Zymosan-induced glycerylprostaglandin and prostaglandin synthesis in resident peritoneal macrophages: roles of cyclo-oxygenase-1 and -2

Abstract

COX [cyclo-oxygenase; PG (prostaglandin) G/H synthase] oxygenates AA (arachidonic acid) and 2-AG (2-arachidonylglycerol) to endoperoxides that are converted into PGs and PG-Gs (glycerylprostaglandins) respectively. In vitro, 2-AG is a selective substrate for COX-2, but in zymosan-stimulated peritoneal macrophages, PG-G synthesis is not sensitive to selective COX-2 inhibition. This suggests that COX-1 oxygenates 2-AG, so studies were carried out to identify enzymes involved in zymosan-dependent PG-G and PG synthesis. When macrophages from COX-1-/- or COX-2-/- mice were treated with zymosan, 20-25% and 10-15% of the PG and PG-G synthesis observed in wild-type cells respectively was COX-2 dependent. When exogenous AA and 2-AG were supplied to COX-2-/- macrophages, PG and PG-G synthesis was reduced as compared with wild-type cells. In contrast, when exogenous substrates were provided to COX-1-/- macrophages, PG-G but not PG synthesis was reduced. Product synthesis also was evaluated in macrophages from cPLA(2alpha) (cytosolic phospholipase A2alpha)-/- mice, in which zymosan-induced PG synthesis was markedly reduced, and PG-G synthesis was increased approx. 2-fold. These studies confirm that peritoneal macrophages synthesize PG-Gs in response to zymosan, but that this process is primarily COX-1-dependent, as is the synthesis of PGs. They also indicate that the 2-AG and AA used for PG-G and PG synthesis respectively are derived from independent pathways.

Figure 1. Formation of PGs and PG-Gs by RPM in response to zymosan

Cultures of RPM from WT, COX-1^−/− or COX-2^−/− mice in 35 mm dishes were pre-incubated for 6 h in the absence (−LPS) or presence (+LPS) of 100 ng/ml bacterial LPS. Cells were washed, transferred to serum-free medium, and then incubated for 2 h with 160 μg of zymosan. The medium was then removed and analysed for PG-Gs (A, B) and PGs (C, D) by LC/MS/MS. Values are given for total product (A, C) or for PGE₂-G or PGE₂ (B, D), the remainder being PGI₂-G or PGI₂. In the absence of zymosan, WT cells pre-incubated in the absence of LPS produced 56±11 pmol of PGs/10⁷ cells and those pre-incubated in the presence of LPS produced 360±60 pmol/10⁷ cells. Formation of PG-Gs was undetectable in the absence of a zymosan stimulus. Asterisk indicates that results are significantly different from WT RPM of the same treatment. The star symbol indicates that results from LPS-pretreated RPM are significantly different from RPM of the same genotype that were not treated with LPS (P<0.05). The results, including values given for PG formation in the absence of zymosan, are the means±S.D. for three separate experiments in which triplicate determinations were made.

Figure 2. AG and AA levels in zymosan-treated RPM

Experimental conditions are exactly as described for Figure 1. Following the 2 h incubation with zymosan, the cells were scraped into methanol and analysed for AG (A) and AA (B) content by LC/MS/MS. Asterisk indicates that results are significantly different (P<0.05) from WT RPM of the same treatment. The star symbol indicates that results from LPS-pretreated RPM are significantly different (P<0.01) from RPM of the same genotype that were not treated with LPS. The results are the means±S.D. for three separate experiments in which duplicate determinations were made.

Figure 3. Expression of COX-1, COX-2 and cPLA_2α in zymosan-treated RPM

RPM cultures were prepared and incubated with LPS and zymosan as described in the legend to Figure 1. Following the zymosan incubation, the medium was removed, and the cells were washed and scraped into lysis buffer for immunoblot analysis of COX-1 (A), COX-2 (B) or cPLA_2α (C) expression. Chemiluminescence was measured using a Fluor-S Max imager (Bio-Rad, Hercules, CA, U.S.A.), and results were normalized to the expression of WT enzyme in each experiment. Results are the means±S.D. for three separate experiments in which single or duplicate determinations were made. Asterisk indicates that results are significantly different from WT RPM at the indicated P value. Example blots are shown for each enzyme. Lane identification numbers are as follows: 1 and 2, WT cells; 3 and 4, COX-2^−/− cells; 5 and 6, COX-1^−/− cells.

Figure 4. Synthesis of PGs by RPM from exogenous AA

Cultures of RPM from WT, COX-1^−/− and COX-2^−/− mice in 35 mm dishes were pre-incubated for 6 h in the absence (−LPS) or presence (+LPS) of 100 ng/ml LPS. Cells were washed and transferred to serum-free medium, containing 1 μM AA. The cells were incubated for 30 min, and the medium was then removed and analysed for PGs by LC/MS/MS. The results are the means±range for two separate experiments in which duplicate determinations were made. Asterisk indicates that results are significantly different from WT RPM of the same treatment. The star symbol indicates that results from LPS-pretreated RPM are significantly different from RPM of the same genotype that were not treated with LPS (P<0.05).

Figure 5. Synthesis of PGs and PG-Gs by RPM from exogenous 2-AG

Experimental conditions are exactly as described in the legend to Figure 4, except that cells were transferred to serum-free medium, containing 1 μM 2-AG. The medium was analysed for PGs (A) or PG-Gs (B) by LC/MS/MS. The results are the means±range for two separate experiments in which duplicate determinations were made. Asterisk indicates that results are significantly different from WT RPM of the same treatment. The star symbol indicates that results from LPS-pretreated RPM are significantly different from RPM of the same genotype that were not treated with LPS (P<0.05).

Figure 6. Time course of substrate release by WT and cPLA_2α^−/− RPM during zymosan phagocytosis

RPM from WT and cPLA_2α^−/− mice in 35 mm dishes were incubated for 6 h in the presence of 100 nM LPS. Cells were then washed and overlaid with serum-free medium. Zymosan (160 μg) was added to each dish, and the cells were incubated for the indicated times. The cells were scraped into methanol for the LC/MS/MS analysis of AA (A) and total AG (B). Results are the means±range for two experiments in which duplicate determinations were made. Asterisk indicates that results are significantly different between WT and cPLA_2α^−/− RPM at the same time point.

Figure 7. Time course of PG and PG-G formation by WT and cPLA_2α^−/− RPM during zymosan phagocytosis

Experimental conditions are exactly as described in the legend to Figure 6. At the indicated times after zymosan addition, the medium was removed for analysis by LC/MS/MS of PG (A) and PG-G (B) content. Results are the means±range for two experiments in which duplicate determinations were made. Asterisk indicates that results are significantly different between WT and cPLA_2α^−/− RPM at the same time point.