A review of the therapeutic potential of the cysteinyl leukotriene antagonist Montelukast in the treatment of bronchiolitis obliterans syndrome following lung and hematopoietic-stem cell transplantation and its possible mechanisms.

Lung and hematopoietic stem cell transplantation are therapeutic modalities in chronic pulmonary and hematological diseases, respectively. One of the complications in these patients is the development of bronchiolitis obliterans syndrome (BOS). The efficacy and safety of available treatment strategies in BOS remain a challenge. A few mechanisms have been recognized for BOS in lung transplant and graft-versus-host disease (GVHD) patients involving the TH-1 and TH-2 cells, NF-kappa B, TGF-b, several cytokines and chemokines, and cysteinyl leukotrienes (CysLT). Montelukast is a highly selective CysLT receptor antagonist that has been demonstrated to exert anti-inflammatory and anti-fibrotic effects in abundant experiments. One area of interest for the use of montelukast is lung transplants or GVHD-associated BOS. Herein, we briefly review data regarding the mechanisms involved in BOS development and montelukast administration as a treatment modality for BOS, and finally, the possible relationship between CysLTs antagonism and BOS improvement will be discussed.

A review of the therapeutic potential and possible mechanism of Montelukast in the treatment of bronchiolitis obliterans syndrome following lung and hematopoietic stem cell transplantationLung and bone marrow transplantation are therapeutic modalities in chronic diseases of the lungs and the blood, respectively. One of the complications in these patients is the development of Bronchiolitis obliterans syndrome (BOS). The efficacy and safety of available treatment strategies in BOS remain a challenge. A few mechanisms for BOS in lung transplant and graft-versus-host disease (GVHD) patients involving many immune components have been recognized. Cysteinyl leukotrienes are products of plasma membrane phospholipids that increase smooth muscle contraction, microvascular permeability, and airway mucus secretion. Montelukast is a highly selective cysteinyl leukotriene receptor blocker demonstrated to exert anti-inflammatory and anti-fibrotic effects. One area of interest for the use of montelukast is in lung transplant- or GVHD-associated BOS. In this article, we briefly review data regarding the mechanisms involved in BOS development and montelukast administration as a treatment modality for BOS. Finally, the possible relationship between cysteinyl leukotriene inhibition and BOS improvement will be discussed.

Redox processes are major regulators of leukotriene synthesis in neutrophils exposed to bacteria Salmonella typhimurium; the way to manipulate neutrophil swarming.

Neutrophils play a primary role in protecting our body from pathogens. When confronted with invading bacteria, neutrophils begin to produce leukotriene B4, a potent chemoattractant that, in cooperation with the primary bacterial chemoattractant fMLP, stimulates the formation of swarms of neutrophils surrounding pathogens. Here we describe a complex redox regulation that either stimulates or inhibits fMLP-induced leukotriene synthesis in an experimental model of neutrophils interacting with Salmonella typhimurium. The scavenging of mitochondrial reactive oxygen species by mitochondria-targeted antioxidants MitoQ and SkQ1, as well as inhibition of their production by mitochondrial inhibitors, inhibit the synthesis of leukotrienes regardless of the cessation of oxidative phosphorylation. On the contrary, antioxidants N-acetylcysteine and sodium hydrosulfide promoting reductive shift in the reversible thiol-disulfide system stimulate the synthesis of leukotrienes. Diamide that oxidizes glutathione at high concentrations inhibits leukotriene synthesis, and the glutathione precursor S-adenosyl-L-methionine prevents this inhibition. Diamide-dependent inhibition is also prevented by diphenyleneiodonium, presumably through inhibition of NADPH oxidase and NADPH accumulation. Thus, during bacterial infection, maintaining the reduced state of glutathione in neutrophils plays a decisive role in the synthesis of leukotriene B4. Suppression of excess leukotriene synthesis is an effective strategy for treating various inflammatory pathologies. Our data suggest that the use of mitochondria-targeted antioxidants may be promising for this purpose, whereas known thiol-based antioxidants, such as N-acetylcysteine, may dangerously stimulate leukotriene synthesis by neutrophils during severe pathogenic infection.

The Multifarious Functions of Leukotrienes in Bone Metabolism.

Leukotrienes (LTs) are derived from arachidonic acid metabolism by the 5-lipoxygenase (5-LO) enzyme. The production of LTs is stimulated in the pathogenesis of rheumatoid arthritis (RA), osteoarthritis, and periodontitis, with a relevant contribution to bone resorption. However, its role in bone turnover, particularly the suppression of bone formation by modulating the function of osteoclasts and osteoblasts, remains unclear. We investigated the effects of LTs on bone metabolism and their impact on osteogenic differentiation and osteoclastogenesis using a 5-LO knockout (KO) mouse model. Results from micro-computed tomography (µCT) analysis of femur from 8-week-old 5-LO-deficient mice showed increased cortical bone and medullary region in females and males and decreased trabecular bone in females. In the vertebra, we observed increased marrow area in both females and males 5-LO KO and decreased trabecular bone only in females 5-LO KO. Immunohistochemistry (IHC) analysis showed higher levels of osteogenic markers tissue-nonspecific alkaline phosphatase (TNAP) and osteopontin (OPN) and lower expression of osteoclastogenic marker tartrate-resistant acid phosphatase (TRAP) in the femurs of 5-LO KO mice versus wild-type (WT). Alkaline phosphatase activity and mineralization assay results showed that the 5-LO absence enhances osteoblasts differentiation and mineralization but decreases the proliferation. Alkaline phosphatase (ALP), Bglap, and Sp7 gene expression were higher in 5-LO KO osteoblasts compared to WT cells. Eicosanoids production was higher in 5-LO KO osteoblasts except for thromboxane 2, which was lower in 5-LO-deficient mice. Proteomic analysis identified the downregulation of proteins related to adenosine triphosphate (ATP) metabolism in 5-LO KO osteoblasts, and the upregulation of transcription factors such as the adaptor-related protein complex 1 (AP-1 complex) in long bones from 5-LO KO mice leading to an increased bone formation pattern in 5-LO-deficient mice. We observed enormous differences in the morphology and function of osteoclasts with reduced bone resorption markers and impaired osteoclasts in 5-LO KO compared to WT osteoclasts. Altogether, these results demonstrate that the absence of 5-LO is related to the greater osteogenic profile. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye.

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes. Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss). We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues. Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

Targeting the Leukotriene Pathway for Colon Cancer Interception.

The role of chronic inflammation and arachidonic acid (AA) metabolism in tumor progression has been well characterized for variety of cancers, with compelling data for colon cancer. Several preclinical and clinical studies primarily focused on inhibiting the cyclooxygenase pathways using NSAIDs and aspirin for colon cancer prevention. However, emerging evidence clearly supports the pro-tumorigenic role of 5-lipoxygenase and its downstream leukotriene pathway within AA metabolism. As discussed in the current issue, targeting the leukotriene pathway by cysteinyl leukotriene receptor antagonist (LTRA) montelukast suppressed formation of aberrant crypt foci (ACF) and cell proliferation in colonic epithelium, suggesting the potential of LTRAs for colon cancer prevention. Although this is a short clinical chemoprevention trial to explore the effects of LTRAs against ACF development, it is a significant and timely study opening avenues to further explore the possibilities of using LTRAs in other inflammation-associated precancerous lesions as well. In this spotlight commentary, we highlight the implications of their data and the opportunities for developing LTRAs as potential candidates for colorectal cancer interception. See related article by Higurashi et al., p. 661.

Montelukast, an Antagonist of Cysteinyl Leukotriene Signaling, Impairs Burn Wound Healing.

BACKGROUND: Burns are severe injuries often associated with impaired wound healing. Impaired healing is caused by multiple factors, including dysregulated inflammatory responses at the wound site. Interestingly, montelukast, an antagonist for cysteinyl leukotrienes and U.S. Food and Drug Administration approved for treatment of asthma and allergy, was previously shown to enhance healing in excision wounds and to modulate local inflammation. METHODS: In this study, the authors examined the effect of montelukast on wound healing in a mouse model of scald burn injury. Burn wound tissues isolated from montelukast- and vehicle-treated mice at various times after burn injury were analyzed for wound areas ( n = 34 to 36), reepithelialization ( n = 14), inflammation ( n = 8 to 9), and immune cell infiltration ( n = 3 to 6) and proliferation ( n = 7 to 8). RESULTS: In contrast to previously described beneficial effects in excision wounds, this study shows that montelukast delays burn wound healing by impairing the proliferation of keratinocytes and endothelial cells. This occurs largely independently of inflammatory responses at the wound site, suggesting that montelukast impairs specifically the proliferative phase of wound healing in burns. Wound healing rates in mice in which leukotrienes are not produced were not affected by montelukast. CONCLUSION: Montelukast delays wound healing mainly by reducing the proliferation of local cells after burn injury. CLINICAL RELEVANCE STATEMENT: Although additional and clinical studies are necessary, our study suggests that burn patients who are on montelukast may exhibit delayed healing, necessitating extra observation.

Cysteinyl Leukotriene Synthesis via Phospholipase A2 Group IV Mediates Exercise-induced Bronchoconstriction and Airway Remodeling.

It is well known that the prevalence of asthma is higher in athletes, including Olympic athletes, than in the general population. In this study, we analyzed the mechanism of exercise-induced bronchoconstriction by using animal models of athlete asthma. Mice were made to exercise on a treadmill for a total duration of 1 week, 3 weeks, or 5 weeks. We analyzed airway responsiveness, BAL fluid, lung homogenates, and tissue histology for each period. In mice that were treated (i.e., the treatment model), treatments were administered from the fourth to the fifth week. We also collected induced sputum from human athletes with asthma and analyzed the supernatants. Airway responsiveness to methacholine was enhanced with repeated exercise stimulation, although the cell composition in BAL fluid did not change. Exercise induced hypertrophy of airway smooth muscle and subepithelial collagen deposition. Cysteinyl-leukotriene (Cys-LT) levels were significantly increased with exercise duration. Montelukast treatment significantly reduced airway hyperresponsiveness (AHR) and airway remodeling. Expression of PLA2G4 (phospholipase A2 group IV) and leukotriene C4 synthase in the airway epithelium was upregulated in the exercise model, and inhibition of PLA2 ameliorated AHR and airway remodeling, with associated lower levels of Cys-LTs. The levels of Cys-LTs in sputum from athletes did not differ between those with and without sputum eosinophilia. These data suggest that AHR and airway remodeling were caused by repeated and strenuous exercise. Cys-LTs from the airway epithelium, but not inflammatory cells, may play an important role in this mouse model.

Peroxiredoxin III Protects Tumor Suppressor PTEN from Oxidation by 15-Hydroperoxy-eicosatetraenoic Acid.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase that coordinates various cellular processes. Its activity is regulated by the reversible oxidation of an active-site cysteine residue by H2O2 and thioredoxin. However, the potential role of lipid peroxides in the redox regulation of PTEN remains obscure. To evaluate this, 15-hydroperoxy-eicosatetraenoic acid (15s-HpETE), a lipid peroxide, was employed to investigate its effect on PTEN using molecular and cellular-based assays. Exposure to 15s-HpETE resulted in the oxidation of recombinant PTEN. Reversible oxidation of PTEN was also observed in mouse embryonic fibroblast (MEF) cells treated with a 15s-HpETE and Lipofectamine mixture. The oxidative dimerization of thioredoxin was found simultaneously. In addition, the absence of peroxiredoxin III aggravated 15s-HpETE-induced PTEN oxidation in MEF cells. Our study provides novel insight into the mechanism linking lipid peroxidation to the etiology of tumorigenesis.

The influence of arachidonic acid metabolites on PPAR and RXR expression in bovine uterine cells.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the superfamily of nuclear receptors. Three isoforms have been described: alpha (PPARα), delta (PPARδ), and gamma (PPARγ). PPARs heterodimerize with retinoid X receptors (RXRs: RXRα, RXRß and RXRγ). PPAR activity can be modulated by several ligands, including arachidonic acid (AA) metabolites. The aims of the study were to determine the effect of AA metabolites (prostaglandin [PG]E2, PGF2α, leukotriene [LT]B4, and LTC4) on mRNA (real-time PCR) and protein expression (Western blotting) of PPARα, PPARδ, and PPARγ, and on mRNA expression of RXRα, RXRß, and RXRγ, in bovine epithelial, stromal, and myometrial primary uterine cells and in bovine stromal cells with silenced PPAR genes (N = 10). All PPAR and RXR isoforms were expressed. Prostaglandins affected expression of PPARs only in stromal cells, whereas LTs modulated PPARγ mRNA expression in epithelial and myometrial primary cells. Blockade of signal transduction through PPARs prevented interactions between AA metabolites and PPARs and changed RXR expression comparing with primary stromal cells. In primary stromal uterine cells, mRNA expression of RXRs was higher than that of PPARs. In uterine stromal cells in which intracellular signaling through PPARs was blocked, RXRs seem to take over the role of PPARs and are pivotal for cell functions. This study revealed the reaction of PPARs and RXRs to agonists which naturally occur in the bovine uterus.

The Orphan Receptor GPR17 Is Unresponsive to Uracil Nucleotides and Cysteinyl Leukotrienes.

Pairing orphan G protein­coupled receptors (GPCRs) with their cognate endogenous ligands is expected to have a major impact on our understanding of GPCR biology. It follows that the reproducibility of orphan receptor ligand pairs should be of fundamental importance to guide meaningful investigations into the pharmacology and function of individual receptors. GPR17 is an orphan receptor characterized by some as a dualistic uracil nucleotide/cysteinyl leukotriene receptor and by others as inactive toward these stimuli altogether. Whereas regulation of central nervous system myelination by GPR17 is well established, verification of activity of its putative endogenous ligands has proven elusive so far. Herein we report that uracil nucleotides and cysteinyl leukotrienes do not activate human, mouse, or rat GPR17 in various cellular backgrounds, including primary cells, using eight distinct functional assay platforms based on labelfree pathway-unbiased biosensor technologies, as well as canonical second-messenger or biochemical assays. Appraisal of GPR17 activity can neither be accomplished with co-application of both ligand classes, nor with exogenous transfection of partner receptors (nucleotide P2Y12, cysteinyl-leukotriene CysLT1) to reconstitute the elusive pharmacology. Moreover, our study does not support the inhibition of GPR17 by the marketed antiplatelet drugs cangrelor and ticagrelor, previously suggested to antagonize GPR17. Whereas our data do not disagree with a role of GPR17 per se as an orchestrator of central nervous system functions, they challenge the utility of the proposed (ant)agonists as tools to imply direct contribution of GPR17 in complex biologic settings.

Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s.

Group 2 innate lymphoid cells (ILC2s) and type 2 helper T cells (Th2 cells) are the primary source of interleukin 5 (IL-5) and IL-13 during type 2 (allergic) inflammation in the lung. In Th2 cells, T cell receptor (TCR) signaling activates the transcription factors nuclear factor of activated T cells (NFAT), nuclear factor κB (NF-κB), and activator protein 1 (AP-1) to induce type 2 cytokines. ILC2s lack a TCR and respond instead to locally produced cytokines such as IL-33. Although IL-33 induces AP-1 and NF-κB, NFAT signaling has not been described in ILC2s. In this study, we report a nonredundant NFAT-dependent role for lipid-derived leukotrienes (LTs) in the activation of lung ILC2s. Using cytokine reporter and LT-deficient mice, we find that complete disruption of LT signaling markedly diminishes ILC2 activation and downstream responses during type 2 inflammation. Type 2 responses are equivalently attenuated in IL-33- and LT-deficient mice, and optimal ILC2 activation reflects potent synergy between these pathways. These findings expand our understanding of ILC2 regulation and may have important implications for the treatment of airways disease.

Lipid mediators foster the differentiation of T follicular helper cells.

Lipid mediators such as leukotrienes and lipoxines broadly regulate innate and acquired immunity, and their dysfunction causes various immune-mediated disorders. We previously reported a salient feature of arachidonate 5-lipoxyganase (Alox5), which is responsible for the production of such lipid mediators, in the regulation of high affinity antibodies in vivo. The aim of this study was to determine the functional significance of Alox5-related lipid mediators during the processes of acquired humoral responses. The results of in vitro experiments using lymphocytes in tonsils and blood specimens showed that lipoxin A4 (LXA4) and leukotriene B4 (LTB4) have the capacity to differentiate naïve CD4+ T cells into T follicular helper (Tfh) cells, which activate naïve B cells to form germinal centers. Such a function of LXA4 was further supported by results of in vitro studies using BML-111 and BOC-2, which are an agonist and an antagonist, respectively, of FPR2 of an LXA4-specific cell-surface receptor. The results suggest that such lipid mediators have a potential role in the development of lymphoid follicles through the regulation of Tfh cell differentiation.

Mecanismos intracelulares induzidos por leucotrienos durante a diferenciação osteogênica / Leukotriene-induced intracellular mechanisms during osteogenic differentiation

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico.(AU)

Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNFα in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.(AU)

In vitro cow uterine response to Escherichia coli, leukotrienes and cytokines.

The aim was to determine the dynamic profile of interactions between Escherichia coli (E. coli) and the actions of leukotrienes (LTs) and TNF and INFγ (cytokines) in the uterus in vitro. Uterine explants (N=6) were incubated for 2, 12 and 24h either as E. coli-treated (106CFU) or non-treated and/or with: LTB4 and C4 (10-6M, for both LTs), LTs receptors antagonists (aLTR; 10-6M) and/or cytokines (each 10ng/ml). Toll Like Receptor 4 (TLR4) mRNA expression increased in explants incubated with E. coli, cytokines and LTs after 2 and 12h and aLTR inhibited the effect of LTs in explants incubated with E. coli (P<0.05). IL-6 mRNA expression was up-regulated in E.coli-treated explants with cytokines after 2h and cytokines with LTs after 12h (P<0.05). E. coli increased prostaglandin (PG)E2 output after all examined time points, and PGF2α and IL-6 levels in E.coli-treated explants after 12 and 24h with cytokines, with LTs (P<0.05). aLTR inhibited LT stimulating action on PGs and IL-6 output in explants incubated with E. coli after 12 and 24h (P<0.05). LTs modify and enhance experimentally induced infection: TLR4 and IL-6 mRNA expression, IL-6 and PGs secretion, and cytokines participate in this process.

Impact of Arachidonic Acid and the Leukotriene Signaling Pathway on Vasculogenesis of Mouse Embryonic Stem Cells.

Embryonic stem (ES) cells can differentiate into various kinds of cells, such as endothelial and hematopoietic cells. In addition, some evidence suggests that inflammatory mediators such as leukotrienes (LTs), which include the 5-lipoxygenase (LOX) family, can regulate endothelial cell differentiation. In the present study, the eicosanoid precursor arachidonic acid (AA) stimulated vasculogenesis of ES cells by increasing the number of fetal liver kinase-1+ vascular progenitor cells as well as vascular structures positive for platelet endothelial cell adhesion protein-1 and vascular endothelial cadherin. The stimulation of vasculogenesis and expression of the rate-limiting enzyme in the LT signaling pathway, 5-LOX-activating protein (FLAP), was blunted upon treatment with the FLAP inhibitors AM643 and REV5901. Vasculogenesis was significantly restored upon exogenous addition of LTs. Downstream of FLAP, the LTB4 receptor (BLT1) blocker U75302, the BLT2 receptor blocker LY255283 as well as the cysteinyl LT blocker BAY-u9773 inhibited vasculogenesis of ES cells. AA treatment of differentiating ES cells increased reactive oxygen species (ROS) generation, which was not affected upon either FLAP or cyclooxygenase-2 inhibition. Prevention of ROS generation by either the free radical scavengers vitamin E and N-(2-mercaptopropionyl)glycine or the NADPH oxidase inhibitor VAS2870 downregulated vasculogenesis of ES cells and blunted the provasculogenic effect of AA. In summary, our data demonstrate that proinflammatory AA stimulates vasculogenesis of ES cells via the LT pathway by mechanisms involving ROS generation.

Pathophysiology of the hepoxilins.

There is increasing evidence from various scientific groups that hepoxilins represent novel inflammatory mediators. In vitro studies have shown that the hepoxilins cause mobilization of intracellular calcium in human neutrophils, cause plasma leakage, and potently stimulate chemotaxis of human neutrophils. In vivo, the hepoxilin pathway is activated in conditions of inflammation, e.g. after pathogen infection, in inflamed conditions (psoriasis, arthritis), and hepoxilins promote inflammatory hyperalgesia and allodynia. Although much work has demonstrated an effect of hepoxilins on neutrophils, the hepoxilin pathway has been demonstrated in a variety of tissues, including the lung, brain, pituitary, pancreatic islets, skin, etc. A genetic defect linked to a deficiency in hepoxilin formation has been described and believed to be responsible for the scaly skin observed in ichthyosis. Despite their biological and chemical instability, the involvement of the hepoxilin pathway in pathology has been demonstrated in vitro and in vivo through either isolation of the hepoxilins themselves (or their metabolites) or implied through the use of stable hepoxilin analogs. These analogs have additionally shown efficacy in animal models of lung fibrosis, cancer, thrombosis and diabetes. Research on these compounds has merely scratched the surface, but results published to date have suggested that the hepoxilin pathway is a distinct and novel pathway leading to inflammation and hepoxilin antagonists may provide the means of controlling early aspects of the acute inflammatory phase. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Effect of polyunsaturated fatty acids and their metabolites on bleomycin-induced cytotoxic action on human neuroblastoma cells in vitro.

In the present study, we noted that bleomycin induced growth inhibitory action was augmented by all the polyunsaturated fatty acids (PUFAs) tested on human neuroblastoma IMR-32 (0.5 × 10(4) cells/100 µl of IMR) cells (EPA > DHA > ALA = GLA = AA > DGLA = LA: ∼ 60, 40, 30, 10-20% respectively) at the maximum doses used. Of all the prostaglandins (PGE1, PGE2, PGF2α, and PGI2) and leukotrienes (LTD4 and LTE4) tested; PGE1, PGE2 and LTD4 inhibited the growth of IMR-32 cells to a significant degree at the highest doses used. Lipoxin A4 (LXA4), 19,20-dihydroxydocosapentaenoate (19, 20 DiHDPA) and 10(S),17(S)-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid (protectin: 10(S),17(S)DiHDoHE), metabolites of DHA, significantly inhibited the growth of IMR-32 cells. Pre-treatment with AA, GLA, DGLA and EPA and simultaneous treatment with all PUFAs used in the study augmented growth inhibitory action of bleomycin. Surprisingly, both indomethacin and nordihydroguaiaretic acid (NDGA) at 60 and 20 µg/ml respectively enhanced the growth of IMR-32 cells even in the presence of bleomycin. AA enhanced oxidant stress in IMR-32 cells as evidenced by an increase in lipid peroxides, superoxide dismutase levels and glutathione peroxidase activity. These results suggest that PUFAs suppress growth of human neuroblastoma cells, augment growth inhibitory action of bleomycin by enhancing formation of lipid peroxides and altering the status of anti-oxidants and, in all probability, increase the formation of lipoxins, resolvins and protectins from their respective precursors that possess growth inhibitory actions.

Multiple-site activation of the cysteinyl leukotriene receptor 2 is required for exacerbation of ischemia/reperfusion injury.

OBJECTIVE: Transgenic overexpression of the human cysteinyl leukotriene receptor 2 (CysLT2R) in murine endothelium exacerbates vascular permeability and ischemia/reperfusion injury. Here, we explore the underlying mechanisms of CysLT2R activation-mediated inflammation and delineate the relative contributions of endogenous murine CysLT2R and the transgene-derived receptor. APPROACH AND RESULTS: We created a novel mouse with only endothelial-expressed CysLT2R (endothelium-targeted overexpression mice [EC]/CysLT2R-knockout mice [KO]) by crossing EC with KO to dissect the role of endothelial CysLT2R in tissue injury. Surprisingly, we discovered that damage in EC/KO mice was not elevated (24% versus 47% EC) after ischemia/reperfusion. We examined vascular permeability and leukocyte recruitment/rolling responses in the cremaster vasculature after cysteinyl leukotriene (cysLT) stimulation. Mice possessing transgenic endothelial CysLT2R overexpression, whether EC or EC/KO, when stimulated with cysLTs, exhibited vascular hyperpermeability, declining leukocyte flux, and a transient increase in slow-rolling leukocyte fraction. Mice lacking endogenous CysLT2R (both KO [20 ± 3 cells/min] EC/KO [24 ± 3]) showed lower-rolling leukocyte flux versus wild-type (38 ± 6) and EC (35 ± 6) mice under unstimulated conditions. EC/KO mice differed from EC counterparts in that vascular hyperpermeability was not present in the absence of exogenous cysLTs. CONCLUSIONS: These results indicate that endothelial and nonendothelial CysLT2R niches have separate roles in mediating inflammatory responses. Endothelial receptor activation results in increased vascular permeability and leukocyte slow-rolling, facilitating leukocyte transmigration. Nonendothelial receptors, likely located on resident/circulating leukocytes, facilitate endothelial receptor activation and leukocyte transit. Activation of both receptor populations is required for injury exacerbation.

Cysteinyl leukotrienes regulate endothelial cell inflammatory and proliferative signals through CysLT2 and CysLT1 receptors.

Cysteinyl leukotrienes (cys-LTs), LTC4, LTD4, LTE4 are potent inflammatory lipid mediators that act through two distinct G-protein-coupled receptors, CysLT1R and CysLT2R. Although cys-LTs are shown to induce vascular leakage and atherosclerosis, the molecular mechanism by which cys-LTs modulate endothelial function is not known. Here, we show that cys-LTs (LTC4 and LTD4) induce robust calcium influx in human umbilical vein endothelial cells (HUVECs) through CysLT2R, but not CysLT1R. Further, cys-LT treatment induced endothelial cell (EC) contraction leading to monolayer disruption via CysLT2R/Rho kinase dependent pathway. Furthermore, stimulation with cys-LTs potentiated TNFα-induced VCAM-1 expression and leukocyte recruitment to ECs through CysLT2R. In contrast, we found that both LTC4 and LTD4 stimulated EC proliferation through CysLT1R. Taken together, these results suggest that cys-LTs induce endothelial inflammation and proliferation via CysLT2R/Rho kinase and CysLT1R/Erk dependent pathways, respectively, which play critical role in the etiology of cardiovascular diseases such as atherosclerosis and myocardial infarction.

Is GPR17 a P2Y/leukotriene receptor? examination of uracil nucleotides, nucleotide sugars, and cysteinyl leukotrienes as agonists of GPR17.

The orphan receptor GPR17 has been reported to be activated by UDP, UDP-sugars, and cysteinyl leukotrienes, and coupled to intracellular Ca(2+) mobilization and inhibition of cAMP accumulation, but other studies have reported either a different agonist profile or lack of agonist activity altogether. To determine if GPR17 is activated by uracil nucleotides and leukotrienes, the hemagglutinin-tagged receptor was expressed in five different cell lines and the signaling properties of the receptor were investigated. In C6, 1321N1, or Chinese hamster ovary (CHO) cells stably expressing GPR17, UDP, UDP-glucose, UDP-galactose, and cysteinyl leukotriene C4 (LTC4) all failed to promote inhibition of forskolin-stimulated cAMP accumulation, whereas both UDP and UDP-glucose promoted marked inhibition (>80%) of forskolin-stimulated cAMP accumulation in C6 and CHO cells expressing the P2Y14 receptor. Likewise, none of these compounds promoted accumulation of inositol phosphates in COS-7 or human embryonic kidney 293 cells transiently transfected with GPR17 alone or cotransfected with Gαq/i5, which links Gi-coupled receptors to the Gq-regulated phospholipase C (PLC) signaling pathway, or PLCε, which is activated by the Gα12/13 signaling pathway. Moreover, none of these compounds promoted internalization of GPR17 in 1321N1-GPR17 cells. Consistent with previous reports, coexpression experiments of GPR17 with cysteinyl leukotriene receptor 1 (CysLTR1) suggested that GPR17 acts as a negative regulator of CysLTR1. Taken together, these data suggest that UDP, UDP-glucose, UDP-galactose, and LTC4 are not the cognate ligands of GPR17.