Limits...
Production of prostaglandin e(2) and i(2) is coupled with cyclooxygenase-2 in human follicular dendritic cells.

Cho W, Kim J, Cho KB, Choe J - Immune Netw (2011)

Bottom Line: 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.In contrast, COX-1 knockdown did not significantly affect PG production induced by lipopolysaccharide (LPS).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, Kangwon National University, Chuncheon 200-701, Korea.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Depletion of COX-2 but not COX-1 prevents LPS-induced production of PGE2 and PGI2. Control, COX-1, or COX-2 siRNA-transfected HK cells (1×105 cells) were cultured for 48 h, followed by further incubation in the presence or absence of LPS (1µg/ml) for another 48 h. The amounts of PGE2 (A) and 6-keto-PGF1α (B) in the culture supernatants were measured by EIA and normalized by protein concentration in each well. Data are presented as means±standard error of the mean (SEM) of duplicates. Representative results of three reproducible experiments. Asterisks indicate significant differences (*p <0.05, **p<0.01). NS, non-significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3275705&req=5

Figure 2: Depletion of COX-2 but not COX-1 prevents LPS-induced production of PGE2 and PGI2. Control, COX-1, or COX-2 siRNA-transfected HK cells (1×105 cells) were cultured for 48 h, followed by further incubation in the presence or absence of LPS (1µg/ml) for another 48 h. The amounts of PGE2 (A) and 6-keto-PGF1α (B) in the culture supernatants were measured by EIA and normalized by protein concentration in each well. Data are presented as means±standard error of the mean (SEM) of duplicates. Representative results of three reproducible experiments. Asterisks indicate significant differences (*p <0.05, **p<0.01). NS, non-significant.

Mentions: To investigate the relative contribution of COX-1 and COX-2 to the production of PGE2 and PGI2, we carried out siRNA technology to knock down COX-1 and COX-2 proteins in HK cells. HK cells were transfected with siRNA duplexes specific to COX-1 and COX-2 and cultured for 48 h, followed by further cultures in the presence or absence of LPS. The silencing of target proteins was demonstrated by immunoblotting. As shown in Fig. 1, COX-2 protein levels were up-regulated by LPS stimulation in control siRNA-transfected HK cells, whereas COX-1 levels were unaffected by LPS treatment. Transfection with COX-1-specific siRNA resulted in significant reduction of COX-1 protein levels regardless of LPS stimulation. Interestingly, LPS-induced COX-2 levels in COX-1 siRNA-transfected cells were markedly higher compared to control cells. Transfection with COX-2-specific siRNA almost completely prevented induction of COX-2 proteins that was triggered by LPS. COX-2 silencing did not significantly affect COX-1 expression levels. These results indicate that COX-1 and COX-2 proteins were successfully knocked down in HK cells by siRNA duplexes. We next measured the concentrations of PGE2 and 6-keto-PGF1α in the culture supernatants after incubation of siRNA-transfected cells in the presence or absence of LPS for 48 h. 6-keto-PGF1α is the hydrolysis product of unstable PGI2. PGE2 and 6-keto-PGF1α concentrations in control cells were increased 4- and 5-folds, respectively, by LPS stimulation (Fig. 2). Similar levels of enhancement were obtained in COX-1 siRNA-transfected cells. In contrast, COX-2 silencing almost completely prevented the PG production to background levels. We observed that knock down of COX-1 resulted in a significant increase of PGE2 but not PGI2 without LPS stimulation (Fig. 2A). Whether this result reflects a preferential coupling of COX-2 with PGES over PGIS is currently unclear. Based upon these results, we conclude that PGE2 and PGI2 production is coupled with COX-2 but not with COX-1 in HK cells.


Production of prostaglandin e(2) and i(2) is coupled with cyclooxygenase-2 in human follicular dendritic cells.

Cho W, Kim J, Cho KB, Choe J - Immune Netw (2011)

Depletion of COX-2 but not COX-1 prevents LPS-induced production of PGE2 and PGI2. Control, COX-1, or COX-2 siRNA-transfected HK cells (1×105 cells) were cultured for 48 h, followed by further incubation in the presence or absence of LPS (1µg/ml) for another 48 h. The amounts of PGE2 (A) and 6-keto-PGF1α (B) in the culture supernatants were measured by EIA and normalized by protein concentration in each well. Data are presented as means±standard error of the mean (SEM) of duplicates. Representative results of three reproducible experiments. Asterisks indicate significant differences (*p <0.05, **p<0.01). NS, non-significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275705&req=5

Figure 2: Depletion of COX-2 but not COX-1 prevents LPS-induced production of PGE2 and PGI2. Control, COX-1, or COX-2 siRNA-transfected HK cells (1×105 cells) were cultured for 48 h, followed by further incubation in the presence or absence of LPS (1µg/ml) for another 48 h. The amounts of PGE2 (A) and 6-keto-PGF1α (B) in the culture supernatants were measured by EIA and normalized by protein concentration in each well. Data are presented as means±standard error of the mean (SEM) of duplicates. Representative results of three reproducible experiments. Asterisks indicate significant differences (*p <0.05, **p<0.01). NS, non-significant.
Mentions: To investigate the relative contribution of COX-1 and COX-2 to the production of PGE2 and PGI2, we carried out siRNA technology to knock down COX-1 and COX-2 proteins in HK cells. HK cells were transfected with siRNA duplexes specific to COX-1 and COX-2 and cultured for 48 h, followed by further cultures in the presence or absence of LPS. The silencing of target proteins was demonstrated by immunoblotting. As shown in Fig. 1, COX-2 protein levels were up-regulated by LPS stimulation in control siRNA-transfected HK cells, whereas COX-1 levels were unaffected by LPS treatment. Transfection with COX-1-specific siRNA resulted in significant reduction of COX-1 protein levels regardless of LPS stimulation. Interestingly, LPS-induced COX-2 levels in COX-1 siRNA-transfected cells were markedly higher compared to control cells. Transfection with COX-2-specific siRNA almost completely prevented induction of COX-2 proteins that was triggered by LPS. COX-2 silencing did not significantly affect COX-1 expression levels. These results indicate that COX-1 and COX-2 proteins were successfully knocked down in HK cells by siRNA duplexes. We next measured the concentrations of PGE2 and 6-keto-PGF1α in the culture supernatants after incubation of siRNA-transfected cells in the presence or absence of LPS for 48 h. 6-keto-PGF1α is the hydrolysis product of unstable PGI2. PGE2 and 6-keto-PGF1α concentrations in control cells were increased 4- and 5-folds, respectively, by LPS stimulation (Fig. 2). Similar levels of enhancement were obtained in COX-1 siRNA-transfected cells. In contrast, COX-2 silencing almost completely prevented the PG production to background levels. We observed that knock down of COX-1 resulted in a significant increase of PGE2 but not PGI2 without LPS stimulation (Fig. 2A). Whether this result reflects a preferential coupling of COX-2 with PGES over PGIS is currently unclear. Based upon these results, we conclude that PGE2 and PGI2 production is coupled with COX-2 but not with COX-1 in HK cells.

Bottom Line: 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.In contrast, COX-1 knockdown did not significantly affect PG production induced by lipopolysaccharide (LPS).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, Kangwon National University, Chuncheon 200-701, Korea.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus