Expression Of Leukotriene B4 Receptor Research Articles

The Role of Leukotriene B4 in Cow Metritis.

Metritis is a common postpartum disease in dairy cows. As a mast cell (MC) mediator, leukotriene B4 (LTB4) is the strongest phagocyte chemokine. It is important in inflammation for the recruitment of immune cells to resist infection. This study investigated the effect of LTB4 in metritis. Twenty Holstein cows 3 to 6 years old and at 6 to 10 days postpartum were selected, ten of which with postpartum metritis were the experimental group, and the other ten of which as healthy cows were the control group. The levels of LTB4, substance P (SP) and vasoactive intestinal peptide (VIP) were measured by ELISA, the expression of LTB4 receptor 2 (BLT2), matrix metalloproteinase (MMP)-2 and MMP-9 mRNA was measured by qPCR, and collagens I and IV were detected by immunohistochemical staining. Concentrations of SP and LTB4 were significantly higher, but those of VIP were significantly lower in the experimental group than those in the control group. The expression of BLT2, MMP-2 and MMP-9 mRNA was significantly higher in the experimental group than that in the control group. The expression of collagen Ⅰ and collagen Ⅳ was significantly lower in the experimental group than that in the control group. In metritis, SP promotes the activation of MC and the synthesis and release of LTB4. Leukotriene B4 chemotactic immune cells promote the high expression of collagenase, which accelerated the hydrolysis of collagen, while the inhibitory effect of VIP on MC was weakened. This may further aggravate the damage to uterine tissue.

Expression of leukotriene B4 receptor 1 defines functionally distinct DCs that control allergic skin inflammation

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a chemotactic G protein-coupled receptor expressed by leukocytes, such as granulocytes, macrophages, and activated T cells. Although there is growing evidence that BLT1 plays crucial roles in immune responses, its role in dendritic cells remains largely unknown. Here, we identified novel DC subsets defined by the expression of BLT1, namely, BLT1hi and BLT1lo DCs. We also found that BLT1hi and BLT1lo DCs differentially migrated toward LTB4 and CCL21, a lymph node-homing chemoattractant, respectively. By generating LTB4-producing enzyme LTA4H knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout (BLT1 cKO) mice, we showed that the migration of BLT1hi DCs exacerbated allergic contact dermatitis. Comprehensive transcriptome analysis revealed that BLT1hi DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression, whereas BLT1lo DCs accelerated T cell proliferation by producing IL-2. Collectively, the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB4-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.

Leukotriene B4 receptors abnormal gene expression is associated with either shorter or longer survival in breast cancer patients depending on the intrinsic tumour molecular subtype

Abstract Background Leukotrienes receptor signaling is involved in tumor development and progression. Expression of leukotriene B4 receptors 1 and 2 (LTB4R and LTB4R2) promotes cell proliferation, survival, migration and metastasis. Aberrant LTB4R gene expression blocks anti-proliferative responses of TGF-β1 in breast cancer (BC) cell lines. LTB4R2 ectopic expression is associated with increased invasiveness of BC cells. Earlier, we reported LTB4R and LTB4R2 genes abnormal demethylation that is potent of initiating ectopic gene expression in a subset of triple-negative BC (TNBC) samples. Methods We acquired the TCGA BRCA level 3 RNA-seq data and clinical datasheet using TCGA data portal. LTB4R/LTB4R2 gene expression cut-off values were defined using maximally selected rank statistics. Kaplan-Meier overall survival (OS) curves were compared with Mantel-Cox (log-rank) method. Results TNBC exhibit a significantly lower survival probability in LTB4R expressing group. Vice versa, in Normal-Like and LumB subtypes absence of LTB4R and LTB4R2 expression respectively is associated with shorter OS (Table). Table: 57P . Overall survival comparison in various molecular BC subtypes. Asteriks (*) stand for p-value less then 0.05. Median OS are presented in days Expression groups/ Molecular groups No subtyping LumA LumB HER2 TNBC Normal-Like Median OS p-value Median OS p-value Median OS p-value Median OS p-value Median OS p-value Median OS p-value LTB4R = high 3945 0.42 3926 0.81 3941 0.059 6456 0.08 7455 0.0029* 4267 0.0034* LTB4R = low 3736 3873 2483 2127 NA 1688 LTB4R2 = high 3945 0.53 3873 0.51 3941 0.026* 6456 0.59 7455 0.74 4267 0.58 LTB4R2 = low 3472 2965 2573 3063 3472 1759 Conclusions Our findings suggest that LTB4R/LTB4R2 gene expression is a new potential BC prognostic biomarker, yet its use requires proper molecular subtyping of the tumors, as far as prognosis would depend upon the intrinsic tumor subtype. This also applies to the potential use of LTB4R/LTB4R2 expression as a marker predictive of tumors sensitivity to leukotriene receptor inhibitors, in case they enter clinical studies. Legal entity responsible for the study Research Centre for Medical Genetics. Funding The research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation. Disclosure All authors have declared no conflicts of interest.

Wogonin suppresses the LPS‑enhanced invasiveness of MDA‑MB‑231 breast cancer cells by inhibiting the 5‑LO/BLT2 cascade

Wogonin, a naturally occurring bioactive monoflavonoid isolated from Scutellariae radix (roots of Scutellariae baicalensis Georgi), has known anticancer effects. However, the molecular signaling mechanism by which wogonin inhibits invasiveness in breast cancer cells remains unclear. In the present study, it was observed that wogonin exerted an inhibitory effect on the lipopolysaccha-ride (LPS)-enhanced invasiveness of MDA-MB-231 cells. In addition, wogonin inhibited the synthesis of interleukin-8 (IL-8) and matrix metallopeptidase-9 (MMP-9), which are critical for promoting invasiveness in MDA-MB-231 cells. Wogonin also suppressed the expression of leukot-riene B4 receptor 2 (BLT2) and the synthesis of its ligand, by inhibiting 5-lipoxygenase (5-LO) in LPS-stimulated MDA-MB-231 cells. Notably, wogonin attenuated the production of IL-8 and MMP-9 by inhibiting the BLT2/extracellular signal-regulated kinase (ERK)-linked cascade. Finally, in vivo, LPS-driven MDA-MB-231 cell metastasis was markedly suppressed by wogonin administration. Overall, the present results suggested that wogonin inhibited the 5-LO/BLT2/ERK/IL-8/MMP-9 signaling cascade and demonstrated that this cascade may be an important target through which wogonin exerts its anticancer effects in breast cancer.

Human articular chondrocytes express functional leukotriene B4 receptors.

Leukotriene B4 (LTB4) is a potent chemoattractant associated with the development of osteoarthritis (OA), while its receptors BLT1 and BLT2 have been found in synovium and subchondral bone. In this study, we have investigated whether these receptors are also expressed by human cartilage cells and their potential effects on cartilage cells. The expression of LTB4 receptors in native tissue and cultured cells was assessed by immunohistochemistry, immunocytochemistry, polymerase chain reaction (PCR) and electron microscopy. The functional significance of the LTB4 receptor expression was studied by Western blotting, using phospho-specific antibodies in the presence or absence of receptor antagonists. In further studies, the secretion of pro-inflammatory cytokines, growth factors and metalloproteinases by LTB4-stimulated chondrocytes was measured by multiplex protein assays. The effects of LTB4 in cartilage signature gene expression in cultured cells were assessed by quantitative PCR, whereas the LTB4-promoted matrix synthesis was determined using 3D pellet cultures. Both receptors were present in cultured chondrocytes, as was confirmed by immunolabelling and PCR. The relative quantification by PCR demonstrated a higher expression of the receptors in cells from healthy joints compared with OA cases. The stimulation of cultured chondrocytes with LTB4 resulted in a phosphorylation of downstream transcription factor Erk 1/2, which was reduced after blocking BLT1 signalling. No alteration in the secretion of cytokine and metalloproteinases was recorded after challenging cultured cells with LTB4; likewise, cartilage matrix gene expression and 3D tissue synthesis were unaffected. Chondrocytes express BLT1 and BLT2 receptors, and LTB4 activates the downstream Erk 1/2 pathway by engaging the high-affinity receptor BLT1. However, any putative role in cartilage biology could not be revealed, and remains to be clarified.

Identification, expression and immunological responses to bacterial challenge following vaccination of BLT1 gene from turbot, Scophthalmus maximus

Leukotriene B4 (LTB4) is well known as a chemoattractant for leucocytes, recent studies also showed its involvement in adaptive immunity. The purpose of this work is to report the cloning, characterization and gene expression of leukotriene B4 receptor (BLT1) in turbot (Scophthalmus maximus), as well as the immunological response to challenge following vaccination with a live attenuated vaccine Vibrio anguillarum MVAV6203. The full cDNA sequence of turbot BLT1 was cloned. The open reading frame consists of 1119bp nucleotides, which translate into 372 amino acid protein. A high conservation of amino acid sequence was found in the seven transmembrane (TM) domains and intracellular loops. The intracellular loop 3 consisting of a unique cluster of basic amino acid residues might be associated with signal transduction. High amino acid similarity and a phylogenetic tree confirmed it as a leukotriene B4 receptor member. The BLT1 gene is expressed in a wide range of tissues with the highest expression in kidney followed by spleen. The expression of turbot BLT1 was significantly up-regulated in spleen, gut and gill after vaccination and in kidney and skin after challenge. These results suggest a potential role of turbot BLT1 in protection against infection.

Purinergic signaling gene network expression in bovine polymorphonuclear neutrophils during the peripartal period

An effective immune response relies on efficient activation of polymorphonuclear neutrophilic leukocytes (PMNL). The PMNL release cellular ATP in response to inflammatory mediators. Although extracellular ATP is rapidly degraded to adenosine, both compounds can readily bind to either the purinergic receptor P1 (adenosine) or P2 (ATP). The P1 and P2 receptors are members of the G-protein-coupled receptor family. The peripartal period is characterized by marked changes in metabolic and inflammatory status that are functionally related with immune responses in the cow. We evaluated the mRNA expression of genes associated with purinergic signaling in PMNL during the peripartal period. Seven multiparous Holstein cows were dried off at d −50 relative to expected parturition and fed a controlled-energy diet (net energy for lactation=1.24Mcal/kg of dry matter) for ad libitum intake during the entire dry period. After calving, all cows were fed a common lactation diet (net energy for lactation=1.65Mcal/kg of dry matter) until 30d in milk. Blood PMNL collected at −10, 3, and 21d in milk were used to study the expression of 22 genes associated with adhesion to endothelium, chemoattractant binding at the plasma membrane, and purinergic signaling. Other blood samples around calving were used to analyze concentrations of insulin, metabolites, and whole-blood phagocytosis. The expression of purinergic receptor P2Y, G-protein coupled, 2 (P2RY2) increased on d 3 and then decreased on d 21. This response suggested that ATP could play a role in the amplification of chemotactic signals. In contrast, the expression of genes encoding cell adhesion [selectin L (SELL) and selectin P ligand (SELPLG)], chemoattractant receptors [complement component 5a receptor 1 (C5AR1), IL-8 receptor α (CXCR1), IL-8 receptor β (CXCR2), and platelet-activating factor receptor (PTAFR)], and adenosine receptors [adenosine A1 receptor (ADORA1) and adenosine A3 receptor (ADORA3)] decreased between −10 and 3d. The decrease coincided with a marked increase in blood nonesterified fatty acids and hydroxybutyrate concentrations, and a decrease in glucose and insulin concentrations. The increase in metabolites also was associated with greater expression of leukotriene B4 receptor (LTB4R) on d 3 and 21 compared with d −10, which is involved in inflammatory prostaglandin synthesis. Most chemoattractant receptors increased by 21d, but cell adhesion genes and blood leukocyte phagocytosis was lower. The expression of adenosine A2a receptor (ADORA2A), which is associated with immunosuppression of PMNL and that of adenosine uptake channels [solute carrier family 29 (nucleoside transporters), member 1 (SLC29A1) and member 2 (SLC29A2)] and the nucleotidase adenosine deaminase (ADA) was greater at 3 and 21d compared with −10d. The reduction in key immune responses, such as cell adhesion and chemotaxis, by bovine PMNL could partly be a function of changes in mRNA expression of genes associated with purinergic signaling.

Expression of Leukotriene B4 receptor 1 on CD8+ T cells is essential for their migration to tumors, immune-surveillance and cancer immunity (P5106)

Abstract The role of Leukotriene B4 (LTB4) receptor 1, BLT1 as a pro-inflammatory mediator in various inflammatory diseases is well known. However, its role in regulating immune responses to tumor is yet unknown. Here, we studied the role of BLT1 in CD8+ T cell mediated anti-tumor response using implantable TC-1 cervical cancer and B16F10 melanoma mice models. BLT1−/− mice had significantly accelerated tumor growth, reduced survival, and decreased CD8+ T cells as well as their effector molecules such as IFN-γ, granzyme-B and IL-2 within tumors as shown by flow cytometry and confocal microscopy, compared to the WT controls. Moreover, the tumor growth patterns in BLT1−/− mice was similar to WT mice that were depleted of their CD8+ T cells. Evaluation of CTL responses by in vivo killing assay revealed no differences in the cytolytic ability between WT and BLT1-/- CD8+ T cells. Adoptive transfer of tumor primed CD8+ T cells from WT but not BLT1-/- mice reduced tumor growth in Rag2-/- mice. In Rag2-/- mice, while the numbers of WT or BLT1-/- CD8+ T cells in dLN remain similar, the recruitment of BLT1-/- CD8+ T cells into tumor was dramatically reduced as compared to WT CD8+ T cells. Analysis of CD8+ T cells from the dLN revealed no difference in the expression of other T cell chemokine receptors. These results demonstrate a previously unidentified, yet crucial role of BLT1 on CD8+ T cell in their effective migration into the tumor and hence in immune-surveillance and anti-tumor immunity.

Regulation of TNFα and IL1β in rheumatoid arthritis synovial fibroblasts by leukotriene B4

Arachidonic acid metabolites, such as leukotriene B4 (LTB4), are known to play an important role in pathogenesis of rheumatoid arthritis (RA). Apart from leukocytes, RA synovial fibroblasts (RASF) produce a broad array of inflammatory mediators to recruit, retain and activate immune cells and resident mesenchymal cells in the joints to promote ongoing inflammation and tissue destruction. To determine how LTB4 may contribute to this process, RASF was cultured from synovial tissues collected from RA patients undergoing total knee replacement. The level of LTB4 in culture medium was determined using ELISA, and expression of LTB4 receptors (BLT1 and BLT2) by RT-PCR. In the presence of exogenous LTB4, mRNA and protein levels of tumor necrosis factor-alpha (TNFalpha) and interleukin 1beta (IL1beta) were determined by real-time PCR and ELISA. Furthermore, we examined the effects of leukotrienes synthesis inhibitors, MK886 and bestatin, on mRNA and protein levels of TNFalpha and IL1beta in RASF. We found that LTB4 was present at a low concentration in the culture medium of RASF, and the major LTB4 receptor expressed in RASF was BLT2. LTB4 synthesis was activated by treatment with LIT (LPS, ionomycin and thapsogargin), and suppressed by MK886 and bestatin. Exogenous LTB4 remarkably increased the expression of TNFalpha and IL1beta at both the mRNA level and the protein level. In contrast, MK886 and bestatin significantly inhibited their expression. These data suggested that LTB4 contributed to RA by regulating the expression of TNFalpha and IL1beta in RASF. BLT2 was probably the major receptor mediating such effects.

Expression of functional leukotriene B 4 receptors on human airway smooth muscle cells

Leukotriene B4 (LTB4) increases in induced sputum and exhaled breath condensate in people with asthma. Furthermore, the T(H)2-type immune response and airway hyperresponsiveness induced by ovalbumin sensitization is markedly suppressed in LTB4 receptor (BLT) 1 null mice. These studies suggest that LTB4 may contribute to asthma pathophysiology. However, the direct effects of LTB4 on human airway smooth muscle (ASM) have not been studied. We sought to determine the expression of LTB4 receptors on human ASM and its functional role in mediating responses of human ASM cells, and the effect of LTB4 on these cells. Immunohistochemistry, RT-PCR, Western blotting, and flow cytometry were used to determine the expression of LTB4 receptors. To determine the effect of LTB4 on human ASM cells, cell proliferation was assessed by counting cells, and chemokinesis was assessed by gold particle phagokinesis assay. We confirmed expression of both BLT1 and BLT2 in human ASM cells in bronchial tissue and in cell culture. LTB4 markedly induced cyclin D1 expression, proliferation, and chemokinesis of human ASM cells. LTB4 also induced phosphorylation of both p42/p44 mitogen-activated protein kinase (MAPK) and downstream PI3 kinase effector, Akt1. However, we observed no induction of c-Jun N-terminal kinase or p38 MAPK. Notably, LTB4-induced migration and proliferation of ASM cells were inhibited by the BLT1 specific antagonist, U75302, and by inhibitors of p42/p44 MAPK phosphorylation (U1026), and PI3 kinase (LY294002). These observations are the first to suggest a role for a LTB4-BLT1 signaling axis in ASM responses that may contribute to the pathogenesis of airway remodeling in asthma.

Time-dependent expression of leukotriene B4 receptors in rat collagen-induced arthritis

Leukotriene B4 acts through its receptors, BLT 1 and BLT 2, however, their expression in rheumatoid arthritis is unknown. In this experiment, BLT 1 and BLT 2 mRNA expressions in the synovium of rats with collagen-induced arthritis (CIA) at days 1, 3, 7 and 14 after CIA onset were analyzed by RT-PCR. The expression of two immunological and inflammatory factors, S100A8 and S100A9, in the synovium of the arthritic rats was also determined at the indicated time. At d14, the differential expressions of BLT 1 and BLT 2 in the synovium, spleen, peripheral blood mononuclear cells (PBMC) and thymus of CIA rats were analyzed. The results showed that, in the synovium of the arthritic rats, the BLT 1 mRNA expression increased after CIA onset, reached the highest value between d1 and d3, and declined afterwards while the BLT 2 expression increased with time and reached its peak at d14. Both S100A8 and S100A9 expression reached the peak levels between d1 and d3, and decreased to lower levels between d7 and d14. For the analyzed tissues from CIA rats at d14, BLT 1 mRNA was expressed in the thymus with the highest level, followed by the spleen, PBMC and synovium. BLT 2 mRNA was expressed in the thymus the highest as well, but followed by the synovium, spleen and PBMC. Since BLT 1 and BLT 2 play distinct roles during CIA, this study may provide basis for new therapies targeting BLT 1 and BLT 2, respectively, for the treatment of arthritic inflammation at different stages.

Blockade of Leukotriene B4 Signaling Pathway Induces Apoptosis and Suppresses Cell Proliferation in Colon Cancer

We investigated whether leukotriene B4 (LTB4) and its signaling pathway play an important role in the progression of human colon cancer via a direct stimulation of cancer cell proliferation. Remarkable expression of LTB4 receptor 1 (BLT1) in human colon cancer tissues was detected by immunohistochemistry, and Western blot analysis revealed the BLT1 expression in cultured human colon cancer cell lines, Caco2 and HT29. The 5-lipoxygenase inhibitor AA-861 and LTB4-receptor antagonist U75302 showed negative effects on survival and proliferation of both Caco2 and HT-29 cells. The inhibition of cell proliferation is due to the apoptosis because nuclear condensation and increased annexin V expression were observed in the cells treated with AA-861 and U75302. Knockdown of BLT1 by small interfering RNA caused the suppression of BLT1 protein, resulting in the inhibition of cancer cell proliferation. Blockade of BLT1 by the receptor antagonist significantly suppresses the LTB4-stimulated extracellular signal-regulated kinase (ERK) activation in colon cancer cells. These results indicate that the blockade of the LTB4-signaling pathway induces apoptosis via the inhibition of ERK activation in colon cancer cells. The LTB4-signaling pathway might be a new therapeutic target for colon cancer.

Leukotriene B4 stimulates human monocyte-derived dendritic cell chemotaxis

Although leukotriene B4 (LTB4) has been reported to stimulate monocytes and neutrophils, its role on dendritic cell (DC) activity has not been examined. Here, we investigated the expression of LTB4 receptor and the effect of LTB4 on human DC chemotaxis. We analyzed LTB4 receptors, BLT1 and BLT2, by using RT-PCR. DCs express BLT2 but not BLT1 mRNA. DCs were chemotactically migrated to LTB4. LTB4-induced DC chemotaxis was completely inhibited by pertussis toxin, indicating the role of Gi proteins. LTB4 induced mitogen activated protein kinase activation and Akt activation. LTB4-induced DC chemotaxis was mediated by extracellular signal-regulated protein kinase and phosphoinositide 3-kinase but not by p38 kinase. BLT2-selevite antagonist, LY255283, almost completely inhibited DC chemotaxis induced by LTB4 but not by Trp-Lys-Tyr-Met-Val-D-Met. Thus human myeloid DCs express functional BLT2 but not BLT1, suggesting a physiological role of LTB4 and BLT2 in regulating DC trafficking during induction of immune responses.