Airway eosinophil migration into lymph nodes in mice depends on leukotriene C4.

BACKGROUND: We previously demonstrated in mice that airway eosinophils traffic from the airway lumen into lung-draining paratracheal lymph nodes. However, mechanisms whereby eosinophils traverse from the lungs and home to paratracheal lymph nodes remain unclear. We investigated roles of cysteinyl leukotrienes in mediating eosinophil trafficking from lungs to paratracheal lymph nodes. METHODS: The expression of CCR7 was determined by flow cytometry. Transwell assays were used to test chemotactic responses of leukotriene C4 synthase-deficient and control airway eosinophils to the chemokine CCL19 ex vivo. Eosinophils from the spleens of IL-5 transgenic mice, fluorescently labeled ex vivo, were intratracheally injected into ovalbumin-sensitized and ovalbumin aerosol-challenged leukotriene C4 synthase-deficient and control mice. Eosinophils were identified by microscopy and flow cytometry in the lungs and paratracheal lymph nodes. RESULTS: Mouse eosinophils expressed CCR7, the receptor for CCL19, and responded chemotactically to CCL19. Leukotriene C4 synthase-deficient eosinophils exhibited impaired chemotaxis to CCL19 that was restored by exogenous leukotriene C4 . The migration of intratracheally injected eosinophils into paratracheal lymph nodes from distal alveolar lung was diminished in leukotriene C4 synthase-deficient mice compared with wild-type mice, with increased retention of eosinophils in the lungs of leukotriene C4 synthase-deficient mice. Exogenous administration of leukotriene C4 restored trafficking of eosinophils to paratracheal lymph nodes in leukotriene C4 synthase-deficient mice. CONCLUSIONS: Our findings that cysteinyl leukotrienes are involved in regulating airway and lung eosinophil migration into paratracheal lymph nodes identify previously unrecognized roles for the cysteinyl leukotrienes in regulating the pulmonary trafficking of eosinophils in experimental allergic asthma.

The responses of pulmonary and systemic circulation and airway to anaphylactic mediators in anesthetized BALB/c mice.

AIMS: Anaphylactic shock sometimes accompanies pulmonary vaso- and broncho-constriction. We previously reported the hemodynamic features of mouse anaphylaxis (Life Sci. 2014; 116: 98-105). However, the effects of anaphylactic chemical mediators on the hemodynamics of in vivo mice are not well known. Furthermore, it is uncertain whether the mediators exert the same directional actions. Therefore, we determined their effects systematically on total peripheral resistance (TPR), pulmonary vascular resistance (PVR), or airway pressure (AWP) in anesthetized mice. MAIN METHODS: We measured directly pulmonary arterial pressure, left atrial pressure, systemic arterial pressure, central venous pressure and aortic blood flow to determine PVR and TPR, as well as AWP, following injections of platelet-activating factor (PAF), histamine, serotonin, leukotriene (LT) C4, and prostaglandin (PG) D2 in anesthetized open-chest artificially ventilated BALB/c mice. KEY FINDINGS: Consecutive administration of any agents increased PVR dose-dependently with the maximal responsiveness being PAF>LTC4>serotonin>>histamine=PGD2. Histamine caused a biphasic PVR response, an initial decrease, which was abolished by L-NAME, followed by an increase at high doses. PAF, serotonin, and histamine decreased TPR dose-dependently, while LTC4 or PGD2 yielded an increase or no change in TPR, respectively. Serotonin, but not the other agents, increased AWP. SIGNIFICANCE: Anaphylactic mediators exert non-uniform actions on the pulmonary and systemic circulation and airway in anesthetized BALB/c mice: PAF, LTC4 and serotonin cause substantial pulmonary vasoconstriction, while histamine biphasic responses of the initial nitric oxide dependent vasodilation followed by vasoconstriction; PAF, serotonin, and histamine, but not LTC4 or PGD2, evoke systemic vasodilatation; only serotonin induces airway constriction.

Pranlukast prevents cysteinyl leukotriene-induced emesis in the least shrew (Cryptotis parva).

Many chemotherapeutic agents activate multiple signaling systems, including potentially emetogenic arachidonic acid metabolites. Of these messengers, the emetic role of the leukotriene family has been neglected. The aims of this study were to test the emetic potential of key leukotrienes (LTA(4), LTB(4), LTF(4), and the cysteinyl leukotrienes LTC(4), LTD(4) and LTE(4)), and to investigate whether the leukotriene CysLT(1) receptor antagonist pranlukast or mixed leukotriene CysLT(1/2) receptor antagonist Bay u9773 can prevent the LTC(4)-induced emesis. Least shrews were injected with varying doses of one of the six tested leukotrienes and vomiting parameters were measured for 30min. LTC(4) and LTD(4) were most efficacious, and significantly increased both the frequency and percentage of animals vomiting at doses from 0.1 and 0.05mg/kg, respectively. The other tested leukotrienes were either weakly emetic or ineffective at doses up to 4mg/kg. The relative emetogenic activities of the cysteinyl leukotrienes (LTC(4)=LTD(4)>LTE(4)) suggest that leukotriene CysLT(2) receptors have a key role in emesis. However, pranlukast dose-dependently, and at 10mg/kg completely, blocked LTC(4)-induced vomiting, implicating a leukotriene CysLT(1) receptor-mediated emetic effect. Bay u9773 dose-dependently reduced the percentage of animals vomiting, but did not significantly reduce vomiting frequency. Fos immunoreactivity, measured subsequent to LTC(4)-induced vomiting to define its putative anatomical substrates, was significantly increased in the enteric nervous system and medullary dorsal vagal complex following LTC(4) (P<0.05) versus vehicle injections. This study is the first to show that some leukotrienes induce emesis, possibly involving both central and peripheral leukotriene CysLT(1) and/or leukotriene CysLT(2) receptors.

IL-4 exacerbates anaphylaxis.

We evaluated whether IL-4, a cytokine critical for inducing allergic responses, also contributes to the effector phase of allergy. Pretreatment of mice with IL-4 or the related cytokine, IL-13, rapidly and dramatically increased the severity of anaphylaxis induced by cross-linking Fc(epsilon)RI or FcgammaRIII. This effect was inhibited by endogenously produced IFN-gamma, was T cell-, B cell-, and common gamma-chain-independent, and required IL-4Ralpha and Stat6. IL-4Ralpha signaling also enhanced anaphylaxis in mice infected with a nematode parasite that stimulates IL-4/IL-13 production. IL-4 exacerbated anaphylaxis by acting synergistically with vasoactive mediators to increase vascular permeability. Synergy between IL-4 and vasoactive mediators during the effector phase of allergic inflammation may both contribute to allergic immunopathology and enhance protective immunity against gastrointestinal worms.

Cysteinyl leukotrienes induce IL-4 release from cord blood-derived human eosinophils.

BACKGROUND: Eosinophils contain preformed stores of IL-4 within their cytoplasmic granules, but physiologic stimuli to release IL-4 from eosinophils are not yet defined. OBJECTIVE: We evaluated whether cysteinyl leukotrienes (CysLTs) could elicit IL-4 release from eosinophils. METHODS: We used a dual-antibody capture and detection assay (EliCell) for IL-4 release and used eosinophils differentiated in vitro from human cord blood-derived progenitors. RESULTS: Leukotriene (LT) C4, LTD4, and LTE4 each elicited the rapid, vesicular transport-mediated, dose- and time-dependent release of IL-4 from eosinophils. Both LTD4 and LTE4 evoked similar and earlier IL-4 release than LTC4. LTC4 did not act directly but only after conversion to LTD4 because an inhibitor of gamma-glutamyl transpeptidase, acivicin, blocked LTC4-induced IL-4 release. MK571 and LY171833, receptor antagonists for CysLT1 and not CysLT2, and pertussis toxin inhibited LTC4-, LTD4-, and LTE4-induced IL-4 release. Cord blood-differentiated eosinophils contained CysLT1 protein detectable by means of immunoblotting. CONCLUSION: CysLTs acting through G(i) protein-coupled and MK571- and LY171833-inhibitable receptors on cord blood-derived human eosinophils can act as autocrine or paracrine mediators to stimulate the rapid, nonexocytotic release of preformed IL-4.

Metabolic and hemodynamic effects of peptide leukotriene C4 and D4 in man.

The time course of the effects of intravenous or intracoronary administration of peptide leukotrienes on metabolic parameters and on systemic and coronary hemodynamics was evaluated in 15 patients with normal coronary arteries. Peptide leukotriene C4 (2 nmol given as a bolus intravenous injection) induced an early fall (at 2 min) in mean arterial pressure (P < 0.02) associated with a rise in heart rate (P < 0.001) and in plasma levels of epinephrine (P < 0.05) and norepinephrine (P < 0.005), but without significant changes in coronary blood flow or coronary vascular resistance. Mean arterial pressure, heart rate, norepinephrine, and epinephrine returned to baseline values 10 min after leukotriene C4 administration. In contrast, at 10 min post leukotriene C4, with coronary blood flow and myocardial oxygen consumption unchanged, an increase in coronary vascular resistance (P < 0.05) and in myocardial oxygen extraction (P < 0.01) was observed, which returned to baseline values at 20 min post leukotriene C4. Peptide leukotriene D4 (3 nmol, given in the left coronary artery) induced an early (20 s) and transient fall in mean arterial pressure (P < 0.001) paralleled by a rise in heart rate and plasma levels of epinephrine and norepinephrine, all of which returned to baseline at 10 min. Coronary vascular resistance increased at 10 and 15 min (P < 0.02 and P < 0.05, respectively) and myocardial oxygen extraction at 15 min (P < 0.02). These results suggest that small doses of peptide leukotrienes induce both an early and transient fall in mean arterial pressure associated with secondary sympathoadrenergic activation, and a late increase in small coronary arteriolar resistance.

Inhibition by actinomycin D of neurogenic mouse ear oedema.

We have investigated the effects of actinomycin D on mouse ear oedema induced by capsaicin, neuropeptides, and established inflammatory mediators. Actinomycin D (0.5 mg/kg, i.v.) significantly (P < 0.01) inhibited ear oedema induced by topical application of capsaicin, while adriamycin (6.0 mg/kg, i.v.) and cycloheximide (6.0 mg/kg, i.v.) had no effect on oedema. The ear oedema induced by intradermal injection of neuropeptides such as mammalian tachykinins, calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP), was markedly (P < 0.05, P < 0.01 or P < 0.001) suppressed by actinomycin D. The drug was also effective (P < 0.01 or P < 0.001) in inhibiting bradykinin (BK)- and compound 48/80-induced ear oedema, but did not inhibit oedema induced by histamine, 5-HT, leukotriene C4 (LTC4), and platelet activating factor (PAF) at a dose of 1 mg/kg. In mast cell-deficient W/WV mice, actinomycin D (1.0 mg/kg, i.v.) failed to inhibit substance P (SP)-induced ear oedema whereas spantide (0.5 mg/kg, i.v.) was an effective (P < 0.01) inhibitor of oedema formation. Furthermore, actinomycin D (10-100 microM) dose-dependently prevented histamine release from rat peritoneal mast cells evoked by SP, compound 48/80, and the ionophore A23182, respectively. These results strongly suggest that an inhibitory effect of actinomycin D on neurogenic inflammation is due primarily to the prevention of mast cell activation mediated by neuropeptides, rather than an interaction with DNA or receptors of neuropeptides.

Increased urinary LTE4 excretion following inhalation of LTC4 and LTE4 in asthmatic subjects.

Urinary leukotriene E4 (LTE4) increases during exacerbations of asthma and following antigen challenge. We determined whether urinary LTE4 excretion reflects sulphidopeptide leukotrienes in the airways of asthmatic patients. Urinary LTE4 concentration was measured prior to and 1.5 and 3.5 h following inhalation of bronchoconstrictive doses of leukotriene C4 (LTC4) or LTE4 in eight asthmatic subjects. Increasing doses of agonist were inhaled until a 35% fall in specific airways conductance (sGaw) was achieved. There was no significant difference between the 53 +/- 3% (mean +/- SEM) fall in sGaw following inhalation of LTC4 (63.1 ng geometric mean, GM, range 5.8-527.5 ng) and the 43 +/- 4% fall in sGaw following inhalation of LTE4 7.94 ng/GM (range 132-3701 ng). The LTE4 excretion rate increased significantly from 2.95 (range 0.6-17.5) ng.h-1 to 4.67 (range 0.8-20) ng.h-1 at 1.5 h following LTC4 inhalation; and from 1.8 (range 0.07-6.7) ng.h-1 to 6.9 (range 2.9-27.3) ng.h-1 at 1.5 h following LTE4 inhalation; and had returned from baseline by 3.5 h. There was a correlation between the dose of LTC4 inhaled and LTE4 excreted in the urine (r = 0.82 and r = 0.72, respectively). The % recovery of LTE4 in the urine, of the total dose of inhaled LTC4 or LTE4 administered, was 6.9 +/- 4.1% and 0.8 +/- 0.3%, respectively. Thus, inhalation of bronchoconstricting doses of LTC4 or LTE4 alter urinary LTE4 excretion in a dose-dependent fashion. This indicates that urinary LTE4 can be used as a marker of sulphidopeptide leukotriene synthesis in the lungs of patients with asthma.

Inactivation of leukotriene C4 in the airways and subsequent urinary leukotriene E4 excretion in normal and asthmatic subjects.

Leukotrienes (LT) are synthesized in the lung during asthmatic reactions and mediate certain inflammatory symptoms. Pulmonary metabolism and clearance of exogenously added peptidoleukotrienes were studied in nonasthmatics, asthmatics, and asthmatics challenged with allergen. [3H]LTC4 and [14C]dextran were instilled into the airways, and bronchoalveolar lavage fluid (BALF) was obtained 15 min later. After comparing the [3H]/[14C] ratio of the instilled solution with that in BALF, 77% of LT were found to have been removed from the airways of nonasthmatics, and 72% of unchallenged asthmatics. Allergen administration to asthmatics 1 min before LT instillation inhibited LT transfer out of the airways by 26%, compared with asthmatics not challenged with allergen. This decrease in the removal of LT from the airways during allergic reactions could potentiate the physiologic effects of LT produced in the airways. The predominant LT in BALF was LTE4, constituting 56% of the LT in asthmatics and 61% in nonasthmatics. The percentage of LTE4 in BALF increased to 87% in allergen-stimulated asthmatics (p < 0.05 compared with the two other groups), this again reflecting decreased transfer of LT out of the lung rather than an increase in metabolism. Urinary excretion of LT metabolites occurred rapidly, the majority being excreted excreted within 6 h after instillation. LTE4, the major urinary LT metabolite identified by high-performance liquid chromatography, was a similar percentage concentration in the three groups and, thus, can be accurately used as an index of LT synthesis.

Inhibition of bronchial hyperresponsiveness to histamine induced by intravenous administration of leukotriene C4 by novel thromboxane A2 receptor antagonists ONO-NT-126 and ONO-8809 in guinea-pigs.

We studied the effect of intravenous administration of leukotriene (LT) C4 on bronchial responsiveness to histamine and airway wall thickening in guinea-pigs. Guinea-pigs were killed and the lungs were fixed in formalin. Slides from paraffin-embedded sections of the lungs were stained and the airways that were cut in transverse sections were measured by tracing enlarged images using a digitizer. Moreover, airway resistance (Raw) was determined by a pulmonary mechanics analyser and we calculated two indices, an index of airway wall thickening and the one of airway hyperresponsiveness to histamine, from changes of baseline-Raw and peak-Raw following intravenous administration of histamine before and after the intravenous administration of LTC4. Intravenous administration of 3 microg/kg LTC4 for 1 hr induced an increase of the relative thickness of the airway wall in peripheral bronchi by the histological examination. In analysis of airway function, intravenous administration of 3 microg/kg LTC4 for 1 hr induced airway hyperresponsiveness to histamine with airway wall thickening. Thromboxane A2 receptor antagonists ONO-NT-126 and ONO-8809 inhibited the LTC4-induced airway hyperresponsiveness to histamine in a dose-dependent manner, but not the airway wall thickening induced by LTC4, suggesting that the effect of LTC4 on bronchial hyperresponsiveness is likely to be mediated through TXA2.