Profiles of eicosanoid production by superficial and proliferative colonic epithelial cells and sub-epithelial colonic tissue

The profile of cyclooxygenase and lipoxygenase products in normal rat colonic epithelium and subepithelium was examined. Colons were thoroughly perfused to eliminate contamination with blood. Two preparations of colonic epithelium were employed. The first consisted of intact colonic crypts and epithelial sheets. The second yielded single cell suspensions of superficial versus proliferative epithelial cells. Lipoxygenase product formation by colonic epithelium as measured by hydroxyeicosatetraenoic acid (HETE) and leukotriene B4 (LTB4) production (5-HETE greater than 12-HETE greater than 15-HETE greater than LTB4) accounted for 58% of the total colonic production of these moieties, whereas epithelium accounted for only 20% of total colonic protein. By contrast, prostaglandin (PG) E2 and PGF2 alpha production occurred predominantly (greater than 97%) in the subepithelial layers. The present studies also demonstrate markedly higher levels of accumulation of lipoxygenase products in proliferative versus superficial epithelial cells, whereas prostaglandin accumulation was greater in superficial cells. Previous studies have supported a role for lipoxygenase and cyclooxygenase products in the control of colonic secretion, inflammatory cell infiltration and proliferative activity. The present results raise the possibility that the striking differences in the sites of production of these products within the colon has functional implications.     

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [¹⁴C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF₂α and TXA₂, as monitored from the formation of its stable degradation product TXB₂ (PGF_2α > TXB₂ > 6-keto-PGF_1α, the stable degradation product of PGI₂=PGD₂=PGE₂=13,14-dihydro-15-keto-PGF_2α). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE₂. PGE₂ and its degradation product 13,14-dihydro-15-keto-PGE₂ accounted for 63% of the PG products formed by submucosal structures (PGE₂ PGD₂ > 13,14-dihydro-15-keto-PGE₂ > PGF_2α=TXB₂=6-keto-PGF_1α > 13,14-dihydro- 15-keto-PGF_2α). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE₂ or PGF_2α. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE₂ degradative capacity but only 23% of total colonic protein. Approximately 15% of [³H] PGE₂ added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE₂ formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.     

Primary colonic epithelial cell culture of the rabbit producing prostaglandins

We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E₂ production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35 % collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10 % fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE₂ and PGF_2α were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF_1α, thromboxane B₂, PGF_2α, PGE₂, and PGD₂ by cultured cells. Quantitatively, 117±7 ng/mg-protein/hour PGE₂ by 7.4±0.7 ng/mg-protein/hour PGF_2α were produced. While hydrocortisone (10⁻⁴-10⁻² M) did not show a significant effect on PGE₂ production, indomethacin (10⁻⁸-10⁻⁶ M), and 5-aminosalicylic acid (2×10⁻⁴-5×10^{−3 M}) inhibited PGE₂ production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE₂ synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.     

Cyclic nucleotide metabolism in rat colonic epithelial cells with different proliferative activities

Cyclic nucleotide metabolism was examined in rat distal colonic epithelial cells with different proliferative activities. Lower crypt cells had DNA synthetic rates 7-10-fold higher than surface cells. Without a phosphodiesterase inhibitor proliferative cells had reduced basal cyclic AMP-, cyclic GMP-, and cyclic AMP-dependent protein kinase activity ratios, as well as blunted cyclic AMP responses to prostaglandin E2 and vasoactive intestinal peptide compared to superficial cells. In the presence of 3-isobutyl-1-methylxanthine, basal cyclic AMP and responses to prostaglandin E2 and vasoactive intestinal peptide of proliferative cells exceeded values in superficial cells. This correlated with higher membrane adenylate cyclase activity in the proliferative cells. By contrast, particulate and soluble guanylate cyclase activities of superficial cells were higher than in proliferative cells. The apparent high Km soluble and particulate cyclic AMP and cyclic GMP phosphodiesterase activities of proliferative cells were 4-7-fold higher than those in superficial cells. Moreover, the apparent low Km soluble activity was absent in superficial cells. Thus, an altered rate of nucleotide degradation may mediate reduced cyclic AMP and cyclic GMP in proliferative versus superficial cells. Dibutyryl cyclic AMP, prostaglandin E2 or vasoactive intestinal peptide inhibited [3H]thymidine incorporation into DNA of colonic segments. Thus, reduced cyclic AMP in lower crypt cells may be a determinant of their greater proliferative activity.     

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [14C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF2 alpha and TXA2, as monitored from the formation of its stable degradation product TXB2 (PGF2 alpha greater than TXB2 greater than 6-keto-PGF1 alpha, the stable degradation product of PGI2 = PGD2 = PGE2 = 13,14-dihydro-15-keto-PGF2 alpha). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE2. PGE2 and its degradation product 13,14-dihydro-15-keto-PGE2 accounted for 63% of the PG products formed by submucosal structures (PGE2 much greater than PGD2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGF2 alpha = TXB2 = 6-keto-PGF1 alpha greater than 13,14-dihydro-15-keto-PGF2 alpha). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE2 or PGF2 alpha. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE2 degradative capacity but only 23% of total colonic protein. Approximately 15% of [3H] PGE2 added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE2 formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.     

A human epithelial cell line, intestine 407, can produce 5-hydroxyeicosatetraenoic acid and leukotriene B4

The present study was carried out to further characterize the role of non-inflammatory cells in the inflammatory process. More specifically, we have investigated whether human epithelial cells can generate inflammatory lipid mediators via activation of the 5-lipoxygenase pathway. The cells were stimulated with the calcium ionophore A23187 (5 μM) for different periods of time, after which the production of eicosanoids was determined by gradient reverse-phase high performance liquid chromatography (RP-HPLC) and rapid spectral detection, permitting continuous ultraviolet spectroscopy. In both non-prelabeled cells and cells prelabeled with [1-^14Carachidonic acid, cell stimulation for 30 min or more resulted in the production of two important 5-lipoxygenase products: 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄). Stimulation for 15 min or less, however, led solely to the formation of 5-HETE. The identities of 5-HETE and LTB₄ were confirmed by HPLC retention times and UV spectra, as well as by gas chromatography-mass spectrometry for 5-HETE and radioimmunoassay for LTB₄. It can therefore be concluded that human epithelial cells in general can produce important inflammatory mediators, which suggests that epithelial cells may play a more active role in the inflammatory process than is normally assumed.     

Differential alteration of lipoxygenase and cyclooxygenase metabolism by rat peritoneal macrophages induced by endotoxin tolerance

Altered macrophage arachidonic acid metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e. endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT)C₄/D₄ and prostaglandin (PG)E₂ production by tolerant cells was greater than that by non-tolerant controls (p<0.001). However, A23187-stimulated i-6-keto-PGF_1α levels were lower in tolerant macrophages compared to controls. Stimulation of prostaglandin and thromboxane (Tx)B₂ synthesis by endotoxin or glucam was significantly less in tolerant macrophages compaared to controls (p<0.05). iLTC₄/D₄ production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in the non-tolerant cells. Synthesis ofb iLTB₄ by control macrophages was stimulated by endotoxin (p<0.01). These results demonstrate that arachidonic acid metabolism via the lipoxygenase and cyclooxygenase pathways in macrophages is differentially altered by endotoxin tolerance.     

Properties of multiple kinetic forms of soluble cyclic nucleotide phosphodiesterase activity of rat colonic mucosa

Soluble phosphodiesterase (EC 3.1.4.1) activity is 3-5-fold lower in superficial colonic epithelial cells compared to that in cells isolated from the lower colonic crypt. Higher phosphodiesterase activity in lower crypt cells is correlated with a 5-fold higher rate of incorporation of [3H]thymidine into DNA in these cells. DEAE-cellulose chromatography of the soluble fraction of superficial and proliferative colonic epithelial cells resulted in separation of three enzyme forms: (1) fraction I, an enzyme which hydrolyzes both cAMP and cGMP with high affinity (apparent Km cAMP = 5 +/- 1 microM, Km cGMP = 2.5 +/- 0.5 microM) and is stimulated 3-6-fold by Ca2+ plus calmodulin; (2) fraction II, a form which hydrolyzes both cAMP and cGMP with low affinity (S0.5 cAMP = 52 +/- 7 microM, S0.5 cGMP = 17 +/- 4 microM), exhibits positive copperativity with respect to substrate and shows cGMP stimulation of cAMP hydrolysis and (3) fraction III, a cAMP-specific form which exhibits biphasic kinetics, a low Km for cAMP (Km cAMP = 5 +/- 1 microM) and does not hydrolyze cGMP. The pattern of distribution of phosphodiesterase activities on DEAE-cellulose was similar in superficial and proliferative colonic epithelial cells. The higher specific activity in proliferative cells was reflected in higher activities of each of the three chromatographically distinct forms of the enzyme. In contrast to epithelial cells, the soluble fraction of homogenates of the submucosa and supporting cells exhibited phosphodiesterase forms I and II and was lacking in the form corresponding to fraction III of epithelial cells.     

Biosynthesis, characterization and inhibition of leukotriene B4 in human whole blood

We have evaluated the biosynthesis, characterization and inhibition of Leukotrien (LT) B₄ in unstimulated and in A23187-stimulated human whole blood. LTB₄ was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37°C for 60 min produced only trace amounts of LTB₄ (0.16±0.05 ng/ml, mean±SD, n=3). LTB₄-like immunoreactivity (ir-LTB₄) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrealed to cyclooxygenasep or lipoxygenase activity. Incubation of human whole blood with A23187 (2–10 μM) resulted in a concentration-dependent stimulation of LTB₄ production. At 10 μM A23187, ir-LTB₄ was 18±2.4 ng/ml (mean±SEM, n=28). In A23187-stimulated serum samples, LTB₄ concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added caused a dose-dependent inhibition of A23187-stimulated ir-LTB₄ production with an IC₅₀ of 17 μM.     

Production of leukotriene B4 and prostaglandin E2 by turbot (Scophthalmus maximus) leukocytes

Production of two eicosanoids derived from lipoxygenase and cyclooxygenase activities: leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), respectively, have been simultaneously determined in turbot (Scophthalmus maximus) blood leucocyte and kidney macrophage supernatants by a reverse phase high performance liquid chromatography (HPLC) system coupled with a Diode–Array detector. Levels of LTB₄ after calcium ionophore challenge were 4.08 ng ml⁻¹ in blood leukocyte supernatants and 0.25 ng ml⁻¹ in kidney macrophage supernatants. The levels found for PGE₂ were 428.23 and 606.67 ng ml⁻¹ in blood leukocytes and kidney macrophage supernatants, respectively. When blood leukocytes were treated with the respective inhibitors for the enzymes implicated on the synthesis of both compounds an inhibition of 90.35% was observed for PGE₂ and 76.44% for LTB₄. The detection limit of the method was 0.15 ng ml⁻¹ for LTB₄ and 50 ng ml⁻¹ for PGE₂.     

Prostaglandin production from homogenates of separated glandular epithelium and stroma from human endometrium

The biosynthesis of prostaglandins by isolated epithelial glandular and stromal cells was studied after collagenase digestion of endometrium collected from women at various stages of the menstrual cycle. Homogenates of the separate cell types were incubated for 60 minutes with 2.08 μg 1¹⁴C arachidonic acid and the products separated by thin layer chromatography. Both glandular and stromal homogenates synthesised PGF_2α. More PGF_2α was synthesised by glandular epithelium separated from both proliferative and secretory endometrium than by stroma. The ratio of PGF_2α/PGE₂ was greater in glands and stroma isolated from secretory than proliferative endometrium. Small but significant amounts of 6-keto-PGF₁α were produced by all cell types. These results suggest that the increased synthesis of PGF₂α from secretory endometrium is due, at least in part, to increased activity of cyclo-oxygenase enzyme in the glandular epithelium.     

The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility

The effects of the lipoxygenase products of arachidonic acid, 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B₄ (LTB₄), on the spontaneous contractility of lower uterine segment human myometrial strips obtained prior to labour have been studied . 5-HETE gave a dose- dependent (10–500ng) increase in both the rate of contractions and overall contractility of myometrial strips while 12-HETE and LTB₄ had no effect at the same concentrations. Prostaglandin F₂ (50ng) contracted all myometrial strips in a similar pattern to 5-HETE but was approximately 10 times more potent. The effect of 5-HETE may be direct or perhaps indirect via interaction with the cyclo-oxygenase pathway. The findings do not disprove the contention that the onset of parturition may be characterized by a switch in arachidonic acid metabolism in intra-uterine tissues from lipoxygenase to cyclo-oxygenase products.     

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation.

Measurements and Main Results

In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E₂ in lung epithelial cells, and a PGE₂ neutralizing antibody blocked the protective effect of epithelial cell co-culture.

Conclusions

We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE₂. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.     

Dual inhibitory effect of Glycyrrhiza glabra (GutGard™) on COX and LOX products

Glycyrrhiza glabra and its phytoconstituents have been known to possess widespread pharmacological properties as an anti-inflammatory, anti-viral, antitumour and hepatoprotective drug. In this study, we examined the inhibitory potential of extract of G. glabra (GutGard™) root and its phytoconstituents (glabridin, glycyrrhizin, and isoliquiritigenin) on both cyclooxygenase (COX) and lipoxygenase (LOX) products in order to understand the mechanism of its anti-inflammatory action. Inhibitory effect of GutGard™ and its phytoconstituents on lipopolysaccharide (LPS) induced prostaglandin E₂ (PGE₂), calcimycin (A23187) induced thromboxane (TXB₂), and leukotriene (LTB₄) release was studied using murine macrophages (J774A.1) and human neutrophil (HL-60) cells. Results revealed that, G. glabra and glabridin significantly inhibited PGE₂, TXB₂ (COX) and LTB₄ (LOX), while, isoliquiritigenin exerted inhibitory effect only against COX products but failed to suppress LOX product. However, glycyrrhizin at the tested concentrations failed to exhibit inhibitory effect on both COX and LOX products. Here, we report for the first time that G. glabra (almost devoid of glycyrrhizin) exhibits anti-inflammatory property likely through the inhibition of PGE₂, TXB₂ and LTB₄ in mammalian cell assay system, which could be influenced in part by glabridin and isoliquiritigenin.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-¹⁴C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF_1α, TxB₂ and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF_2α, PGE₂, PGD₂, LTB₄ and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB₄, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB₄ is considerably increased after adrenalectomy. In the spleen, PGD₂ and 12-HETE are decreased after adrenalectomy.The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA₂ inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

5-aminosalicylic acid inhibits leukotriene B4ω-hydroxylase activity in human polymorphonuclear leukocytes

ω-oxidation is regarded as the major pathway for the metabolism and inactivation of leukotriene B₄ (LTB₄). To investigate the action of 5-aminosalicylic acid (5-ASA) on LTB₄ω-hydroxylase activity, we incubated human polymorphonuclear leukocytes (PMNLs) with ³H-labeled LTB₄ after pre-incubation with various concentrations of 5-ASA. ω-oxidation metabolites were separated by high performance liquid chromatography and each radioactivity was measured by a liquid scintilation counter. LTB₄ω-hydroxylase activity was inhibited by 5-ASA in a concentration-dependent fashion. The 50% inhibitory dose was about 50 μmol/l, which is within the concentration range found in the colonic mucosa. Our findings may be valuable in elucidating the potentially critical aspect of 5-ASA treatment in ulcerative colitis (UC).     

Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-¹⁴C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D₂, thromboxane (TX) B₂, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15- hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE₂ was also detected but neither 6-keto-PGF_1α nor PGF_2α were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB₄) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD₂, TXB₂ and PGE₂ was further confirmed by analyzing [³H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB₄ and LTC₄ by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD₂, TXB₂ and PGE₂ from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10⁻⁶ M).     

Metabolism of arachidonic acid by caruncular and allantochorionic tissues in cows with retained fetal membranes (RFM)

The metabolism of arachidonic acid (AA) by caruncular and allantochorionic tissues and its regulation was studied in normal cows (n=13) and those with retained fetal membranes (RFM; n=9). Tissues were taken via the vagina about 6 hours postpartum and incubated for 6 hours in minimum essential medium containing tritiated AA alone or in the presence of oxytocin, platelet activating factor (PAF), epidermal growth factor (EGF) or ionophore calcium (A23187). The metabolites of AA were separated by reverse phase-high pressure-liquid chromatography. Tissue concentrations of prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂) and plasma 13,14-dihydro-15-keto-PGF_2α (PGFM) concentration were also measured by radioimmunoassay. For caruncular tissue, less thromboxane B₂ (TXB₂) and more 6-keto prostaglandin F_1α (PGIM) was synthesized in tissue from the animals with RFM than in the controls. Oxytocin, PAF, EGF and A23187 increased only PGIM production in the control animals; A23187 also decreased TBX₂ synthesis. For the allantochorion, more PGE₂, leukotriene B₄ (LTB₄) and PGIM and less TXB₂, PGF_2α and hydroxyecosatetranoic acids (HETE) was synthesized in tissue from cows with RFM than from animals that delivered normally. All of the substances used in this study increased PGIM, PGF_2α and LTB₄ and decreased TXB₂ production by the allantochorionic tissue in control animals. The metabolism of AA by the allantochorionic tissue seems quantitatively under hormonal control. The metabolism of AA at the level of both maternal and fetal components of the placenta in cows with RFM differed from that seen in animals that expelled the membranes normally.     

Leukocytic functions in burn-injured patients

Anergy associated with an increase in suppressor helper T cell (T_c) ratio and a decrease in natural killer (NK) is one main cause of death following thermal injury (Tl). Recently, in vitro studies have shown that LTB₄ can induce human T_c to exert suppressor cell activity, and incubation of lymphocytes with LTB₄ for 24 hours significantly suppressed NK cell activity. Thus, we undertook an investigation of both AA metabolism and immunologic response in 20 patients who suffered 40–90% total body surface area (TBSA) burns. Cyclooxygenase (CO:RIA) and lipoxygenase (LO;HPLC det.) metabolites and superoxide (O₂^.−) production were measured in stimulated polymorphonuclear cells (PMNL) (A 23187 ± AA for icosanoid release; phorbol myristate acetate for O₂^.− production). Lyso-paf-acether (P-LPA) was measured in plasma samples. Ca²⁺-dependent K⁺ permeability in PMNL was measured by the cell K⁺ release induced by A 23187. T_c and T_c subsets were determined using monoclonal antibodies (OKT₃₊, OKT₄₊ and OKT₈₊). A biphasic sequential release of the different substances (leukocytic icosanoids and O₂^.− was monitored: increase ( 36–48 h after Tl) and decrease ( 72 h after Tl). The increase in AA stimulation was more transient than that of O₂^.−. The decline in the release of AA metabolites and O₂^.− production was associated with the anergic phase (decrease OKT₄₊/OKT₈₊ ratio) and with the clinical outcome of the patients. The decrease in LTB₄ and other LO metabolites could explain the impairment of neutrophil chemotaxis. Ca²⁺-dependent K⁺ permeability increased early up to 2 or 3 times normal.In order to go further with the mechanism of inhibition of LTB₄ and O₂^.− release, the effect of Tl plasma was assayed on normal leukocytes: a 10 min incubation with such plasma was sufficient to abolish LTB₄ secretion. A less important inhibition was observed with O₂^.− release (−32%) and Ca²⁺-dependent K⁺ permeability (−30%). Plasma inhibition seems to be due to a thermolabile factor(s) [protein(s): “suppressive factor(s) of membrane activation ”SFMA] which is (are) under active investigation using gel-filtration chromatography and fast protein liquid chromotography (FPLC). Among the SFMAs, certain acute phase proteins could play a key role: i.e., incubation (10 min) of normal PMNL with ceruloplasmin (1 mg/ml) abolished LO products and O₂^.− release.