Douglas C. Kuhn

Neutrophil attractant/activating peptide-1/interleukin-8: Association with histologic chorioamnionitis, preterm delivery, and bioactive amniotic fluid leukoattractants†‡

OBJECTIVES The goals of this study were (1) to determine immunoreactive neutrophil attractant/activating peptide-1/interleukin-8 levels in amniotic fluid from patients with preterm labor and (2) to compare neutrophil attractant/activating peptide-1/interleukin-8 levels, amniotic fluid culture, Gram stain, and the leukotaxis bioassay for their ability to predict histologic chorioamnionitis and clinical outcome. STUDY DESIGN Amniotic fluid was collected by amniocentesis from 55 patients with idiopathic preterm labor and three patients with preterm labor and clinical chorioamnionitis. Gram stain, culture (aerobic, anaerobic, and Mycoplasma species), leukotaxis bioassay, and a commercially available neutrophil attractant/activating peptide-1/interleukin-8 enzyme-linked immunosorbent assay (sensitivity 1 ng/ml) were performed on the amniotic fluid samples. Placentas and chorionic membranes were evaluated for evidence of histologic chorioamnionitis in patients delivered preterm. RESULTS All patients with detectable leukoattractants by the leukotaxis bioassay had neutrophil attractant/activating peptide-1/interleukin-8 levels above the threshold of the assay. The presence of amniotic fluid neutrophil attractant/activating peptide-1/interleukin-8 is a more sensitive marker for histologic chorioamnionitis and delivery before 34 weeks than is amniotic fluid culture (100% vs 59%, p < 0.01; and 95% vs 56%, p < 0.01, respectively). Also, of patients in idiopathic preterm labor those without amniotic fluid leukoattractants (group 1) had the lowest amniotic fluid levels, followed by patients with amniotic fluid leukoattractants and a negative culture (group 2) and patients with amniotic fluid leukoattractants and a positive culture (group 3) who had the highest levels (group 1 vs group 2, p < 0.001; group 2 vs group 3, p < 0.01). CONCLUSIONS Amniotic fluid neutrophil attractant/activating peptide-1/interleukin-8, like the leukotaxis assay, is an accurate antepartum predictor of histologic chorioamnionitis and subsequent early delivery in patients with preterm onset of labor. This study supports the role of neutrophil attractant/activating peptide-1/interleukin-8 in the recruitment of neutrophils into chorionic membranes and placenta during developing intrauterine infection.

Placental essential fatty acid transport and prostaglandin synthesis.

The studies reported here demonstrate two important aspects of placenta EFA transport and metabolism. (1) A mechanism exists within the placenta for the selective incorporation of 20:4 omega 6 into phosphoglycerides and the export of those phosphoglycerides to the fetal circulation. This mechanism allows the selective sequestering of 20:4 omega 6 in the fetoplacental unit and may provide the fetus with important performed structural membrane components. (2) Placental PG synthesis is directed mostly to the maternal circulation and stimulated placental PG synthesis is directed totally to the maternal circulation. This mechanism may protect the fetus from fluctuations in maternal and placental PG synthesis and may direct stimulated placental PG synthesis to a target organ, the myometrium. The perfused human placental model provides a valuable method for the study of a variety of biochemical phenomena in a whole human organ and its use may further elucidate the role of this tissue in the maintenance of pregnancy, the transport of EFA to the developing fetus and the involvement of placental PG synthesis in fetal development and parturition.

Alterations in Transfer and Lipid Distribution of Arachidonic Acid in Placentas of Diabetic Pregnancies

Placental tissue from nondiabetic term pregnancies and pregnancies complicated by maternal insulindependent diabetes mellitus (IDDM) was perfused in vitro to compare the transfer and lipid distribution of arachidonic acid (AA). Radiolabeled albumin-bound AA was administered into the maternal afferent circulation, and samples of fetal and maternal effluent were collected at 10-min intervals. Perfused placental tissue was collected at the end of each experiment. The effluent was analyzed for total radioactivity, and extracts were subjected to thin-layer chromatography for the assessment of radioactivity associated with various lipid fractions. Placental AA uptake was significantly increased in perfused tissue from diabetic pregnancies (0.88 vs. 1.72 nM · min−1 · g−1 in nondiabetic and IDDM, respectively; P < 0.01), as was AA transfer (0.22 vs. 0.42 ml/min in nondiabetic and IDDM, respectively; P < 0.01). However, transfer of the highly diffusible marker substance antipyrine was significantly reduced in IDDM placentas (1.79 vs. 2.49 ml/min in IDDM and nondiabetic, respectively; P < 0.01). Compared with nondiabetic placentas, incorporation of AA into triglyceride was significantly increased in both maternal and fetal effluents and in placental tissue from IDDM pregnancies, whereas the percentage of AA remaining unesterified was reduced in both placental tissue and fetal effluent. Incorporation of AA into phosphoglycerides was significantly reduced in placental tissue but increased in fetal effluent in placentas from IDDM pregnancies. The results of these studies suggest that transfer and lipid distribution of AA are significantly altered in placentas from IDDM pregnancies. These findings may be relevant to the increased incidence of abnormal fetal growth and development associated with IDDM pregnancies.

Eicosanoid production and transfer in the placenta of the diabetic pregnancy

The metabolism of arachidonic acid (AA) and the transfer of its metabolites was determined in in vitro perfused placental tissue from normal pregnancies and those complicated by maternal insulin-dependent diabetes mellitus (IDDM). 14C-labelled AA was recirculated in the fetal circulation for 60 min while 3H-AA was recirculated in the maternal circulation. Placental effluent was subjected to high performance liquid chromatography (HPLC) and analysis of dual-label scintillation counts. Placentae from IDDM pregnancies converted 3-6 times more radiolabelled AA to eicosanoids than did normal placentae. In addition, the transfer of eicosanoids into the opposing circulation was doubled in placentae from IDDM pregnancies compared to normal placentae. The predominant direction of eicosanoid transfer in both groups of placentae was in the fetal-to-maternal direction. The relative amounts of eicosanoids produced was also altered in placentae from IDDM pregnancies. Increased amounts of thromboxane (Tx) B2 and hydroxyeicosatetraenoic acids (HETEs) were present in both circulations of placentae from IDDM pregnancies. Levels of 6-keto prostaglandin F1a (6KPGF1a) were significantly reduced in both circulations in placentae from IDDM pregnancies. Thus, the ratio of TxA2 to PGI2 and the ratio of HETEs to PGI2 were both significantly increased in placentae from IDDM pregnancies. These results suggest an imbalance in eicosanoid production which may be relevant to abnormal placental structure and function in IDDM pregnancies.

Induction of nitric oxide and nitric oxide synthase mRNA by silica and lipopolysaccharide in PMA‐primed THP‐1 cells

Nitric oxide (NO), a nitrogen‐free radical, plays an important role in mediating inflammatory reaction and cytotoxicity of tissue. To determine whether NO was involved in silica‐induced pulmonary tissue damage, we studied the effects of silica on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression by THP‐1 cells, a monocyte‐like cell line with properties of the pulmonary alveolar macrophage. Experimental results showed that silica elicited a marked stimulation of nitric oxide production in a time‐dependent manner by THP‐1 cells in vitro following the priming of these cells with the phorbol ester PMA. Both nitric oxide synthase inhibitor N‐monomethyl‐L‐arginine (NMMA) and xanthine oxidase inhibitor allopurinol can partially suppress silica‐induced NO production in PMA‐primed THP‐1 cells. Northern blot analysis indicated that, after 2 h of silica exposure, PMA‐primed THP‐1 cells began to express iNOS mRNA, which reached peak expression at 8 h. Endotoxin treatment of these cells produced a similar effect. These results indicated that silica is a potent inducer of NO production in macrophages and its ability to induce tissue damage may partially be attributed to its ability to initiate excessive production of nitric oxide from macrophages.

Aspects of in vitro placental perfusion: Effects of hyperoxia and phenol red

The study of a number of parameters of placental function indicated that the perfused human placental lobe maintained its structural and functional integrity when PO2 levels in buffer perfusate were near physiological values, despite low O2 consumption. High O2 content in the perfusate may reduce placental transfer either through a direct vasoconstrictor effect or in combination with the destruction of vascular cyclo-oxygenase, resulting in the reduced synthesis of the vasodilator prostacyclin. A similar mechanism may be involved in the reduction of placental transfer observed in the presence of phenol red. These studies suggest that aspects of in vitro methodologies which may relate to prostaglandin production deserve careful consideration and further study.

An inhibitor of thromboxane production attenuates tumor necrosis factor release by activated human alveolar macrophages

Tumor necrosis factor alpha (TNF alpha) and thromboxane A2 (TXA2) are major products of the activated alveolar macrophage and serve as key mediators of lung injury. In order to determine if the synthesis of TXA2 and the release of TNF alpha are associated, the production of these inflammatory agents by the human alveolar macrophage (AM), as a result of activation by lipopolysaccharide (LPS), was assessed in the absence and presence of the thromboxane synthase inhibitors UK 38,485 (Dazmegrel) and OKY 046. UK 38,485 and OKY 046 inhibited both LPS-stimulated TXA2 production and TNF alpha release in a dose-dependent manner. Prostaglandin E2 (PGE2) production was not increased by UK 38,485 or OKY 046. Neither LPS nor UK 38,485 had any effect on LTB4 production by AM. Neither UK 38,485 or OKY 046 had any effect on LPS-stimulated interleukin-1 beta release. However, the TXA2 mimetic, U46619, did not stimulate TNF alpha release by AM either in the absence or presence of UK 38,485. These findings suggest that 1) UK 38,485 and OKY 046 are inhibitors of both TXA2 production and TNF alpha release by activated human AM, 2) UK 38,485 probably does not exert its inhibitory action on TNF alpha release through effects on eicosanoid production and 3) the possibility that TNF alpha- and TXA2-induced lung injury may be subject to amelioration by imidazole-based compounds should be further evaluated.

Cyclooxygenase inhibition reduces placental transfer: Reversal by carbacyclin

The effect of the cyclooxygenase inhibitors, indomethacin and ibuprofen, on diffusional transfer in the human placenta was assessed with the dual-perfused isolated placental lobe. Antipyrine, a freely diffusible substance, was used as an indicator of placental transfer efficiency. Each inhibitor (100 mumol/L) was perfused for 30 minutes after a baseline period, resulting in a significant reduction in antipyrine clearance. During a subsequent washout period, ibuprofen-inhibited antipyrine clearance returned to baseline values, whereas indomethacin-inhibited clearance remained reduced. An additional 30 minutes perfusion of 500 mumol/L of ibuprofen resulted in a further reduction in antipyrine clearance compared with 100 mumol/L of ibuprofen, suggesting a dosage-related effect. The perfusion of each inhibitor caused a reduced production of 6-keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin) in the fetal circulation. The simultaneous perfusion of carbacyclin, a prostacyclin analogue, at 100 nmol/L and 1 mumol/L resulted in a dosage-dependent reversal of the effects of ibuprofen (500 mumol/L) on antipyrine clearance. The results indicate that the inhibition of cyclooxygenase activity reduces placental transfer and that the effects of these inhibitors are reversed by carbacyclin. This study suggests that the use of cyclooxygenase inhibitors during pregnancy could compromise the developing fetus by reducing placental transfer.

Characterization of alveolar macrophage eicosanoid production in a non-human primate model of mineral dust exposure

The relative activation of eicosanoid production which results from the exposure of the alveolar macrophage (AM) to mineral dusts is thought to be a key factor in the pathophysiology of occupational lung disease. We compared in vitro basal and silica-stimulated production of prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) by AM from normal humans and non-human primates (Macaca nemestrina). In addition, we instilled mineral dusts directly into one lung of the non-human primate and evaluated AM eicosanoid production at two week intervals following dust instillation. Unstimulated AM from humans produce more PGE2 and TXA2 than do AM from M. nemestrina. However, in vitro exposure of AM from both species to silica dust produced a qualitatively similar increase in TXA2 production accompanied by no change in PGE2 production. Sequential analysis of AM eicosanoid production following a single bolus exposure to bituminous or anthracite coal dusts, titanium dioxide (TiO2) dust or crystalline silica showed marked variability among individual non-human primates in qualitative and quantitative aspects of dust-induced eicosanoid production. However, the rank order of potency of the different dusts (silica > anthracite > bituminous) correlated with epidemiological evidence relating the type of dust mined to the incidence of pneumoconiosis. These studies suggest that the non-human primate may serve as a model for the study of both the role of eicosanoids in the etiology of dust-induced occupational lung disease and the biochemical basis for individual variability in the response of lung cells to mineral dust exposure.

Bacterial Endotoxin Primes the Human Alveolar Macrophage for Subsequent Stimulation by Silica but Silica Does Not Prime for Stimulation by Bacterial Endotoxin

Abstract It has been suggested that prior exposure to mineral dust may attenuate the ability of the alveolar macrophage (AM) to respond to subsequent provocative stimuli, thus compromising the host defense mechanism. On the contrary, prior exposure of the AM with lipopolysacharride (LPS) has been shown to sensitize the AM for subsequent stimuli, thereby enhancing the reactivity of the AM. Since AM-derived eicosanoids serve as important mediators of the lungs response to AM activation, we evaluated the relative effects of LPS and silica dust on the production of several key eicosanoids when these activating agents were administered in sequence. Prior exposure of AM to LPS, followed by exposure to silica dust, led to an enhanced production of thromboxane A2 (TXA2) and leukotriene B4 (LTB4) when results were compared to AM stimulated with silica alone. In contrast, prior exposure of AM to silica dust reduced the ability of AM to respond to LPS, since levels of PGE2 and TXA2 produced by silica-primed AM subs...