15-Hydroxy-eicosatetraenoic acid (15-Hete) inhibits carragheenan-induced experimental arthritis and reduces synovial fluid leukotriene B4 (LTB4)

15-Hydroxy-eicosatetraenoic acid (15-HETE), a product of arachidonic acid, has no proinflammatory capacity, but can inhibit the formation and the chemotactic response of neutrophils to leukotriene B₄ (LTB₄), a potent mediator of inflammation. The purpose of the present study was to determine whether intraarticular administration of 15-HETE in carragheenan-induced acute arthritis might decrease the levels of LTB₄ in synovial fluid and modify the arthritis. A bilateral acute knee joint arthritis was established in 7 dogs by intraarticular injections of carragheenan every third day. To the right joints, 15-HETE was administered both concomitantly with the carragheenan injections and continously via an osmotic pump. In samples of synovial fluid obtained on day 0, 3 and 10 PGE₂ and LTB₄ were determined using reversed phase high performance liquid chromatography combined with radioimmunoassays and neutrophil chemokinesis. In the presence of 15-HETE the clinical severity of arthritis was significantly reduced and the volume synovial effusate was decreased on an average by 42%. Furthermore, the relative number of neutrophils in histological sections of synovial tissue was decreased by 58%. Intaarticular carragheenan injection induced LTB₄ formation, and maximum levels were obtained on day 3 (279.2 ± 148.2 pg/joint). PGE₂ was also present on a day 3, but maximum levels were detected on day 10 (9.5 ± 4.8 ng/joint). In joints injected with both carragheenan and 15-HETE the levels of LTB₄ on days 3 and 10 were inhibited by 90% and 83%, respectively. For PGE₂ a small but significant decrease was found on both day 3 and on day 10. These results show that LTB₄ may be an important mediator of acute arthritis induced by carragheenan in dogs, and that intraarticular administration of 15- HETE can modify this arthritis by inhibiting LTB₄ formation.     

Tumor necrosis factor alpha stimulates arachidonic acid metabolism in human osteoblastic osteosarcomal cells

The effects of tumor necrosis factor alpha (TNF-α) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [³H]AA. TNF-α differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-α (10⁻⁸ M) significantly increased the cyclooxygenase pathway, with prostaglandin E₂ (PGE₂) being a major product. However, at the lowest concentration (10⁻¹⁰ M) of TNF-α, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-α was increased to 10⁻⁹ M leukotriene B₄ (LTB₄), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10⁻⁶ M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-α (10⁻⁸ M) and A23187 (10⁻⁶ M) caused an inhibitory effect on each agent-induced PGE₂ or 15-HETE production.     

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.     

Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells

The role of individual eicosanoids of the arachidonic acid (AA) cascade in the growth control of A549 human lung adenocarcinoma cells has been studied. Cyclooxygenase and lipoxygenase metabolites of [¹⁴C]AA incorporated were actively synthesized in the cultures of tumor cells with full confluence unaccomplished. In such cultures inhibitors of AA metabolism (indomethacin and esculetin) and also a lipoxygenase metabolite of AA, 15-hydroxyeicosatetraenoic acid (15-HETE), significantly suppressed the incorporation of [³H]thymidine and biosynthesis of prostaglandin E₂(PGE₂). Other lipoxygenase metabolites of AA (5-HETE and 12-HETE) had no effect on these parameters. The basic fibroblast growth factor (bFGF) had practically no affect on the growth of A549 cells and the PGE₂ production in cultures with 5% fetal calf serum (FCS); however, in the presence of 0.5% FCS this factor significantly increased the number of tumor cells. The growth-stimulating effect of bFGF was completely abolished by a cyclooxygenase inhibitor indomethacin. The data suggest a key role of PGE₂ in the growth control of A549 cells with an active synthesis of cyclooxygenase and lipoxygenase metabolites of AA, its importance in realization of the mitogenic effect of bFGF, and specific features of 15-HETE as a down-regulator of the PGE₂-dependent proliferation.     

Esterification of monohydroxyfatty acids into the lipids of a macrophage cell line

Cells of a mouse macrophage-like tumor cell line, J774.2, were incubated with 0.6μM radiolabeled mono- and di-hydroxyfatty acids. Monohydroxyfatty acid products of the neutrophil and platelet lipoxygenase pathways (5-HETE, 15-HETE, and 12-HETE) were rapidly taken up (42–64% of the counts cell associated at 1 min) and esterified into triglycerides and phospholipids. 5-HETE and 12-HETE were found in triglycerides and distributed among phospholipid classes while 50% of added 15-HETE was esterified into phosphatidyl inositol. Treatment of phospholipids from cells incubated with 5-HETE, 12-HETE, and 15-HETE with phospholipase A₂ resulted in release of the respective monohydroxyfatty acid. HHT, a monohydroxyfatty acid product of the cyclooxygenase pathway, was taken up and esterified more slowly than the lipoxygenase products. In addition, HHT was not released when the phospholipids from cells incubated with HHT were treated with phospholipase A₂. LTB₄, a dihydroxyfatty acid product of neutrophil lipoxyegnase, was not taken up by J774.2 cells. The unique patterns of uptake and intracellular distribution of the different monohydroxyfatty acids suggests that the enzymes involved in the esterification of these compounds have substrate specificity and may also relate to the specific biologic effects of the compounds.     

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B₄ were compared, on a molar basis, with those LTC₄, LTD₄, LTE₄, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD₂, PGE₁, PGF_2α, PGI₂, 6-oxo-PGF_1α, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series.LTB₄ similar to LTC₄ and LTD₄ on GPP, in relation to potency and contractions induced, but differed from LTE₄ in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB₄ produced contractions which were resistant to FPL 55712 (1.9μM) and, when given repeatedly, caused tachyphylaxis in GPP,LTB₄ was considerable more active on GPP than the other substances investigated. Further, PGD₂, PGF_2α and PGI₂ contracted GPP, the order of potency being PGD₂ > PGF_2α ? PGI₂ whereas PGE₁ and PGE₂ relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD₄ displaying the highest activity, LTB₄ was inactive on this tissue. 5-HETE and 6-oxo-PGF_1α were inactive on both GPP and GPISM.On the basis of differential effects of LTB₄ on GPP and GPISM, this assay repressents a simple and selective means to distinguish LTB₄-like materials from other naturally-occuring substances likely to be generated in inflammatory fluids.     

Growth Inhibitory Effect of Polyunsaturated Fatty Acids (PUFAs) on Colon Cancer Cells via Their Growth Inhibitory Metabolites and Fatty Acid Composition Changes

Background

Colorectal cancer is common. Polyunsaturated fatty acids (PUFAs) exert growth-inhibitory and pro-apoptotic effects on colon cancer cells. Metabolites of PUFAs such as prostaglandins (PGs), leukotrienes (LTs) and lipoxins (LXs) play a significant role in colon cancer.

Methods

Human colon cancer LoVo and RKO cells were cultured with different concentration of PUFAs and 5-fluorouracil (5-FU) in vitro. Cell morphological changes, fatty acid composition, formation of PGE2, LTB4 and LXA4 and expression of COX-2, ALOX5, PGD synthase (PGDS), microsomal prostaglandin E synthase (mPGES) were assessed in LoVo and RKO cells when supplemented with PUFAs and 5-FU.

Results

PUFAs and 5-FU inhibited growth of LoVo and RKO cells to the same extent at the doses used and produced significant alterations in their shape. As expected, higher concentrations of supplemented PUFAs were noted in the cells compared to control. LA, GLA, AA, ALA and EPA supplementation to LoVo cells suppressed production of PGE₂, LTB₄,and ALOX5, mPGES expression, but enhanced that of LXA₄; whereas DHA enhanced PGE₂ and LXA₄ synthesis but decreased LTB₄ formation and COX-2, ALOX5, mPGES expression. In contrast, 5-FU enhanced formation of PGE₂, LTB₄ and mPGES expression, but suppressed LXA₄ synthesis and COX-2 expression. PGE₂, LTB₄ synthesis and ALOX5 expression was suppressed by LA, GLA, ALA and DHA; whereas AA, EPA and 5-FU enhanced PGE₂ but paradoxically AA decreased and EPA and 5-FU enhanced LTB4 synthesis in RKO cells. All the PUFAs tested enhanced, while 5-FU decreased LXA₄ formation in RKO cells; whereas GLA, AA, and 5-FU augmented while LA, ALA, EPA and DHA enhanced COX-2 expression in RKO cells.

Conclusions

Tumoricidal action of PUFAs on colorectal LoVo and RKO cancer cells in vitro was associated with increased formation of LXA₄, decreased synthesis of PGE₂ and LTB₄ and suppressed expression of COX-2, ALOX5, mPGES, whereas 5-FU produced contrasting actions on these indices.     

RETRACTED ARTICLE: Anti-Inflammatory Effects of Chronic Aspirin on Brain Arachidonic Acid Metabolites

Pro-inflammatory and anti-inflammatory mediators derived from arachidonic acid (AA) modulate peripheral inflammation and its resolution. Aspirin (ASA) is a unique non-steroidal anti-inflammatory drug, which switches AA metabolism from prostaglandin E₂ (PGE₂) and thromboxane B₂ (TXB₂) to lipoxin A₄ (LXA₄) and 15-epi-LXA₄. However, it is unknown whether chronic therapeutic doses of ASA are anti-inflammatory in the brain. We hypothesized that ASA would dampen increases in brain concentrations of AA metabolites in a rat model of neuroinflammation, produced by a 6-day intracerebroventricular infusion of bacterial lipopolysaccharide (LPS). In rats infused with LPS (0.5 ng/h) and given ASA-free water to drink, concentrations in high-energy microwaved brain of PGE₂, TXB₂ and leukotriene B₄ (LTB₄) were elevated. In rats infused with artificial cerebrospinal fluid, 6 weeks of treatment with a low (10 mg/kg/day) or high (100 mg/kg/day) ASA dose in drinking water decreased brain PGE₂, but increased LTB₄, LXA₄ and 15-epi-LXA₄ concentrations. Both doses attenuated the LPS effects on PGE₂, and TXB₂. The increments in LXA₄ and 15-epi-LXA₄ caused by high-dose ASA were significantly greater in LPS-infused rats. The ability of ASA to increase anti-inflammatory LXA₄ and 15-epi-LXA₄ and reduce pro-inflammatory PGE₂ and TXB₂ suggests considering aspirin further for treating clinical neuroinflammation.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

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).     

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.     

The effect of n-3 polyunsaturated fatty acids on leukotriene B₄ and leukotriene B₅ production from stimulated neutrophil granulocytes in patients with chronic kidney disease

The proinflammatory leukotriene B₄ (LTB₄) may be of importance in the progression of chronic kidney disease (CKD). We investigated whether n-3 polyunsaturated fatty acids (PUFA) decrease LTB₄ and increase the formation of the less inflammatory leukotriene B₅ (LTB₅) in patients with CKD.Fifty-six patients with CKD stage 2-5 were randomised to 2.4 g n-3 PUFA or olive oil for 8 weeks. Compared to controls, n-3 PUFA significantly decreased release of LTB₄ (p<0.001) and 5-hydroxyeicosatetraenoic acid (5-HETE) (p<0.01) and significantly increased release of LTB₅ (p<0.001) and 5-hydroxyeicosapentaenoic acid (5-HEPE) (p<0.001) from stimulated neutrophil granulocytes. Kidney function evaluated by creatinine clearance and proteinuria did not improve. In conclusion, n-3 PUFA supplementation for 8 weeks in patients with CKD stage 2-5 significantly decreased LTB₄ and 5-HETE and significantly increased LTB₅ and 5-HEPE. No effect was seen on kidney function.     

Cyclical binding,processing, and functional interactions of neutrophils with leukotriene B4

Leukotrene (LT) B₄ activates human polymorphonuclear neutrophils. (PMN) by binding to plasmalemmal receptors. It stimulates PMN to raise cytosolic calcium and degranulate. Both responses end within 15–30 sec. However, in < 15 sec, LTB₄-treated PMN lose the ability to respond further to LTB₄; decrease the affinity and number of high affinity receptors available for binding LTB₄ sequester LTB₄ in plasmalemma-associated sites that are inaccessible to a releasing buffei regi i men; and begin internalizing LTB₄. Over the next 90 min, the cells increasingly internalize LTB₄ and convert it to less potent metabolites; release the metabolites; recover LTB₄ binding sites; and become fully sensitive to LTB₄. Contrastingly, during the entire 90 min incubation with LTB₄. PMN retained the capacity to bind and respond normally to a second stimulus platelet-activating factor. We therefore suggest the following model. LTB₄ receptors, when ligand-bound, initiate function but rapidly lose this capacity as they lower their ligand binding affinity and sequester, internalize, or otherwise uncouple from transducing elements. These LTB₄ receptor changes contribute to terminating PMN responses and producing a stimulus-selective state of desensitization. During the desensitization period, PMN progressively process and metabolize LTB₄. This removes LTB₄ from the environment, thereby allowing PMN to recover functional receptors for and sensitivity to the ligand.     

Hepatocytes are a rich source of novel aspirin-triggered 15-epi-lipoxin A4

Novel aspirin (ASA)-triggered 15-epi-lipoxins (ATL) comprise newpotent bioactive eicosanoids that may contribute to the therapeutic effect of this drug. ATL biosynthesis is initiated by ASA acetylation of cyclooxygenase (COX)-2 and was originally identified during theinteraction of leukocytes with either endothelial or epithelial cells.Here, we examined ATL biosynthesis in rat hepatocytes either alone orin coincubation with nonparenchymal liver cells (NPC) and in liverhomogenates from ASA-treated rats. Rat hepatocytes and CC-1 cells, arat hepatocyte cell line, displayed COX-1 but not COX-2 mRNA expressionand predominantly produced thromboxane A₂(TXA₂) and15-hydroxyeicosatetraenoic acid (15-HETE). In these cells, ASA shiftedthe arachidonic acid metabolism fromTXA₂ to 15-HETE in aconcentration-dependent manner. In contrast, neither indomethacin,ibuprofen, valeryl salicylate, nor nimesulide was able to trigger15-HETE biosynthesis. SKF-525A, a cytochromeP-450 inhibitor, significantly reducedthe effect of ASA on 15-HETE biosynthesis. Furthermore, phenobarbital,a potent inducer of cytochrome P-450activity, further increased ASA-induced 15-HETE production. ASAtreatment of hepatocyte-NPC coincubations resulted in the generation ofsignificant amounts of ATL. In addition, in vivo experimentsdemonstrated augmented hepatic levels of 15-epi-lipoxin A₄ in ASA-treated rats. Takentogether and considering that ASA is hydrolyzed on its first passthrough the portal circulation, these data indicate that, during ASA'sconsumption, liver tissue generates biologically relevant amounts ofATL by COX-2-independent mechanisms.

     

Arachidonic acid and 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid modulate human polymorphonuclear neutrophil activation by monocyte derived neutrophil activating factor

Exposure of human polymorphonuclear neutrophils (PMN) to human monocyte derived neutrophil activating factor(s) (NAF) resulted in a concentration-dependent extracellular release of granule constituents. NAF also induced the generation of 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid [Leukotriene B4 (LTB4)] by PMNs which was enhanced in the presence of exogenous arachidonic acid (AA). In contrast to its enhancing effect on LTB4 production, AA inhibited NAF-stimulated PMN degranulation. 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a product of the 15-lipoxy-genation of AA in PMNS, caused a concentration-dependent suppression of degranulation and LTB4 generation by PMNs in contact with NAF. 15-HETE also inhibited the rise in cytosolic-free calcium [( Ca2+]i) observed in NAF activated PMNs. These data suggest that AA and a 15-lipoxygenase product modulate the NAF-associated activation pathway in human PMNs.     

15(S)-HETE modulates LTB(4) production and neutrophil chemotaxis in chronic bronchitis

We evaluated the levels of15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of15-lipoxygenase (15-LO) mRNA in induced sputum obtained from 10 controland 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reversephase high-performance liquid chromatography separationfollowed by specific RIA. 15-LO mRNA expression was determined byprimed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than incontrol subjects. The percentage of cells expressing 15-LO mRNA wassignificantly higher in chronic bronchitis than in control subjects(P < 0.01). Double staining for specific cell typemarkers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did notshow evidence for 15-LO expression, suggesting that expression of 15-LOin neutrophils takes place on migration into the airways. Because15(S)-HETE inversely correlated with the percentage of neutrophils insputum of chronic bronchitis subjects, we studied the effect of15(S)-HETE on leukotriene B₄ (LTB₄) productionin vitro and evaluated the concentration of LTB₄ in inducedsputum and the contribution of LTB₄ to the chemotacticactivity of induced sputum samples ex vivo. The results obtainedindicate that macrophages and neutrophils present within the airways ofchronic bronchitis subjects express 15-LO mRNA; increased basal levelsof 15(S)-HETE may contribute to modulate, through the inhibition of5-lipoxygenase metabolites production, neutrophil infiltration andairway inflammation associated with chronic bronchitis.

     

Delayed-onset synergism between leukotriene B4 and prostaglandin E2 in human skin

The time-course of cutaneous inflammatory responses to LTB₄ and PGE₂ both alone and in combination has been studied in 10 healthy volunteers. LTB₄ induced a transient wheal and flare response in some subjects, maximal at 15 minutes and succeeded by an erythematous, indurated lesion at 2–4 hours. PGE₂ elicited a wheal and erythema response which resolved within 1–2 hours. Combination of LTB₄ and PGE₂ produced acute wheal and erythema responses which did not differ significantly from the summation of responses to the individual constituents of the mixture or from responses to a two-fold increase in the concentration of either component. Wheal and erythema responses persisted, however, with significant potentiation of responses 4 hours after injection. As both leukotrienes and prostaglandins are generated in acute allergic reactions, the effects of these mediators in combination could contribute to persisting and late-onset responses to allergen, in both the skin and lung. In particular, sustained responses to the combination of LTB₄ and PGE₂ might be important in the pathogenesis of inflammatory skin diseases such as psoriasis.     

Biosynthesis and biological activity of leukotriene B5

Several studies indicate that increased intake of eicosapentaenoic acid (EPA) in the diet may lead to decreased incidence of thrombotic events. Most investigators agree that this is achieved by competitively inhibiting the conversion of arachidonic acid (AA) to thromboxane A₂ in the platelets. The effect of high EPA-intake on the formation of prostacyclin is less clear. However, EPA is a good substrate for lipoxygenase enzymes which results in formation of hydroperoxy- and hydroxy-acids, and, in some cases, leukotrienes. The biological activities of the leukotrienes derived from arachidonic acid suggest that they mediate or modulate some symptoms associated wth inflammatory and hypersensitivity reactions. In order to clarify the possible effect of dietary manipulation of inflammatory processes, leukotriene B₅ (LTB₅) was prepared and its biological activities assessed. LTB₅ was biosynthesised by incubating EPA with glycogen-elicited polymorphonuclear neutrophils (PMN) from rabbits in the presence of the divalent cation ionophore, A23187. The LTB₅ was extracted from the incubate using minireverse phase extraction columns (Sep-pak) and purified by reverse-phase high pressure liquid chromatography (RP-HPLC). The purity of the product assessed by repeat RP-HPLC and straight phase (SP) HPLC was greater than 95%. Ultra-violet spectrophotometry of the product confirmed its purity and also provided assessment of the yield. The biological activity of LTB₅ was assessed and compared with that of LTB₄ in the following tests: aggregation of rat neutrophils, chemokinesis of human PMN, lysosomal enzyme release from human PMN and potentiation of bradykinin-induced plasma exudation. In all these tests. LTB₅ was considerably less active (at least 30 times) than LTB₄.     

Tissue differences in vascular permeability induced by leukortriene B4 and prostaglandin E2 in the rat

The activity of synthetic LTB₄ and PGE₂, in increasing vascular permeability was tested simultaneously in seventeen different organs in the rat. Rats were injected in the aortic arch through a cannula in the carotid artery with ¹²⁵-I-albumin, ⁵¹Cr-erythrocytes, and ⁵⁷Co-EDTA. The rats were then injected through the carotid artery cannula with LTB₄, PGE₂ or a combination of LTB₄ and PGE₂. Eight minutes later the rats were killed and the activity of ¹²⁵I, ⁵¹Cr, and ⁵⁷Co measured in different organs. Changes in vascular permeability were infered from changes in the ratios of the isotope activities. LTB₄ (15 μg/kg) induced enhanced permeability in caecum, small bowel, skin, fat pad, stomach, pancreas, and aorta, but not in the heart, brain, colon, testes, diaphragm, forelimb, cremaster muscle, lung, kidney or eye. A lower dose of LTB₄, 3 μg/kg, enhanced vascular permeability in caecum, small bowel, skin, stomach, and aorta. PGE₂ (1 μg/kg) enhanced vascular permeability only in the caecum. A combination of LTB₄ (3 μg/kg) and PGE₂ (1 μg/kg) was more potent than either alone. Rats depleted of neutrophils with anti-neutrophil serum were less sensitive to LTB₄ than intact rats. These findings suggest that the vasculatures of different tissues in the rat vary markedly in their susceptibility to LTB₄ induced increases in permeability.     

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.