The Pivotal Role of 5-Lipoxygenase-Derived LTB4 in Controlling Pulmonary Paracoccidioidomycosis

Leukotrienes (LTs) produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. However, studies published in the literature regarding these mediators are contradictory and it remains uncertain whether these lipid mediators play a role in host defense against the fungal pathogen Paracoccidioides brasiliensis. To determine the involvement of LTs in the host response to pulmonary infection, wild-type and LT-deficient mice by targeted disruption of the 5-lipoxygenase gene (knockout mice) were studied following intratracheal challenge with P. brasiliensis yeasts. The results showed that infection is uniformly fatal in 5-LO-deficient mice and the mechanisms that account for this phenotype are an exacerbated lung injury and higher fungal pulmonary burden. Genetic ablation or pharmacological inhibition of LTs resulted in lower phagocytosis and fungicidal activity of macrophages in vitro, suggesting that deficiency in fungal clearance seems to be secondary to the absence of activation in 5-LO^−/− macrophages. Exogenous LTB₄ restored phagocytosis and fungicidal activity of 5-LO^−/− macrophages. Moreover, P. brasiliensis killing promoted by LTB₄ was dependent on nitric oxide (NO) production by macrophages. Taken together, these results reveal a fundamental role for 5-LO-derived LTB₄ in the protective response to P. brasiliensis infection and identify relevant mechanisms for the control of fungal infection during the early stages of the host immune response.     

Control of experimental pulmonary tuberculosis depends more on immunostimulatory leukotrienes than on the absence of immunosuppressive prostaglandins

Prostaglandins (PGs) and leukotrienes (LTs) are produced in Mycobacterium tuberculosis (Mtb)-infected lungs and have immune suppressive and protective effects, respectively. Considering that both of these mediators are produced during mycobacterial infection, we investigated the specific and relative biological importance of each in regulating host response in experimental tuberculosis. Administration of celecoxib, which was found to reduce lung levels of PGE₂ and increase LTB₄, enhanced the 60-day survival of Mtb-infected mice in 14%. However administration of MK-886, which reduced levels of LTB₄ but did not enhance PGE₂, reduced 60-day survival from 86% to 43% in Mtb-infected mice, and increased lung bacterial burden. MK-886 plus celecoxib reduced survival to a lesser extent than MK-886 alone. MK-886- and MK-886 plus celecoxib-treated animals exhibited reduced levels of the protective interleukin-12 and gamma-interferon. Our findings indicate that in this model, the protective effect of LTs dominates over the suppressive effect of PGs.     

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.     

The Leukotriene B4/BLT1 Axis Is a Key Determinant in Susceptibility and Resistance to Histoplasmosis

The bioactive lipid mediator leukotriene B₄ (LTB₄) greatly enhances phagocyte antimicrobial functions against a myriad of pathogens. In murine histoplasmosis, inhibition of the LT-generating enzyme 5-lypoxigenase (5-LO) increases the susceptibility of the host to infection. In this study, we investigated whether murine resistance or susceptibility to Histoplasma capsulatum infection is associated with leukotriene production and an enhancement of in vivo and/or in vitro antimicrobial effector function. We show that susceptible C57BL/6 mice exhibit a higher fungal burden in the lung and spleen, increased mortality, lower expression levels of 5-LO and leukotriene B₄ receptor 1 (BLT₁) and decreased LTB₄ production compared to the resistant 129/Sv mice. Moreover, we demonstrate that endogenous and exogenous LTs are required for the optimal phagocytosis of H. capsulatum by macrophages from both murine strains, although C57BL/6 macrophages are more sensitive to the effects of LTB₄ than 129/Sv macrophages. Therefore, our results provide novel evidence that LTB₄ production and BLT₁ signaling are required for a histoplasmosis-resistant phenotype.     

Antigen-Induced leukotriene relaese from rat lung

Chopped lung from inbred hyperreactive rats was challenged with antigen following active on passive sensitization and supernatants were assayed for the presence of leukotrienes (LTs) by radioimmunoassay. Dose-related increases in the release of LTC₄- and LTB₄-immunoreactive material were obtained with significantly more material being released following passive sensitization. Chromatographic analysis indicated the presence of LTB₄, LTC₄ and LTE₄. When LT release inbredred rats was compared to Sprague-Dawley or Fischer rats, the amounts released were as follows: Inbred > Sprague-Dawley > Fischer. It was concluded that the release of LTs in the three strains correlated with the degree of non-specific bronchial hyperreactivity.     

Antigen-Induced leukotriene relaese from rat lung in vitro

Chopped lung from inbred hyperreactive rats was challenged with antigen following active on passive sensitization and supernatants were assayed for the presence of leukotrienes (LTs) by radioimmunoassay. Dose-related increases in the release of LTC₄- and LTB₄-immunoreactive material were obtained with significantly more material being released following passive sensitization. Chromatographic analysis indicated the presence of LTB₄, LTC₄ and LTE₄. When LT release inbredred rats was compared to Sprague-Dawley or Fischer rats, the amounts released were as follows: Inbred > Sprague-Dawley > Fischer. It was concluded that the release of LTs in the three strains correlated with the degree of non-specific bronchial hyperreactivity.     

Malnutrition promotes prostaglandin over leukotriene production and dysregulates eicosanoid-cytokine crosstalk in activated resident macrophages

We previously described a murine model of malnutrition that mimicked features of moderate human malnutrition, and led to increased dissemination of Leishmania donovani. In this study, we investigated the effect of malnutrition on macrophage production of cytokines, prostaglandins (PGs), and leukotrienes (LTs). Using either LPS or calcium ionophore A23187 as a stimulus, macrophages from the malnourished mice produced a 3-fold higher PG/LT ((PGE₂+6-keto-PGF_1α)/(LTB₄+cysteinyl leukotrienes)) ratio than macrophages from well-nourished mice. LPS-stimulated macrophages from the malnourished mice produced decreased levels of TNF-α, GM-CSF, and IL-10, but similar levels of IL-6 and NO compared to well-nourished mice. A complex crosstalk between the eicosanoids and cytokines in the LPS-stimulated macrophages from the malnourished mice was evident by the following: (1) high levels of PG secretion despite low levels of TNF-α; (2) supplemental IL-10 modulated the excessive PG production; (3) GM-CSF rectified the PG/LT ratio, but did not correct the abnormal cytokine profile; and (4) inhibitors of cyclooxygenase decreased the PG/LT ratio, but did not affect TNF-α. Thus, in this model of malnutrition, there is a relative increase in anti-inflammatory PGs compared to pro-inflammatory LTs, which may contribute to immunodeficiency.     

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.     

A simple and sensitive radioreceptor assay for leukotrienes

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [³H]leukotriene D₄ (LTD₄) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD₄. The values for fifty percent inhibition of bound [³H]LTD₄ was 1.5 nM for LTD₄, 45 nM for LTC₄ and 24 nm for LTE₄. LTB₄ at 3.0 × 10⁻⁵ M had no effect on [³H]LTD₄ binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC₄ and LTD₄. However, in the presence of 20 nM serine-borate this method was highly specific for LTD₄. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD₄. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient ‘r’ of 0.992. The assay was also validated by quantitation of LTs released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD₄ than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD₄ specifically or LTC₄ plus LTD₄ simultaneously in one cell system.     

Production of leukotrienes and prostaglandins in the rat uterus during periimplantation period

We have measured by radioimmunoassay the production of leukotrienes (LTC₄ and LTB₄) and prostaglandins (PGE₂ and PGF_2α) in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC₄ or LTB₄ remained unaltered on days 1–3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE₂ and PGE_2α showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: (1) vasodilators and (2) agents that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation .     

Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD₄. This material however had a different retention time from LTD₄ when subjected to HPLC and co-chromatographed with synthetic LTE₄. In addition to LTE₄ a substance which had properties indistinguisable from those of LTB₄ when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB₄. The adventitia and intima were the richest source of LTE₄, the adventitia releasing slightly more than the intima. The output of LTB₄ and LTE₄ was inhibited by 6,9-deepoxy-6,9-(phenylimino)-Δ^6,8 prostaglandin I₁ (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE₄.     

Leukotriene receptor antagonists,LY293111 and ONO-1078, protect neurons from the sPLA2-IB-induced neuronal cell death independently of blocking their receptors

In the ischemic brain, leukotrienes (LTs) are increased and their receptor antagonists protect neurons. However, it has not yet been sufficiently clarified how antagonists for LT receptors exhibit neuroprotective effects. In the present study, we evaluated protective effects of receptor antagonists for LTB₄ (LY293111) and cysteinyl LTs (ONO-1078) in the primary culture of rat cortical neurons. The group IB secretory phospholipase A₂ (sPLA₂-IB)-induced neuronal cell death had been established as the in vitro model for cerebral ischemia. sPLA₂-IB triggered the influx of Ca²⁺ into neurons via L-type voltage-dependent calcium channel (L-VDCC). Subsequently, the enzyme produced eicosanoids including LTB₄ before neuronal cell death. Neither administration of LTB₄ nor cysteinyl LTs such as LTC₄, LTD₄ and LTE₄ killed neurons. However, both LY293111 and ONO-1078 significantly prevented neurons from the neurotoxicity of sPLA₂-IB, suggesting that the two LT receptor blockers protected neurons through alternative pathways beside LT receptors. An L-VDCC blocker does not only inhibit the influx of Ca²⁺ into neurons but also rescues neurons from the sPLA₂-IB-induced neuronal cell death. The two LT receptor antagonists also blocked the sPLA₂-IB-induced Ca²⁺ influx significantly. Thus, LTs exhibited no neurotoxicity, but their receptor antagonists protected neurons directly in the in vitro ischemic model. Furthermore, the suppression of L-VDCC appeared to be involved in the neuroprotective effects of LY293111 and ONO-1078 independent of blocking their receptors.     

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.     

Cyclooxygenase and lipoxygenase metabolite generation in nasal polyps

A role of prostaglandins (PGs) and leukotrienes (LTs) in the pathogenesis of nasal polyps has been recently suggested. Cyclooxygenase (CO) products (thromboxane B₂, PGE₂ and 6-keto PGF₁ alpha) and lipoxygenase (LO) products (LTB₄ and LTC₄) were investigated by radioimmunoassay in polyps, hypertrophic turbinates and nasal mucosa from 14 patients with non-allergic (n = 6), allergic chronic rhinitis (n = 6) and aspirin-sensitive asthma (ASA) (n = 2), who underwent polypectomy. In all tissues CO metabolite levels were found higher than LO products (P < 0.01). Nasal polyps showed a significantly lower (P < 0.05) arachidonic acid (AA) metabolism in comparison to nasal mucosa. In polyps of allergic patients significantly higher LTB₄ levels (P < 0.001) and a tendency to produce higher amounts of CO products in comparison to non-allergic subjects were observed, whereas in turbinates of non-allergic patients LT levels were significantly higher in comparison to those of allergic ones (P < 0.01). In ASA patients a decreased ratio was found supporting the hypothesis of an imbalance of AA metabolism in this syndrome. These findings seem to indicate that the occurrence of nasal polyps may represent the result of different chronic inflammatory stimuli, regulated in part by AA metabolites.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A₄, B₄, C₄, D₄ and E₄ have potent myotropic activity on guinea-pig lung parenchymal strip . The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB₄, the tissues became desensitized to LTB₄ but were still responsive to histamine, LTA₄, LTC₄, LTD₄ and LTE₄. When LTD₄ was infused continuously, the lung strips contracted to LTB₄ and histamine but were no longer responsive to LTA₄, LTC₄, LTD₄ and LTE₄. Furthermore, FPL-55712 (10 ng ml⁻¹− 10 ug ml⁻¹) produced dose-dependent inhibitions of LTA₄, LTC₄, LTD₄ and LTE₄ without inhibiting the contraction to LTB₄ and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB₄ and another for LTD₄; LTA₄, LTC₄ and LTE₄ probably act on the LTD₄ receptor.     

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.     

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.     

Analysis of leukotriene B4 in human lung lavage by HPLC and mass spectrometry

Although leukotrienes are beleived to mediate symptoms of human lung disease, there is little direct evidence of their existence in the lung. This is due to the difficulty in obtaining lung samples, the small amounts of leukotrienes typically present in such samples and the problems associated with purifying and analyzing leukotrienes in complex biological samples. In this study, lung lavagates were collected and analyzed for leukotrienes. The methods in this analysis included solid phase extraction using a C-18 reverse phase cartridge followed by HPLC using a new photodiode array detector which provides full UV spectra of eluting compounds. Lung lavage fluid from a patient with chronic pulmonary disease contained a compound with a UV spectra of LTB₄ which was found to elute with synthetic [³H]-LTB₄. This compound was confirmed as PTB using gas chromarography/mass spectrometry in the negative ion-chemical ionization mode. The inclusion of oxygen-18 LTB₄ as an internal standard allowed approximate quantitation of the amount of LTB₄ present in this 5 ml lung lavagate as 40–50 ng.     

Eicosanoid Profiling in an Orthotopic Model of Lung Cancer Progression by Mass Spectrometry Demonstrates Selective Production of Leukotrienes by Inflammatory Cells of the Microenvironment

Eicosanoids are bioactive lipid mediators derived from arachidonic acid¹ (AA), which is released by cytosolic phospholipase A₂ (cPLA₂). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA₂ knockout vs wild-type mice, we demonstrated that prostaglandins (PGE₂, PGD₂ and PGF_2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB₄, LTC₄, LTD₄, LTE₄) production required cPLA₂ expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.     

Quantitative measurement of cysteinyl leukotrienes and leukotriene B₄ in human sputum using ultra high pressure liquid chromatography-tandem mass spectrometry

The role of leukotrienes (LTs) in airway inflammatory diseases, such as asthma, has been extensively reported. The measurement of LTs in sputum supernatants, which is commonly done via enzyme immunoassays (EIAs), may prove to be useful for assessing airway inflammation. Despite the many advantages of EIA, these methods suffer from a lack of selectivity. Therefore, a selective and reliable method for the analysis of LTs in human sputum is needed. In this study we developed and validated a sensitive and specific method using ultra high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS), to measure simultaneously cysteinyl leukotrienes (CysLTs) and leukotriene B₄ (LTB₄) in human sputum. Sputum supernatants obtained by ultracentrifugation were stabilized by protease inhibitors, spiked with stable isotopic internal standards, and subjected to solid phase extraction (SPE) and UHPLC separation. Multiple reaction monitoring (MRM) transitions were optimized and measured on a mass spectrometer. The limit of detection (LOD) for LTE₄ and LTB₄ was 9.8 and 19.5 pg/mL, respectively. The lower limit of quantitation (LLOQ) for LTE₄ and LTB₄ was 19.5 and 39.0 pg/mL, respectively. The dynamic range of the LTE₄ assay was from 9.8 to 5000 pg/mL, whereas for the LTB₄ assay was from 19.5 to 10,000 pg/mL. The intra- and inter-day % coefficient of variation (%CV) was <6.5% and <10%, for both LTE₄ and LTB₄, respectively. Spike recovery ranged from 105% to 111% for both analytes. In addition, twenty-two sputum samples were analyzed for cysLTs and LTB₄. Fourteen of these samples were purchased commercially and eight were collected during the course of a clinical trial. LTB₄ was detectable in all samples tested and it ranged from 79 to 7220 pg/mL. LTE₄ was detectable in most of the sputum samples (12.3–891 pg/mL), whereas LTC₄ and LTD₄ were below limit of detection for majority of sputum samples. The in vitro conversion of LTC₄ and LTD₄ into LTE₄ was observed. The measurement of LTB₄ was sensitive to low pH and high temperature. The use of UHPLC–MS/MS method will allow a more accurate and reliable quantitation of LTs in human sputum, which in turn, may lead to a better understanding of the role of LTs in airway disease pathways and the application in associated clinical treatments.