No abstract

We and others have previously demonstrated that human alveolar macrophages produce more PGE2 in response to lipopolysaccharide (LPS) than do blood monocytes. We hypothesized that this observation was due to a greater increase in prostaglandin H synthase-2 (PGHS-2) enzyme mass in the macrophage compared to the monocyte. To evaluate this hypothesis, alveolar macrophages and blood monocytes were obtained from healthy nonsmoking volunteers. The cells were cultured in the presence of 0 to 10 ,g/ml LPS. LPS induced the synthesis of large amounts of a new 75-kD protein in human alveolar macrophages, and a lesser amount in monocytes. Synthesis of this protein required more than 6 h and peaked in 24 to 48 h; the protein reacted with an anti-PGHS-2 antibody prepared against mouse PGHS-2. Associated with synthesis of the protein was a marked increase in LPS-stimulated and arachidonic acid-stimulated synthesis of PGE2 by alveolar macrophages compared to monocytes. Cells not exposed to LPS contained only PGHS-1 and synthesized very little PGE2 during culture or in response to exogenous arachidonic acid. An LPSinduced mRNA, which hybridized to a human cDNA probe for PGHS-2 mRNA, was produced in parallel with production of this new protein and was produced in much greater amounts by alveolar macrophages compared to blood monocytes. This mRNA was not detectable in cells not exposed to LPS. In contrast, both types of cells contain mRNA, which hybridizes to a cDNA probe for PGHS-1. This mRNA did not increase in response to LPS. LPS also had no effect on PGHS-1 protein.These data demonstrate that PGE2 synthesis in human alveolar macrophages and blood monocytes correlates to the mass of PGHS-2 in the cell. We conclude that the greater ability of the macrophage to synthesize PGE2 in response to LPS is due to greater synthesis of PGHS-2 by the macrophage. (J. Clin. Invest. 1994. 93:391-396.)

Our previous work demonstrated that hypoxia decreases transcription of the human prostaglandin H synthase-2 (PGHS-2) gene during exposure to lipopolysaccharide (LPS), resulting in decreased prostaglandin E2 (PGE2) synthesis (J. Biol. Chem. 269:32979-32984, 1994). Because PGE2 is reported to inhibit interleukin 1 (IL-1) and tumor necrosis factor (TNF), it is likely that hypoxia, through changes in PGE2, will alter IL-1 and TNF release from the human alveolar macrophage. In addition, like PGHS-2, the TNF and IL-1 promoters contain oxidant-sensitive elements which might be altered by hypoxia. Therefore, we hypothesized that LPS-induced release of TNF and IL-1 would be altered by hypoxia. To test this, human alveolar macrophages were cultured for 24 h with 0 to 1 microgram/ml LPS in a room-air incubator with 5% CO2 or a hypoxia incubator continuously perfused with 5% CO2/95% N2 (O2 < 0.05%). With room air, LPS increased IL-1 beta mRNA and increased IL-1 beta protein release into the culture medium in a dose-dependent manner. Hypoxia increased the LPS-stimulated release of IL-1 beta 30% above that of room-air controls. However, immunoblots showed that hypoxia caused no change in intracellular IL-1 beta compared with room-air controls. There was also no change in LPS-induced IL-1 beta message with hypoxia. The inhibitor of IL-1, IL-1RA, was apparently decreased by hypoxia, but this decrease was not statistically significant. TNF-alpha mRNA and release of protein also increased during LPS exposure in room air. Hypoxia markedly increased LPS-induced TNF-alpha message and release of TNF-alpha compared with LPS-exposed room-air controls. Consistent with our prior observations, hypoxia decreased LPS-induced PGHS-2 message and protein, and also the PGHS-2 product, PGE2. Because PGE2 is reported to inhibit the expression of IL-1 and TNF genes, we inhibited PGE2 synthesis with indomethacin during culture in room air; the result was an increase in the release of IL-1 and TNF. In additional studies, adding PGE2 inhibited TNF release from the hypoxia cells to values near those of room-air controls. In summary, hypoxia increases the release of the cytokines IL-1 beta and TNF-alpha. This increase may be due to decreased PGE2 synthesis during hypoxia. These results demonstrate that the response of the human alveolar macrophage to hypoxia is complex. Hypoxia increases the LPS-stimulated release of the inflammatory cytokines IL-1 and TNF, whereas synthesis of PGHS-2, which generates the anti-inflammatory prostaglandin PGE2 is decreased.

Pericentral and periportal hepatocytes differ in their capacity to eliminate and velocity of eliminating bile acids and other organic anions. We wonder whether differences in the distribution of anion transporters (ntcp [M77479], besp [NM_031760], mrp2 [NM_012833], oatp1 [NM_017111], oatp2 [NM_131906]) cause the differences in bile acid excretion. Therefore, we analyzed the distribution of these anion transporters in periportal and pericentral cells by immunohistology, their mRNA by quantitative PCR, and regulating nuclear factors (NF-kappaB, HNF1, HNF3, HNF4, FXR, PXR) by gel shift assay. We did not find any differences in nuclear factors or regarding the proteins that could explain the zonal differences in anion transport.

No abstract