Detection of leukotriene C4-liked immunoreactivity in tear fluid from subjects challenged with specific allergen

Tear fluid was obtained from allergic subjects from control eyes and eyes challenged with specific allergen and levels of leukotriene C4 (LTC4)-immunoreactivity determined by radioimmunoassay. Formal identification of the leukotrienes released was not possible but the levels of LTC4-immunoreactive material in allergen-challenged tear fluid (4.9 +/- 2.3 ng/ml, n = 9) were significantly higher (p less than 0.01) than those in control tear fluid (0.07 +/- 0.06 ng/ml, n = 9). These results provide evidence that leukotrienes, which account for the biological activity of slow reacting substance of anaphylaxis, may be released in allergic reactions in vivo in man.     

Release of slow-reacting substance of anaphylaxis and leukotriene C4-like immunoreactivity from guinea pig colonic tissue

Colonic mucosa, muscularis propria and subserosa from ovalbumin-sensitized guinea pigs were incubated and challenged with antigen in vitro. Slow-reacting substance of anaphylaxix (SRS-A) was determined biologically as well as radioimmunologically in terms of leukotriene (LT) C₄-like immunoreactivity. Before antigenic challenge release of immunoreactive LTC₄ by all tissues was below or close to the detection limit of the radioimmunoassay. After addition of antigen colonic mucosa released considerable amounts of LTC₄-like immunoreactivity, while muscularis propria and subserosa were less active. The biological activity of the SRS-A formed after challenge was antagonized by FPL 55712. Contrary to LTC₄-like immunoreactivity release of 6-keto-prostaglandin (PG) F1_α was predominant in the subsero and smaller amounts were released from the smooth muscular and mucosal layers. Synthesis of SRS-A and LTC₄-like immunoreactivity, respectively, as well as synthesis of 6-keto-PGF_1α was inhibited by the dual inhibitor of lipoxygenase and cyclooygenase BW755C. The results suggest a role for LTs as local mediators of inflammatory reactions in colonic disease states, particularly those with possible involvement of immunological processes.     

Isolation of leukotriene C4 from human seminal fluid

LTC₄ was isolated and characterized from seminal fluid of seven human volunteers. A compound with a similar retention time of that of synthetic LTC₄ was obtained using reverse-phase high performance liquid chromatography. The ultraviolet absorbance of the extracted substance was identical to synthetic LTC₄. Furthermore this compound contracted the guinea pig ileum and lung parenchymal strip. Its effects were antagonized by the leukotriene antagonist FPL55712. It was concluded that LTC₄ is present in human seminal fluid in very small amounts (about 100 ng/ejaculate). The possible physiological functions of LTC₄ in the reproductive tract area discussed.     

Release of leukotriene C4 from guinea pig trachea

Immunological (ovalbumin) and non-immunological (calcium ionophore A23187) stimulation of guinea pig trachea induces a prolonged contraction that is enhanced by indomethacin (8.5 μM) and inhibited by nordihydroguaiaretic acid (50 μM) pretreatment of the tissue. The mediator released by the above stimuli was identified as leukotriene C₄ by reverse-phase high performance liquid chromatography, and quantitated by bioassay. Indomethacin, and/or arachidonic acid (32.8 μM) did not enhance the release, whereas nordihydrolguaiaretic acid reduced the contraction and release of LTC₄. The results demonstrate the hitherto unproved capability of the large airways to synthesize leukotrienes and emphasize the importance of examining their role in asthma.     

Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C₄ (LTC₄) binding sites were identified in membrane preparations from human fetal lung. Specific binding of [³H]-LTC₄ represented 95 percent of total binding, reached steadystate within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC₄. Binding assays were performed at 4°C under conditions which prevented metabolism of [³H]-LTC₄ (80 mM serineborate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC₄ standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (K_d) of 26 + 6 nM and a density (B_max) of 84 ± 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [³H]-LTC₄ was displaced by LTC₄ and its structural analogs with inhibition constants (K_j) of 10 to 30 nM, whereas LTD₄, diastereoisomers of LTD₁, LTE₄ and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC₄. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [³H]-LTC₄ in human fetal lung membranes.     

Detection of leukotriene C4 and D4 in the exudate of rat carrageenin-induced pleurisy

Rat carrageenin-induced pleurisy was used as an acute exudative inflammatory model. The crude ethanol extract of the pleural fluid at 5 hr after carrageenin injection caused the very slow contraction of guinea-pig ileum, which was antagonized by FPL 55712 (1 μg/ml). The ethanol extract was cleaned by LH-20 and was rendered for separation of LTC₄ and LTD₄ by reversed-phase high-performance liquid chromatography (HPLC). Two peaks which showed the same retention time on HPLC as those of LTC₄ and LTD₄ had the contractile activity of guinea-pig ileum and the ratios of the contractile activity to the height on HPLC agreed with those of synthetic LTC₄ and LTD₄. Two peaks of Δ⁶-trans-LTB₄, 5S,12R-(E,E,E,Z)-diHETE and 5S, 12S-(E,E,E,Z)-diHETE, were detected, but the appreciable amount of LTB₄ was smaller than that of each Δ⁶-trans-LTB₄ in the pleural fluid at 5 hr.     

Agonist specific desensitization of leukotriene C4-stimulated PGI2 biosynthesis in human endothelial cells

Leukotriene C₄ (LTC₄) and, to a lesser extent, leukotriene D₄ (LTD₄) concentration dependently stimulate prostacyclin (PGI₂) biosynthesis in cultured human umbilical vein endothelial cells. PGI₂ biosynthesis was quantitated by radioimmunoassay and its structure confirmed by gas chromatography/mass spectrometry. Preincubation of endothelial cells with LTC₄ resulted in desensitization to subsequent LTC₄ stimulation. However, PGI₂ biosynthesis in response to thrombin, PGH₂ and arachidonic acid was not inhibited by preincubation with LTC₄. The C-6-sulfidopeptide leukotriene receptor level antagonist FPL-55712 attenuates LTC₄, but not thrombin-stimulated PGI₂ biosynthesis. These data suggest that human umbilical vein endothelial cells have a C-6-sulfidopeptide leukotriene receptor, and that stimulation of this receptor results in PGI₂ biosynthesis.     

Leukotriene A4: Enzymatic conversion to leukotriene C4

An unstable epoxide, leukotriene A₄ (5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C₄. In the present work synthetic leukotriene A₄ was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C₄ and 11-$\text{trans}$-leukotriene C₄ were the main products with SRS activity. It was not established whether the 11-$\text{trans}$-compound was formed by isomerization at the leukotriene A₄ or C₄ stage.     

Involvement of leukotriene C4 in PAF-induced death in mice

The role of leukotrienes (LTs) in the pathogenesis of platelet-activating factor (PAF)-induced death in mice was reinvestigated, since previously reported results are in conflict. A novel 5-lipoxygenase inhibitor, E6080, and a leukotriene antagonist, LY17883, protected mice from PAF-induced death in a dose-dependent manner, while the well-known 5-lipoxygenase inhibitor, AA861, was less effective than E6080. After the intravenous injection of PAF in mice, immunoreactive leukotriene C4 (i-LTC4), which was co-eluted with authentic LTC4 in HPLC, was significantly increased in bronchoalveolar lavage fluid (BALF). Oral administration of E6080 suppressed the increase in i-LTCM4. The results suggest that LTs may play an important role in PAF-induced lethality in mice.     

Functional expression of single-chain antibody to leukotriene C4

Leukotriene C₄ (LTC₄) is synthesized by binding of glutathione to LTA₄, an epoxide derived from arachidonic acid, and further metabolized to LTD₄ and LTE₄. We previously prepared a monoclonal antibody with a high affinity and specificity to LTC₄. To explore the structure of the antigen-binding site of a monoclonal antibody against LTC₄ (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC₄ comparable to mAbLTC. The scFvLTC also bound to LTD₄ and LTE₄ with 48% and 17% reactivities, respectively, as compared with LTC₄ binding, whereas the antibody showed almost no affinity for LTB₄.     

Interaction between leukotriene C4 and histamine in the guinea-pig

Lipoxygenase metabolites have proposed as potential chemical mediators of the bronchial hyperractivity which characterizes asthma (2,6). In addition to the possibility that leukotrienes (LTs) sensitize airways smooth muscle to the contractile actions of other mediators such as histamine (1–3), a number of studies have provided evidence for LT-induced enhancement of bronchoconstriction by a vagal dependent mechanism (4–6). In the present study the effects of exposure of the airway to LTC₄ on subsequent responsiveness to histamine have been investigated in both and experiments. LTC₄, in a concentration eliciting threshold contractile responses of the isolated trachea (1.7 nM), had no effect on either the EC₅₀ or maximal contractile response to histamine. At a concentration eliciting an approximately EC₅₀ contractile response, LTC₄ (10 nM) shifted the histamine concentration-response curve rightwards altering the maximum response. In anaesthetized, mechanically ventilated guinea pigs LTC₄ (0.1–0.4 nMole/kg, i.v.) injected 20 s beforehand, failed to alter histamine (9–36 nMole/kg, i.v.)-induced bronchoconstriction whereas, under the same conditions, LTD₄ (0.05–0.2 nMole/kg, i.v.) dose-dependently enhanced histamine-induced bronchoconstriction. On the other hand, LTC₄ or LTD₄ (16 uM, 30 s) aerosols potentiated histamine (9.36 nMole/kg, i.v.) in a concentration-dependent manner (Table). Both LTC₄ and LTD₄ aerosols enahance airway reactivity to histamine whereas only LTD₄ has this action when administered intravenously. Neither LTC₄ nor LTD₄ (6) enhances the contractile effects of histamine on isolated airways smooth muscle. It is concluded that the broncho-constriction enhancing action of these leukotrienes may be indirectly mediated.     

Bioconversion of leukotriene C4 by rat glomeruli and papilla

Since leukotriene C₄ (LTC₄) may be locally synthesized by bone marrow-derived cells infiltrating the kidney in inflammatory renal diseases we examined the in vitro metabolism of exogenously added |³H| LTC₄ by rat glomeruli and papilla using radiometric HPLC. Homogenized as well as intact glomeruli converted |³H| LTC₄ mainly into |³h| LTE₄ (83%) and, at a smaller extent, into |³H| LTD₄ (4%). Intact |³H| LTC₄ represented 13% of the sum of radioactive leukotrienes. Addition of L-cysteine resulted in accumulation of LTD₄. In contrast, there was nearly no conversion of |³H| LTC₄ (87% ntact) in the presence of homogenized papilla. The metabolism of |³H| LTC₄ by the glomeruli was time- and temperature- dependent. The 10,000 g supernatant and pellet of homogenized glomeruli both retained the ability to metabolize |³H| LTC₄. The papillary 10,000 g supernatant was inactive, as found for the total homogenate, whereas the papillary 10,000 g pellet separated from its supernatant could transform |³H| LTC₄ into its metabolites, LTD₄ and LTE₄. Addition of increasing amounts of papillary 10,000 g supernatant to homogenized glomeruli progressively protected |³H| LTC₄ from its bioconversion. These results demonstrate that both glomeruli and papilla possess the γ-glutamyl transpeptidase and dipeptidase necessary to process LTC₄. However, the enzyme activity of the papilla is unmasked only when the inhibitor present in the 10,000 g supernatant is separated from the enzyme present in the pellet.     

Ethanol stimulates formation of leukotriene C4 in rat gastric mucosa

Ethanol-induced gastric mucosal damage is characterized by microcirculatory changes such as statis and plasma leakage. Sluggish blood flow and statis have also been observed after administration of exogenous leukotriene (LT) C₄. The effect of ethanol on the release of LTC₄ from rat gastric mucosa was therefore investigated. It was found that intragastric instillation of ethanol increases gastric mucosal release of LTC₄ in a dose- and time-dependent manner parallel to the production of gastric lesions. The lipoxugenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-ulcer drug carbenoxolone (CX) inhibited mucosal release of LTC₄ and simultaneously protected against gastric damage caused by ethanol. It is concluded that increased formation of LTC₄ and /or other 5-lipoxygenase-derived products of arachidonate metabolism may be involved in ethanol-induced gastric damage. Furthermore, inhibition of the 5-lipoxygenase pathway may be an important mechanism of action of gastric protective drugs.     

Autoradiographic localization of leukotriene C4 and D4 binding sites in guinea-pig lung

Binding of [3H] leukotriene C4 and D4 to guinea-pig lung sections was charaterised and binding sites were localized by autoradiography. Both leukotrienes bound to guinea-pig lung sections and membranes with high affinity and with similar charateristics to binding in a membrane preparation. Autoradiography revealed that the distribution of LTC4 and D4 binding sites was markedly different. Smooth muscle and epithelium of central and peripheral airways were densely labelled with [3H]LTC4; vascular smooth muscle and alveolar walls were also labelled. With [3H]LTD4, however, there was no detectable labelling of airways or vessels but subtantial labelling of alveolar walls. This lends futher support that LTC4 and LTD4 binding sites differ and may not be identical with functional receptors.     

Species difference in increased vascular permeability by synthetic leukotriene C4 and D4

The activity of synthetic LTC₄ was tested in guinea-pig ileum and was 200 times more potent than histamine in contraction of the ileum (3 × 10^?11 M- 3 × 10^?9 M). The activities of LTC₄ and LTD₄ in increased vascular permeability in guinea pigs, rats and rabbits were compared with those histamine, bradykinin and prostaglandin (PG) E₂. LTC₄ was approximately equipotent to bradykinin on a molar basis in guinea pigs and rats and 5–100 times more potent than histamin. LTD₄ was about 10 times more potent than LTC₄ in guinea pigs and as equipotent to LTC₄ in rats. On the contrary, in rabbits, neither LTC₄ (upto 30 nmole/site) nor LTD₄ (1 nmole/site) induced the dye exduation. These results show that species difference is present in activity of LTC₄ and LTD₄ in vascular permeability. Furthermore, in guinea pigs, the vascular permeability increased by LTC₄ was not affected after pretreatment with pyrilamine (2.5 mg/kg, i.v.), and LTC₄ and LTD₄ did not potenciate the activity of bradykinin in vascular permeability.     

Leukotriene B4 and prostaglandin E2-like activity in synovial fluid in osteoarthritis

LTB4 and PGE2-like activity in synovial fluid samples from patients with osteoarthritis of the knee joint were determined and found to be significantly higher than in samples obtained from normal patients. The results suggest that leukotrienes and prostaglandins may have a role in the pathogenesis of osteoarthritis.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Secretogogue responses of leukotriene C4, D4: Comparison of potency in canine trachea

By the use of close arterial injection of leukotrienes into the circulation supplying the upper cervical canine trachea, it has been possible to assess the secretogogue effects of leukotriene C₄, and D₄ on mucus secretion. Both LTC₄ and LTD₄ increased mucus secretion over baseline levels by a statistically significant level (p = < 0.05). LTD₄ was more potent than C₄ with relative potencies of 2500, 320, 630, and 500 based on hillock formation (a measure of secretion) at 1, 2, 3, and 4 minutes after injection. The overall difference in potency in this animal model of mucus production was LTD₄ > C₄ by 1000-fold.     

Defective inhibition by dexamethasone of leukotriene B4 and C4 production by leukocytes in patients with cystic fibrosis

To investigate the effects of glucocorticoids on leukotriene (LT) generation in patients with cystic fibrosis (CF), we evaluated calcium ionophore A23187-induced LTB₄ and LTC₄ production by leukocytes with and without pretreatment with dexamethasone. The CF patients were in good condition and did not have acute infection. There were no significant differences in LTB₄ and LTC₄ production without dexamethasone pretreatment between the CF patients and controls. However, the ratios of LTB₄ and LTC₄ production by leukocytes preincubated with dexamethasone to those of leukocytes without dexamethasone pretreatment were significantly higher in the CF patients than in the controls (both p < 0.05). Our data suggest that the response of LTB₄ and LTC₄ production to dexamethasone is disturbed in patients with CF. The generation of LTs may be enhanced due to a disturbance in glucocorticoid suppression.