inhibition of COX-2 upregulation required 24h of pretreatment with IL-4, implying that newly transcribed molecules mediate the inhibitory signals downstream of STAT6. Interestingly, unphosphorylated STAT6 proteins were constitutively expressed in the nucleus, and depletion of STAT6 impaired background level expression of COX-2 and PGs. Our results highlight the crucial roles of TGF-β and IL-4 in the regulation of PG production, which lead us to suggest that T cells play an important role in FDC production of PGs.

Production of Prostaglandin E2and I2Is Coupled with Cyclooxygenase-2 in Human Follicular Dendritic Cells

BackgroundProstaglandins (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.MethodsTo 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.ResultsBoth 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).ConclusionThe 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.

Application of the human follicular dendritic cell line, HK, as a novel experimental system to analyse osteoblast function

Application of the human follicular dendritic cell line, HK, as a novel experimental system to analyse osteoblast function

Prostacyclin production is not controlled by prostacyclin synthase but by cyclooxygenase-2 in a human follicular dendritic cell line, HK

We have recently demonstrated that human follicular dendritic cells (FDCs) strongly express prostacyclin synthase. The purpose of this study is to investigate the production mechanism of prostacyclin using the established human FDC line, HK. The levels of PGIS protein expression did not vary during the different stages of the cell cycle. We stimulated HK cells with various inflammatory cytokines but, none of the tested stimuli modulated PGIS expression significantly. However, incubation of HK cells with tumor necrosis factor (TNF)-α gave rise to a significant increase in the protein level of cyclooxygenase (COX)-2. Furthermore, elevated levels of prostacyclin secretion stimulated by TNF-α were markedly down-regulated by indomethacin and a selective COX-2 inhibitor. These results suggest that the production of prostacyclin in FDC is controlled by the regulation of upstream COX-2 but not by terminal PGIS protein production. This study has important implications for the development of new anti-inflammatory drugs.

Follicular Dendritic Cells Produce IL-15 That Enhances Germinal Center B Cell Proliferation in Membrane-Bound Form

Factors that control the survival and proliferation of Ag-stimulated B cells within the germinal center (GC) are crucial for humoral immune responses with high affinity Abs against infectious agents. The follicular dendritic cell (FDC) is known as a key cellular component of the GC microenvironment for GC-B cell survival and proliferation. In this study, we report that IL-15 is produced by human FDC in vivo and by an FDC cell line, FDC/HK cells, in vitro. IL-15 is captured by IL-15Ralpha on the surface of FDC/HK cells. The surface IL-15 is functionally active and augments GC-B cell proliferation. Because GC-B cells have the signal-transducing components (IL-2/15Rbetagamma), but not a receptor for binding of soluble IL-15 (IL-15Ralpha), IL-15 signaling is possibly transduced by transpresentation from FDCs to GC-B cells via cell-cell contact. Together, these results suggest that IL-15 from FDC, in membrane-bound form, plays an important role in supporting GC-B cell proliferation, proposing a new target for immune modulation as well as treatment of B cell tumors of GC origin.

Functional effects of TNF-alpha on a human follicular dendritic cell line: persistent NF-kappa B activation and sensitization for Fas-mediated apoptosis.

Follicular dendritic cells (FDC) play crucial roles in germinal center (GC) formation and differentiation of GC B cells. FDC functions are influenced by cytokines produced in the GC. Among the GC cytokines, TNF is known to be essential for the formation and maintenance of the FDC network in the GC. We found that TNF is a mitogenic growth factor to an established FDC-like cell line, HK cells. Differing from most cell types which become desensitized to TNF action, HK cells exhibited persistent TNF signaling, as demonstrated by prolonged and biphasic NF-kappaB activation even after 3 days of TNF treatment. As a result, antiapoptotic genes including TNFR-associated factors 1 and 2, and cellular inhibitor of apoptosis proteins 1 and 2 were persistently induced by TNF, leading to the protection against TNF-mediated cell death. However, TNF pretreatment enhanced Fas-mediated apoptosis by up-regulating surface Fas expression in an NF-kappaB-dependent pathway. During the GC responses, proliferation followed by FDC death has not been documented. However, our in vitro results suggest that FDCs proliferate in response to TNF, and die by Fas-mediated apoptosis whose susceptibility is enhanced by TNF, representing a mode of action for TNF in the maintenance of FDC networks by regulating the survival or death of FDC.

Follicular dendritic cell accessory activity crosses MHC and species barriers.

Productive follicular dendritic cell (FDC)-B cell interactions appear to involve critical ligand-receptor interactions. Immune complexes (IC) on FDC activate complement and provide FDC with a complement-derived CD21 ligand (CD21L), which bind CD21, while antigen in the IC binds on the B cell-BCR. Further, FDC-FcgammaRIIB binds Fc regions of antibodies in IC and reduces coligation of BCR and FcgammaRIIB minimizing an inhibitor of B cell activation. Given that Fc receptors and complement receptors bind immunoglobulins and complement fragments of other species, we reasoned that FDC accessory activity should cross MHC and species barriers. This prediction was tested using memory lymphocytes from OVA-immune mice and TT-immune humans in combination with FDC from murine lymph nodes and human tonsils. Human and murine FDC converted IC into potent immunogens (specific antibody increased from background to thousands of ng / ml). MHC barriers did not restrict this activity and human FDC worked with murine lymphocytes and murine FDC worked with human lymphocytes. Furthermore, stimulation via MHC-dependent allogeneic or zenogeneic mechanisms did not promote antibody production by FDC. Polyclonal responses stimulated by lipopolysaccharide and pokeweed mitogen were also promoted (10 - 100-fold) and anti-CD21 blocked FDC activity. These results substantiate the hypothesis that FDC are necessary for strong recall responses and that FDC-CD21L is critical.

Differential Induction of Cytokine Genes and Activation of Mitogen-Activated Protein Kinase Family by Soluble CD40 Ligand and TNF in a Human Follicular Dendritic Cell Line

Follicular dendritic cells (FDC)3 play crucial roles in germinal center (GC) formation and differentiation of GC B cells. Many aspects of FDC function are influenced by contact with B or T cells, and by cytokines produced in the GC, which involve stimulation of CD40 and TNF-alpha receptors on FDC. In this study, using an established FDC line, HK cells, we compared the effects of CD40 and TNF receptor triggering on cytokine induction and activation of mitogen-activated protein kinase family. We show that HK cells spontaneously produced IL-6, M-CSF, and G-CSF mRNA. Both the soluble form of CD40 ligand (sCD40L) and TNF increased the level of M-CSF and G-CSF mRNA. While TNF strongly induced IL-6 mRNA, its expression was not affected by sCD40L treatment, differing from the strong IL-6 induction in other cell types upon CD40 stimulation. In addition, sCD40L treatment resulted in activation of extracellular signal-related kinase 1 and 2 (ERK1/2) and p38 without significant increase in c-Jun N-terminal kinase (JNK) activity. Lack of JNK activation differs in that most B cells respond to CD40 stimulation by inducing JNK activity strongly, suggesting distinct characteristics of CD40 signaling in FDC. Compared with the effects of sCD40L, TNF was capable of inducing JNK activity in addition to the activation of ERK1/2 and p38. Furthermore, the proximal signaling elements activated by TNF differed from those activated by sCD40L, in that TNF did not require PMA-sensitive protein kinase C isoforms in the activation of ERK and p38, whereas sCD40L did. However, signals activated by these stimuli converged on cytokine gene expression in a synergistic manner, which may have implication in augmenting FDC function during GC reaction.

Expression of the lymphotoxin beta receptor on follicular stromal cells in human lymphoid tissues.

The lymphotoxin beta receptor (LTbetaR), and its ligand, LTalpha1beta2, have been proposed to play a key role in the development and organization of lymphoid tissues. The LTbetaR is expressed on a variety of human primary and transformed cells, but strikingly absent on T or B lymphocytes and primary monocytes or peripheral dendritic cells, although LTbetaR is detected on some myeloid leukemic lines. In the developing thymus LTbetaR is prominent along the trabeculae and into the medulla upto corticomedullary junction. In the spleen, LTbetaR is prominently expressed by cells in the red pulp and along the borders of red and white pulp which colocalizes with reticular stromal cells. The LTbetaR is expressed on a human follicular dendritic cell line, FDC-1, and signals expression of CD54 when ligated with the LTalpha1beta2 complex. These results support the concept that directional interactions between LTalpha1beta2 bearing lymphocytes and LTbetaR bearing stromal cells are involved in the organization of lymphoid tissue.

Activation of B lymphocyte maturation by a human follicular dendritic cell line, FDC-1.

Previously, we described the characteristics of a cell line that is derived from a low density fraction of human tonsillar cells and, on the basis of a number of criteria, is related to follicular dendritic cells (FDC). This line, FDC-1, binds B lymphocytes and not T lymphocytes, and promotes anti-Ig- or anti-CD40-induced B cell proliferation. In this work, we show that culturing B cells with small numbers of FDC-1 cells leads to significant production of IL-6 and of both IgM and IgG. As few as 50 to 100 FDC-1 augmented B cell IgM production by 10- to 100-fold. Although fixed FDC-1 cells, unlike live FDC-1 cells, do not stimulate Ig production, cell contact is not required for all FDC-dependent Ig production. Supernatants from cultured FDC-1 cells can also stimulate B cells to produce IgM, suggesting that FDC produce a soluble B cell stimulating factor(s). Augmentation of FDC-dependent IgM production by either IL-6 or IL-7 and augmentation of FDC-dependent IgG production by IL-4 does require FDC-1 cells to be in contact with B cells. When the effects of FDC-1 cells were compared with those of epithelial cell lines and human foreskin fibroblasts (HFF), both FDC-1 cells and HFF induced B cells to produce IgM. FDC-1, unlike HFF, were positive for CD40, CD54, CD73, CD74, and nerve growth factor receptor (NGFR), and unlike HFF, but like certain stromal cells, FDC-1 cells also expressed smooth muscle actin, and a novel marker for stromal cells, BST-1. The possible relationship of FDC-1 cells and FDC in general to a fibroblast/stromal cell lineage is discussed.

Annual Address Delivered at the Royal College of Physicians, London

Arachidonic acids are subjected to the enzymatic transformation by PGH2 synthase or lipoxygenase upon the liberation from the membrane phospholipid. Using in vitro and in vivo experimental models, we have investigated the cellular and molecular mechanisms in the production and function of arachidonic acid metabolites. Here we report that a downstream product of 15-lipoxygenase, 15-S-hydroxyeicosatetraenoic acid (15-S-HETE) is not produced by human follicular dendritic cell (FDC)-like cells but exerts a regulatory effect on prostaglandin production by this type of cells. 15-S-HETE appears to derive from endothelial cells. FDC-like cells, which were isolated from human tonsil, do not express 15-lipoxyenase or 5-lipoxygenase but metabolize 15-S-HETE into downstream products. Immunoblotting analysis and results with chemical inhibitors imply that 15-S-HETE acts on FDC-like cells via PPAR-γ and leukotriene B4 receptor 2. 15-S-HETE enhances or inhibits the expression of PGH2 synthase and resultant prostaglandin production in FDC-like cells, which depends on the types of co-stimulation. Based on these data, we suggest a novel cellular and molecular pathway of immune regulation mediated by arachidonic acid metabolites.