Whajung Cho

Human Follicular Dendritic Cells Interact with T Cells via Expression and Regulation of Cyclooxygenases and Prostaglandin E and I Synthases

PGE2 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 PGE2 and PGI2 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-α, TGF-β, 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 PGE2 and PGI2. Interestingly, IL-4 almost completely abrogated the stimulatory activity of TNF-α, TGF-β, and LPS in PG production. Furthermore, the up-regulation of PGE2 and PGI2 production was markedly down-regulated by indomethacin and a selective COX-2 inhibitor. PGI2 analog and PGE2 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 PGE2 and PGI2 production, suggesting an important implication for development and use of anti-inflammatory drugs.

IL-4 and IL-13 suppress prostaglandins production in human follicular dendritic cells by repressing COX-2 and mPGES-1 expression through JAK1 and STAT6

Originally discovered as a B cell growth and differentiation factor, IL-4 displays a variety of functions in many different cell types. Germinal center T cells are abundant producers of IL-4. In a recent report, we demonstrated that IL-4 inhibits prostaglandins (PGs) production in follicular dendritic cell (FDC)-like cells, HK. To understand the inhibitory mechanisms of IL-4, its effects on the biosynthesis of enzymes in charge of PG production were assessed in this study. Although IL-4 did not affect COX-1 expression, it specifically inhibited LPS-induced COX-2 biosynthesis at mRNA and protein levels. Protein expression of mPGES-1, a downstream enzyme of COX-2, was also markedly diminished by IL-4 but not by IL-10, maximizing the inhibitory activity. Next, we attempted to identify the early signaling molecules that led to this inhibition of COX-2 expression. Although IL-4 induced tyrosine phosphorylation of JAK1 and TYK2, RNA interference experiments revealed that only JAK1 was responsible for the IL-4-stimulated STAT6 phosphorylation. Knocking down JAK1 and STAT6 ablated the inhibitory effect of IL-4 on COX-2 expression and significantly reduced production of PGE(2) and prostacyclin. Similar results were obtained with IL-13. Pharmacologic inhibitors of ERK and p38 mitogen-activated protein kinases inhibited the COX-2 upregulation. However, IL-4 did not affect LPS-induced phosphorylation of ERK and p38. These results stress the essential roles of JAK1 and STAT6 in the early signaling pathway of IL-4 and IL-13 leading to suppression of COX-2 expression and repression of PG production by HK cells. Our results suggest that T cells via IL-4 play a regulatory role in PG generation in FDC. IL-4 therapeutics may be applied to immune disorders where normal and ectopic expression of germinal center reactions needs to be regulated.

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 24 h 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.

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.

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.

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.

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.