A paracrine effect of 15 (S)-hydroxyeicosatetraenoic acid revealed in prostaglandin production by human follicular dendritic cell-like cells.

Lipid mediators play active roles in each stage of inflammation under physiological and pathologic conditions. We have investigated the cellular source and functions of several prostanoids in the immune inflammatory responses using follicular dendritic cell (FDC)-like cells. In this study, we report a novel finding on the role of 15(S)- hydroxyeicosatetraenoic acid (HETE). Our observation of 15(S)-HETE uptake by FDC-like cells prompted to hypothesize that 15(S)-HETE might have a regulatory role in the other branch of eicosanoid production. The effects of 15(S)-HETE on COX-2 expression and prostaglandin (PG) production were analyzed by immunoblotting and specific enzyme immunoassays. The addition of 15(S)-HETE resulted in elevated levels of COX-2 expression and PG production. The enhanced PG production was not due to growth stimulation of FDC-like cells since 15(S)-HETE did not modulate FDC-like cell proliferation by the culture period of PG measurement. Peroxisome proliferator-activated receptor gamma (PPARγ) seems to mediate the augmenting activity as the antagonist GW9662 dose- dependently prevented 15(S)-HETE from increasing PG production. In addition, PPARγ protein expression was readily detected in FDC-like cells. These effects of 15(S)-HETE were displayed in the combined addition with IL-1ß. Based on these results, we suggest that 15(S)-HETE is an inflammatory costimulator of FDC acting in a paracrine fashion.

Activated human B cells stimulate COX-2 expression in follicular dendritic cell-like cells via TNF-α.

In spite of the potential importance of cyclooxygenase (COX)-2 expression in the germinal center, its underlying cellular and molecular mechanisms are largely unknown. COX-2 is the key enzyme generating pleiotropic prostaglandins. Based on our previous findings, we hypothesized that lymphocytes would stimulate COX-2 expression in follicular dendritic cell (FDC) by liberating cytokines. In this study, we examined the effect of tonsillar lymphocytes on COX-2 expression in FDC-like cells by immunoblotting. B but not T cells induced COX-2 protein in a time- and dose-dependent manner. Sub-fractionation analysis of B cell subsets revealed that activated but not resting B cells were responsible for the COX-2 induction. Confocal microscopy of frozen tonsils demonstrated that FDCs indeed express COX-2 in situ, in line with the in vitro results. To identify the stimulating molecule, we added neutralizing antibodies to the coculture of FDC-like cells and B cells. COX-2 induction in FDC-like cells was markedly inhibited by TNF-α neutralizing antibody. Finally, the actual production of TNF-α by activated B cells was confirmed by an enzyme immunoassay. The current study implies an unrecognized cellular interaction between FDC and B cells leading to COX-2 expression during immune inflammatory responses.

Positive feedback effect of PGE2 on cyclooxygenase-2 expression is mediated by inhibition of Akt phosphorylation in human follicular dendritic cell-like cells.

Prostaglandins (PGs) are bioactive lipid mediators generated from the phospholipids of cell membrane in response to various inflammatory signals. To understand the potential role of PGs in PG production itself during immune inflammatory responses, we examined the effect of PGE2, PGF2α, and beraprost on COX-2 expression using follicular dendritic cell (FDC)-like HK cells isolated from human tonsils. Those three PGs specifically augmented COX-2 protein expression in a dose-dependent manner after 4 or 8h of treatment. The enhancing effect was also reflected in the actual production of PGs and the viable cell recovery of germinal center B-cells. To investigate the underlying molecular mechanism, we examined the impact of PI3K inhibitors on PG-induced COX-2 expression. Interestingly, COX-2 induction by PGE2 and beraprost, but not PGF2α, was enhanced by wortmannin and LY294002. In line with this result, Akt phosphorylation was inhibited by PGE2 and beraprost but not by PGF2α. The distinct effect of PGE2 and beraprost from PGF2α was reproduced in Akt-knockdowned HK cells. Our current findings imply that PGE2 and PGI2 stimulate COX-2 expression in FDC by inhibiting Akt phosphorylation. Additional studies are warranted to determine the potential role of Akt as a therapeutic target in patients with inflammatory disorders.

Factors influencing the difference between forecasted and actual drug sales volumes under the price-volume agreement in South Korea.

This study analyzed factors contributing to increases in the actual sales volumes relative to forecasted volumes of drugs under price-volume agreement (PVA) policy in South Korea. Sales volumes of newly listed drugs on the national formulary are monitored under PVA policy. When actual sales volume exceeds the pre-agreed forecasted volume by 30% or more, the drug is subject to price-reduction. Logistic regression assessed the factors related to whether drugs were the PVA price-reduction drugs. A generalized linear model with gamma distribution and log-link assessed the factors influencing the increase in actual volumes compared to forecasted volume in the PVA price-reduction drugs. Of 186 PVA monitored drugs, 34.9% were price-reduction drugs. Drugs marketed by pharmaceutical companies with previous-occupation in the therapeutic markets were more likely to be PVA price-reduction drugs than drugs marketed by firms with no previous-occupation. Drugs of multinational pharmaceutical companies were more likely to be PVA price-reduction drugs than those of domestic companies. Having more alternative existing drugs was significantly associated with higher odds of being PVA price-reduction drugs. Among the PVA price-reduction drugs, the increasing rate of actual volume compared to forecasted volume was significantly higher in drugs with clinical usefulness. By focusing the negotiation efforts on those target drugs, PVA policy can be administered more efficiently with the improved predictability of the drug sales volumes.

Suppressor of cytokine signaling 1 is a positive regulator of TGF-ß-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-ß-induced cyclooxygenase (COX)-2 augmentation in follicular dendritic cell-like cells. TGF-ß 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-ß-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-ß-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.

Interferon-γ stimulates human follicular dendritic cell-like cells to produce prostaglandins via the JAK-STAT pathway.

IFN-γ plays a critical role in the regulation of innate and adaptive immunity. Paying attention to the emerging role of prostaglandins (PGs) as immune regulators, we attempted to establish the effect of IFN-γ on PG production in human follicular dendritic cell-like HK cells and the underlying signaling pathway by using RNA interference technology. IFN-γ induced COX-2 protein expression in HK cells in a time- and dose-dependent manner, which was not observed in peripheral blood monocytes. Although IFN-γ induced phosphorylation of STAT1, STAT3, and STAT5, only STAT1 was essential for the COX-2 augmentation. The JAK kinases responsible for IFN-γ-triggered STAT1 phosphorylation were JAK1 and JAK2, which were also required for the COX-2 induction. The essential requirement of JAK1 and JAK2 was verified by confocal microscopic analysis, since STAT1 phosphorylation and nuclear translocation were impaired in HK cells with these two kinases knocked down. Finally, we demonstrated that JAK1, JAK2, and STAT1 were indispensable for the actual enhancement of PG production in response to IFN-γ stimulation. These results provide a novel insight into our understanding of IFN-γ under inflammatory conditions and support the emerging concept of PGs as important immune regulators.

Retinoic acid acts as a selective human IgA switch factor.

Retinoic acid (RA) is known to have several functions that lead to a potent mucosal IgA response. Nevertheless, its exact role in human IgA synthesis has yet to be elucidated. Thus, we investigated the role of RA in promoting IgA isotype switching in human B cells. We found that RA increased IgA production and the expression of germ-line IgA1 and IgA2 transcripts (GLTα1 and GLTα2). This induction occurred alongside an increase in the frequency of IgA1-secreting B cell clones, as assessed by limiting dilution analysis. Under the same conditions, RA did not increase IgM and IgG production. Am80, an agonist of RA receptor α (RARα), increased IgA production. In addition, RA activity was abrogated by LE540, an antagonist of RAR, suggesting that the RAR pathway is involved in RA-induced IgA production. Taken together, these results indicate that RA induces IgA isotype switching mainly through RARα in human B cells.

Human follicular dendritic cells promote germinal center B cell survival by providing prostaglandins.

Evidence for the immunoregulatory function of lipid molecules in addition to proteins is accumulating. Based on our previous reports on the production of prostaglandin E2 (PGE2) and prostacyclin by human follicular dendritic cells (FDCs), we hypothesized that these prostaglandins (PGs) have a regulatory effect on the survival, proliferation, and differentiation of germinal center B (GC) cells. We observed that PGE2 and a prostacyclin analog (beraprost) specifically enhanced the viable recovery of GC B cells in dose-dependent manners. FDC-like cells also mimicked the effect of PGE2, which was significantly inhibited in the presence of indomethacin. The viable recovery was due to the prevention of cell death by PGE2 and beraprost but did not result from augmented proliferation of GC B cells. The effect of PGE2 and beraprost was manifest when they were added at early times of the culture. Interestingly, we found that the combined addition of a pan-caspase inhibitor and a necroptosis inhibitor gave rise to a similar result to PGE2. Finally, PGE2 and beraprost enhanced the generation of both CD20⁻CD38⁺ plasma cells and CD20⁺CD38⁻ memory B cells from GC B cells. Our current data suggest that FDCs play an important function of sustaining GC B cell survival by providing PGs. Our data also implies that selective cyclooxygenase inhibitors administered during infection or vaccination may have an adverse effect on the course of humoral immune response.

Beraprost enhances production of antigen-specific IgG isotypes without modulating germinal center B cell generation and the affinity maturation.

Immune regulating functions of lipid mediators are being recognized. Prostaglandins (PGs) are derived from phospholipid by cyclooxygenase-2 (COX-2) in inflammatory tissues. Based upon our previous data that imply an immune modulating activity of prostacyclin, we hypothesized that PGs promote the humoral immune responses in vivo. To test this hypothesis, we examined the effect of a prostacyclin analog beraprost and PGE2 on the antibody responses that were induced by immunizing mice with keyhole limpet hemocyanin (KLH). Beraprost was used due to the extremely unstable property of prostacyclin. Our results showed that beraprost indeed promoted the production of anti-KLH antibodies, which was dose-dependent and specific to beraprost because PGE2 did not modulate the antibody response compared with controls. The enhancing effect of beraprost was reproduced in the secondary responses, suggesting that memory B cell generation during the primary response was significantly affected by beraprost. Analysis of the isotypes of anti-KLH antibodies revealed that beraprost stimulated the production of IgA and IgG subisotypes but not IgM. However, germinal center B cell generation and the affinity of anti-KLH antibodies were not affected by beraprost administration. To confirm these results we immunized COX-2 knockout mice with KLH and analyzed whether the results with wild-type mice were reflected in the absence of PGs. The primary and secondary antibody responses were significantly impaired in the KO animals. The levels of anti-KLH IgG subisotypes and IgA were severely reduced in KO mice whereas those of IgM were comparable to controls. These results reveal an unrecognized function of PG in the humoral immune responses.

Human follicular dendritic cells promote the APC capability of B cells by enhancing CD86 expression levels.

Follicular dendritic cells (FDCs) are an essential cellular component of the germinal center (GC) and are believed to exert regulatory effects on the various stages of GC reactions. According to our previous reports, human FDCs express prostacyclin synthase, and prostacyclin analogues augment adhesion and co-stimulatory molecules on the surface of activated B cells. These findings prompted us to investigate whether FDCs would contribute to the antigen-presenting capability of B cells by using the well-established FDC-like cells, HK cells, and tonsillar B cells. Our results show that HK cells significantly enhance the expression levels of CD54, CD80, and CD86 on the surface of activated B cells. The enhancing effect of HK cells on CD86 is impeded by indomethacin and an EP4 antagonist, implying that a certain prostaglandin is mediating the up-regulation. Prostacyclin indeed recapitulates the enhancing effect on CD86, which is inhibited by EP4 as well as IP antagonists. B cells co-cultured with HK cells exhibit an augmented APC activity, which is inhibited by CD86 neutralization. These results reveal another unrecognized function of human FDC.

Beraprost enhances the APC function of B cells by upregulating CD86 expression levels.

Lipid mediators are emerging as important regulators of the immune system. Based on our previous result that shows strong expression of prostacyclin synthase in the germinal center, we investigated whether prostacyclin would regulate the APC function of B cells. Owing to the very short half-life of prostacyclin in experimental conditions, we used a more stable analog, beraprost. Beraprost increased the amounts of the costimulatory molecule CD86 but not CD80 on the surface of activated B cells in time- and dose-dependent manners. However, the enhancing effect of beraprost was not observed on memory B cells, centroblasts, and centrocytes. Beraprost required BCR and CD40 signals to upregulate CD86 expression levels. Other prostanoids such as PGE(2), 6-keto-PGF(1α), and PGF(2α) failed to alter CD86 expression levels, whereas other prostacyclin analogs were as potent as beraprost. Results carried out with receptor antagonists revealed that beraprost enhanced CD86 levels by binding to prostacyclin receptor IP and by increasing intracellular cAMP concentrations. Beraprost-treated B cells potently stimulated allogeneic T cells, which was significantly abolished by CD86 neutralization. Our data imply an unrecognized cellular and molecular mechanism about the germinal center reactions.

Production of prostaglandin e(2) and i(2) 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 E(2) (PGE(2)) and prostaglandin I(2) (PGI(2)) 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 PGE(2) and PGI(2) production in HK cells, we performed siRNA technology to knock down COX enzymes and examined the effect on PG production. RESULTS: Both PGE(2) and PGI(2) 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.

Human follicular dendritic cells interact with T cells via expression and regulation of cyclooxygenases and prostaglandin E and I synthases.

PGE(2) inhibits mature T cell proliferation and protects T cells from activation-induced cell death (AICD). We have previously demonstrated that human follicular dendritic cells (FDC) strongly express PGI synthase. In this study, the hypothesis that FDC have regulatory roles on germinal center T cells by controlling production of PGE(2) and PGI(2) was tested. Confocal microscopic analyses of human tonsil tissues revealed that FDC indeed expressed PGE synthase in addition to PGIS. To confirm these results, we studied the regulation mechanism of PG production in FDC, using an established human FDC-like cell line, HK. Specifically in response to TNF-alpha, TGF-beta, and LPS, protein expression of cyclooxygenase (COX)-2 and downstream PGE synthase was up-regulated with coordinate kinetics, whereas COX-1 and PGIS were constitutively expressed. The increase of these enzymes was reflected in actual production of PGE(2) and PGI(2). Interestingly, IL-4 almost completely abrogated the stimulatory activity of TNF-alpha, TGF-beta, and LPS in PG production. Furthermore, the up-regulation of PGE(2) and PGI(2) production was markedly down-regulated by indomethacin and a selective COX-2 inhibitor. PGI(2) analog and PGE(2) inhibited proliferation and AICD of T cells in dose- and time-dependent manners. Finally, coculture experiments revealed that HK cells indeed inhibit proliferation and AICD of T cells. Put together, these results show an unrecognized pathway of FDC and T cell interactions and differential mechanisms for PGE(2) and PGI(2) production, suggesting an important implication for development and use of anti-inflammatory drugs.