Abstract
Gram-negative bacterial endotoxin lipopolysaccharide (LPS) triggers the production of inflammatory cytokines, reactive oxygen species (ROS), and prostaglandins (PGs) by pulmonary macrophages. Here, we investigated if ROS influenced PGs production in response to LPS treatment in mouse bone marrow-derived macrophages (BMDM). We observed that pretreatment of BMDM with two structurally unrelated ROS scavengers, MnTMPyP and EUK-134, not only prevented LPS-induced ROS accumulation, but also attenuated the LPS-induced PGD2, but not PGE2, production. Conversely LPS-induced PGD2, but not PGE2, production, was potentiated with the cotreatment of BMDM with H2O2. These data suggest that ROS differentially regulate PGD2 and PGE2 production in BMDM. In addition, selective inhibition of the ROS generator NADPH oxidase (NOX) using either pharmacologic inhibitors or its p47phox subunit deficient mouse BMDM also attenuated LPS-induced PGD2, but not PGE2 production, suggesting the critical role of NOX-generated ROS in LPS-induced PGD2 production in BMDM. We further found that both hematopoietic PGD synthase (H-PGDS) siRNA and its inhibitor HQL-79, but not lipocalin PGDS (L-PGDS) siRNA and its inhibitor AT-56, significantly attenuated LPS-induced PGD2 production, suggesting that H-PGDS, but not L-PGDS, mediates LPS-induced PGD2 production in BMDM. Furthermore, data from our in vitro cell-free enzymatic studies showed that coincubation of the recombinant H-PGDS with either MnTMPyP, EUK-134, or catalase significantly decreased PGD2 production, whereas coincubation with H2O2 significantly increased PGD2 production. Taken together, our results show that LPS-induced NOX-generated ROS production differentially and specifically regulates the H-PGDS-mediated production of PGD2, but not PGE2, in mouse BMDM.
Footnotes
This work was supported in part by National Institutes of Health [Grants 1R01-HL083218, 3R01-HL083218-01A2S1, P50-AT000155, and 5R01-HL075557] to L.X., R.B.v.B., or J.W.C.; Merit Review [Grant 1I01BX000108] to J.W.C. from Jesse Brown VA Medical Center; Campus Research Board [Grant S06-118] and Faculty Scholarship Support Grants to L.X. from the University of Illinois at Chicago.
- Received June 22, 2012.
- Accepted October 15, 2012.
- U.S. government work not protected by U.S. copyright.
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