Bronchoconstrictor effects of leukotriene B4 in the guinea pig in vivo

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous $\text{in vitro}$ studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

Leukotriene B4 potentiates airway muscle responsiveness and

We studied the effects of leukotriene B₄ (LTB₄) on guinea pig airway muscle responsiveness and . Responsiveness in vivo was assessed by measuring specific airway resistance (SRaw) upon intravenous acetylcholine infusion in 5 unanesthetized, spontaneously breathing guinea pigs. We found that aerosolized LTB₄, in a concentration that itself had no effect on baseline SRaw, caused a substantial increase in bronchial reactivity to i.v. ACh within 8 min of its administration. Responsiveness was assessed by measuring isometric contraction of the guinea pig trachealis upon stimulation by either chemical or electrical field stimuli. These studies showed that a concentration of LTB₄ that itself did not cause contraction, potentiated airway muscle contraction to ACh and KCl, but not to norepinephrine. This effect of LTB₄ was substantially reduced by nifedinpine. Our data suggests that amounts of LTB₄ that are themselves non-contractile or in , may directly potentiate the responsiveness of airway smooth muscle to other bronchoconstrictors.     

NaCN inhibits the stereospecific depletion of LTB4 by guinea pig polymorphonuclear leukoxytes

Caseinate elicited suspension of guinea pig peritoneal PMNs synthesized LTB₄, 6t-LTB₄, 12-epi-6t-LTB₄ and 5HETE after incubations with A23187 and arachidonic acid. Concentrations of LTB₄ peaked in 3 minutes and were then rapidly depleted. 6t-LTB₄ and 12-epi-6t-LTB₄ also peaked in concentrations in 3 min but were depleted slower than LTB₄. NaCN inhibited the depletion of LTB₄ in a dose dependent fashion without dramatically affecting biosythesis.     

In vitro and in vivo chemotaxis of guinea pig leukocytes toward leukotriene B4 and its w-oxidation products

Leukotriene B₄ (LTB₄), 20-OH-LTB₄, and 20-COOH-LTB₄ were studied for their relative activities towards guinea pig peritoneal eosinophils and neutrophils during in vitro chemotaxis in modified Boyden chambers. The leukotrienes were also injected into guinea pig skin, and the cellular infiltrate in 4 hour biopsies was evaluated histologically. Eosinophils migrated more actively than neutrophils towards LTB₄in vitro, while in vivo, more neutrophils were observed. 20-OH-LTB₄, was markedly less active than LTB₄in vivo and in vitro, and 20-COOH-LTB₄ was barely active at all. Crude ionophore-stimulated neutrophil supernatants (ECF) were more active towards eosinophils than towards neutrophils, both in vivo and in vitro, compared to the pure leukotrienes. The data confirm the potent chemotactic properties of LTB₄ for eosinophils and neutrophils, with less activity of its w-metabolites.     

Leukotrienes cause eosinophil emigration into conjunction tissue

The ability of LTB₄, LTC₄, the 5S,6R and 5R,6S LTD₄ stereoisomers, and LTE₄ to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological andl ight microscopy techniques. LTD₄ and LTE₄ demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10ng to 1000ng), while there was only a minimal response to LTC₄. LTB₄ produced marked eosinophil inflitrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB₄. The 5R,6S LTD₄ stereoisomer did not evoke any leukocyte infiltrantion. The SRS-A antagonist, FPL 55712, abolished pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10μg to 1000μg. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.     

Correlation between the myotropic activity of leukotriene A4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A₄ and D₄ displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD₄ was much higher than that of LTA₄. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD₄ was very active whereas LTA₄ was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA₄ by the smooth muscle preparations was a prerequisite to its biological activity. LTA₄ was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP-HPLC). Homogenates of lung parenchyma rapidly transformed LTA₄ to LTB₄, LTC₄, LTD₄ and LTE₄. Incubation of LTA₄ with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB₄ and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA₄ undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activites of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA₄ is related to the tissue levels of enzymes which catalyse its biotransformation.     

-alkoxyphenol leukotriene B4 receptor antagonists

A series of -alkoxyphenols containing a tetrazole acid sidechain have been prepared as antagonists of leukotriene B₄ receptors. These compounds were tested as receptor antagonists of human neutrophil and guinea pig lung membrane leukotriene B₄ receptors. Compounds in this series were found to be up to 18-fold more potent than LY255283. These results indicate that the acyl group of the 1,2,4,5 substituted hydroxyacetophenone class of LTB₄ antagonists is not critical to antagonist potency.     

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B₄ (LTB₄) analog, 20-trifluoromethyl LTB₄ (20CF₃−LTB₄), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF₃−LTB₄ was equipotent to LTB₄ as a chemoattractant (EC₅₀, 3 nM), produced 50% of maximal activity of LTB₄, and competed with [H] LTB₄ for binding to intact human neutrophil LTB₄ receptors. In contrast to chemotactic activity, 20CF₃−LTB₄ in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 μM on LTB₄-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB₄, 20CF₃−LTB₄ induced neutrophil desensitization to degranulation by LTB₄. The results indicate that hydrogen atoms at C-20 of LTB₄ are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF₃−LTB₄ is a partial agonist for chemotaxis and an antagonist for degranulation syggests that different LTB₄ receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB₄ can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

Comparative biological activities of synthetic leukotriene b4 and its ω-oxidation products

Synthetic leukotriene B₄ (LTB₄) and its ω-oxidation products, 20 OH-LT₄ and 20 COOH-LTB₄, were tested for their ability to induce the aggregation of rat neutrophils $\text{in}$$\text{vitro}$, to contract the guinea pig parenchymal strip $\text{in}$$\text{vitro}$ and to cause vascular permeability changes in rabbit skin $\text{in}$$\text{vivo}$. 20 OH-LTB₄ had 10, 100 and 20% of the activity of LTB₄ in the neutrophil aggregation, parenchymal strip and vascular permeability assays respectively. 20 C00H-LTB₄ was inactive $\text{in}$$\text{vivo}$ and showed <1% of the activity of LTB₄$\text{in}$$\text{vitro}$. These results show that while ω-oxidation is a route for biological inactivation of LTB₄, 20 OH-LTB₄ still retains significant biological activity.     

Leukotriene B4, polymorphonuclear leukocytes and inflammatory exudates in the rat

Leukocyte numbers and Leukotriene B₄- (LTB₄-) and LTC₄-immunoreactivity were measured in inflammatory exudates obtained from sponges impregnated with several irritants implanted subcutaneously in the rat. Sponges containig 1% uric acid, carragennan or zymosan were implanted for 5h and compared to saline sponges. Increases in leukocyte numbers and LTB₄-immunoreactivity were found in the presence of irritants, the highest concentrations being observed in the presence of zymosan. The presence of LTB₄ was confirmed by liquid chromatographic (HPLC) analysis. A time course study was carried out with zymosan-impregnated sponges and the maximal rate of leukocyte infiltrations was found to coincide with the maximal levels of LTB₄-immunoreactivity. The LTC₄-immunoreactivity was low and following analysis by HPLC was concluded to be unrelated to leukotrienes. The levels of LTB₄-immunoreactivity, but not the numbers of leukocytes, were elevated compared to corresponding controls in sponges containing 0.01% ionphore A23187 (untreated rats) or in sponges containing zymosan (rats pretreated with indomethacin; 3 and 10 mg/kg p.o.). Impregnation of sponges with 3 × 10⁻⁶M LTB₄ but not 3 × 10⁻⁷M LTB₄ induced a significant leukocyte migration. It was concluded that LTB₄ can induced leukocyte migration into sponge exudates in the rat but that measurements of LTB₄ in such exudates can not be correlated with the degree of leukocyte infiltration.     

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.     

Further studies on the mechanism of action of leukotrienes and histamine on guinea pig lung parenchyma role of calcium, phospholipase and methyltransferase

The contractile activity of leukotriene B₄ (LTB₄), leukotriene D₄ (LTD₄) and histamine on strips of guinea pig lung parenchyma was shown to be dependent on the calcium concentrations of the Krebs solution. The calcium channel blocker verapamil (2.0 to 15uM) had an additive effect on the inhibitory activity of low calcium (0.1 mM) on contractions of guinea pig parenchyma to leukotrienes and histamine. Cobalt chloride, a divalent cation, also produced dose-dependent reductions of the myotropic activities of LTB₄, LTD₄ and histamine. An antagonist of calmodulin, triflouperazine (1–200 uM), dose-dependently inhibited the contractile activity of the three agonists on the parenchyma strip. The IC₅₀ of this compound for inhibition of histamine was much lower (2–3uM) than the IC₅₀ for inhibition of leukotrienes (75 uM). Valinomycin, a potassium ionophore, also interfere with the contractile activities of leukotrienes and histamine whereas a blocker of sodium channel, tetrodotoxin, had no effect on the activity of these agonists. Furthermore, an inhibitor of methyltransferase, 3-deazaadenosine, significantly diminished the responses of the parenchyma to leukotrienes and histamine. These results confirmed the important role of extracellular and intracellular calcium in the myotropic activity of leukotrienes and histamine in guinea pig lungs and showed that compunds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness.     

The Role of Selectins and CD18 in Leukotriene B4-Mediated White Blood Cell Emigration in Human Skin Grafts Transplanted on SCID Mice

The purpose of this study was to examine the role of selectins and CD18 cell adhesion molecules (CAMs) in inflammation induced by injection of leukotriene B₄(LTB₄) into human skin. To accomplish this, the expression of CAMs and the ability of specific antibodies against CAMs to block white blood cell (WBC) transmigration were studied in an in vivo model consisting of human skin transplanted onto mice with the severe combined immune deficiency (SCID) mutation. The results indicate that LTB₄-induced WBC transmigration in the human/SCID model is rapid and pronounced; however, it is not accompanied by a significant upregulation of the baseline expression of endothelial P-selectin, E-selectin, ICAM-1 or VCAM-1. An anti-murine CD18 mAb markedly inhibited white cell infiltration (89% inhibition) confirming the importance of β₂integrins in the process. The role of selectins was also examined. MEL-14, a bioactive antibody against murine L-selectin inhibited transmigration by 66%. A significant, but smaller, effect (39% inhibition) was observed by blocking E-selectin function. These results indicate that LTB₄-induced inflammation does not require upregulation of endothelial CAM expression and, in contrast to TNFα-induced transmigration, is only partially blocked by anti-E-selectin antibodies.     

Expression of 5-lipoxygenase in specialized epithelial cells of nasopharyngeal-associated lymphoid tissue

Leukotrienes are lipid mediators that are produced primarily by certain types of leukocytes. The synthesis of the leukotriene LTB₄ is initiated by the enzyme 5-lipoxygenase and completed by LTA₄ hydrolase. Epithelial cells constitutively express LTA₄ hydrolase but normally lack 5-lipoxygenase. In this study, we report that the stratified squamous epithelial cells from inflamed or hyperplastic tissues of palatine and pharyngeal tonsils (nasopharyngeal-associated lymphoid tissue) express 5-lipoxygenase protein. The localization of 5-lipoxygenase was indicated by immunohistochemical staining and presence confirmed by immunoblot. Positive staining for 5-lipoxygenase in infiltrating leukocytes in inflamed tissues served as internal positive controls for immunohistochemical staining. Staining for 5-lipoxygenase in appendix tissue was negative for epithelial cells while positive for polymorphonuclear leukocytes, indicating that 5-lipoxygenase expression is not a general feature of epithelial cells in mucosa-associated lymphoid tissue. In tonsils, 5-lipoxygenase staining was pronounced in broad regions but reduced or absent in others, suggesting regional regulation of expression. Epithelial cells of tonsils were also positive for 5-lipoxygenase activating protein and leukotriene A₄ hydrolase, indicating a capacity to produce LTB₄. Taken together, these results suggest that the specialized epithelial cells of the mucosa-associated lymphoid tissue of human tonsils can synthesize LTB₄. This lipid mediator may serve to modulate the function of cells within the lymphoid tissue as well as promote an inflammatory response.     

A new method to evaluate anti-allergic effect of food component by measuring leukotriene B<Subscript>4</Subscript> from a mouse mast cell line

Leukotrienes (LTs), chemical mediators produced by mast cells, play an important role in allergic symptoms such as food allergies and hay fever. We tried to construct an evaluation method for the anti-LTB₄ activity of chemical substances using a mast cell line, PB-3c. PB-3c pre-cultured with or without arachidonic acid (AA) was stimulated by calcium ionophore (A23187) for 20 min, and LTB₄ production by the cells was determined by HPLC with UV detection. LTB₄ was not detected when PB-3c was pre-cultured without AA. On the other hand, LTB₄ production by PB-3c pre-cultured with AA was detectable by HPLC, and the optimal conditions of PB-3c for LTB₄ detection were to utilize the cells pre-cultured with 50 µM AA for 48 h. MK-886 (5-lipoxygenase inhibitor) completely inhibited LTB₄ production, but AACOCF₃ (phospholipase A₂ inhibitor) slightly increased LTB₄ production, suggesting that LTB₄ was generated from exogenous free AA through 5-lipoxygenase pathway. We applied this technique to the evaluation of the anti-LTB₄ activity of food components. PB-3c pre-cultured with 50 µM AA for 48 h was stimulated with A23187 in the presence of 50 µM soybean isoflavones (daidzin, genistin, daidzein, and genistein), equol, quercetin, or kaempferol. Genistein, equol, quercetin, and kaempferol strongly inhibited LTB₄ production without cytotoxicity. These results suggest that a new assay system using PB-3c is convenient to evaluate LTB₄ inhibition activity by food components. This method could be utilized for elucidation of the mechanisms of LTB₄ release suppression by food components such as flavonoids and the structure–activity relationship.     

Evidence for a dual pathway in platelet activating factor-induced aggregation of rat polymorphonuclear leucocytes

The purpose of this study was to determine the role, if any, of Leukotriene B₄ (LTB₄) in Platelet Activating Factor (PAF)-induced aggregation of rat polymorphonuclear leucocytes (PMNs). Exposure of rat PMNs to 10⁻⁷ M PAF resulted in the release of 4.5 ± 0.7 ng/10⁷ cells of LTB₄ measured by radioimmunoassay. However, the maximum aggregation of PMNs achieved by exposure to LTB₄ (10⁻⁷M) was only 50% of that produced by maximally aggregating concentrations of PAF (10⁻⁷M). 5-Lipoxygenase inhibitors, BW755c and Nafazatrom at concentrations that completely abolished LTB₄ synthesis inhibited the aggregation induced by PAF only by 40% and 50% respectively. Furthermore, desensitisation experiments revealed that the aggregatory response of PMNs to PAF was only partially refractory to prior treatment with LTB₄ whereas the aggregatory response to LTB₄ was completely refractory to prior treatment with PAF. These results suggest that PAF-induced aggregation of rat PMNs is in part mediated by LTB₄ and in part directly by an as yet unidentified mechanism.     

Inflammation and pain sensitivity: Effects of leukotrienes D4, B4 and prostaglandin E1 in the rat paw

Leukotrienes (LT's) and prostaglandins (PG's) have been proposed as mediators of vascular permeability change in inflammatory reactions. Also, prostaglandins, especially of the E-type, have been shown to enhance pain responses. In the present studies in rat, the effects of LTB₄ and LTD₄ on edema and pain thresholds were examined in combination with PGE₁ and/or brewer's yeast. Subplantar injections of LTD₄ or LTB₄ induced small increases in paw thickness which were potentiated by the co-administration of PGE₁. LTD₄ alone had no significant effect on the development of the yeast paw edema. LTB₄ was found to reduce significantly the yeast edema and this reduction could be reserved by administration PGE₁. A small but significant decrease in pain threshold was caused by PGE₁ and this was significant enhanced in the presence of LTD₄. LTB₄, like PGE₁, was found to cause slight hyperalgesia but no synergy between the two agents was observed. LTD₄ was found to have no effect on the initial hypoalgesia or subsequent development of hyperalgesia caused by brewer's yeast. Both LTB₄ and PGE₁, however, prevented the initial hypoalgesia and significantly reduced tha latency for development of yeast induced hyperalgesia. These effects of LTB₄ are discussed in terms of possible release of cyclooxygenase products.     

Characterization of leukotriene A4 and B4 bioysnthesis

We have studied LTA₄ and LTB₄ synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB₄ from arachidonic acid are localized in the soluble fraction (100, 000 x g supernatant) of these cells. The formation of LTA₄ and LTB₄ is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 μM, the synthesis of LTB₄ leveled off at 30 μM and of LTA₄ at 100 μM while 5-HETE had not reached a plateau at 300 μM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA₄ and only a relatively small amount of LTB₄. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA₄ to LTB₄. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA₄ synthesis did not produce any additional LTA₄ or LTB₄. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.     

Corticosteroid suppression of lipoxin A4 and leukotriene B4from alveolar macrophages in severe asthma

Background

An imbalance in the generation of pro-inflammatory leukotrienes, and counter-regulatory lipoxins is present in severe asthma. We measured leukotriene B₄ (LTB₄), and lipoxin A₄ (LXA₄) production by alveolar macrophages (AMs) and studied the impact of corticosteroids.

Methods

AMs obtained by fiberoptic bronchoscopy from 14 non-asthmatics, 12 non-severe and 11 severe asthmatics were stimulated with lipopolysaccharide (LPS,10 μg/ml) with or without dexamethasone (10^-6M). LTB₄ and LXA₄ were measured by enzyme immunoassay.

Results

LXA₄ biosynthesis was decreased from severe asthma AMs compared to non-severe (p < 0.05) and normal subjects (p < 0.001). LXA₄ induced by LPS was highest in normal subjects and lowest in severe asthmatics (p < 0.01). Basal levels of LTB₄ were decreased in severe asthmatics compared to normal subjects (p < 0.05), but not to non-severe asthma. LPS-induced LTB₄ was increased in severe asthma compared to non-severe asthma (p < 0.05). Dexamethasone inhibited LPS-induced LTB₄ and LXA₄, with lesser suppression of LTB₄ in severe asthma patients (p < 0.05). There was a significant correlation between LPS-induced LXA₄ and FEV₁ (% predicted) (r_s = 0.60; p < 0.01).

Conclusions

Decreased LXA₄ and increased LTB₄ generation plus impaired corticosteroid sensitivity of LPS-induced LTB₄ but not of LXA₄ support a role for AMs in establishing a pro-inflammatory balance in severe asthma.     

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.