register interest

Professor Luzheng Xue

Research Area: Immunology
Technology Exchange: Cellular immunology, Drug discovery, Flow cytometry and Microscopy (EM)
Scientific Themes: Immunology & Infectious Disease
Keywords: GPCR, inflammation, T cells, signal transduction, translational medicine and allergy
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The main aim of our research is to understand the molecular and cellular mechanisms mediating inflammatory diseases and to translate our findings into therapeutic concepts to treat these diseases. The focus of current projects is on the novel functions and signalling mechanisms of eicosanoid receptors, particularly the receptor for prostaglandin D2 (PGD2) CRTH2, that play a critical role in the pathogenesis of allergic inflammatory diseases such as asthma, rhinitis and dermatitis.

  • Exploration of the novel functions of CRTH2 on pro-inflammatory responses in in vitro immune cells
  • Analysis of the signalling pathways downstream of CRTH2 that promote pro-inflammatory responses
  • Development of novel therapeutic strategies for the treatment of inflammatory diseases
  • Identification of novel GPCR receptors, that promote pro-inflammatory responses, as drug targets in inflammatory diseases
  • Identification of biomarkers for diagnosing and differentiating sub-phenotypes of inflammatory diseases

Name Department Institution Country
Professor Paul Klenerman Experimental Medicine Division Oxford University, Peter Medawar Building United Kingdom
Prof Graham Ogg (RDM) Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Dr Roy Pettipher Oxagen Ltd United Kingdom
Professor Ian Pavord FMedSci FRCP Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Professor Ling-Pei Ho Experimental Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Dr Guy Brown University of Cambridge United Kingdom
Professor Hergen Spits Tytgat Institute for Liver and Intestinal Research University of Amsterdam Netherlands
Professor Evi Kostenis University of Bonn Germany
Hilvering B, Hinks TSC, Stöger L, Marchi E, Salimi M, Shrimanker R, Liu W, Chen W, Luo J, Go S et al. 2018. Synergistic activation of pro-inflammatory type-2 CD8+ T lymphocytes by lipid mediators in severe eosinophilic asthma. Mucosal Immunol, 11 (5), pp. 1408-1419. | Show Abstract | Read more

Human type-2 CD8+ T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia-a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

Hinks TSC, Batty P, Klenerman P, Pavord ID, Xue L. 2017. Cytometric Gating Stringency Impacts Studies of Type 2 Innate Lymphoid Cells in Asthma. Am J Respir Cell Mol Biol, 57 (6), pp. 745-747. | Read more

Tinnevelt GH, Kokla M, Hilvering B, van Staveren S, Folcarelli R, Xue L, Bloem AC, Koenderman L, Buydens LMC, Jansen JJ. 2017. Novel data analysis method for multicolour flow cytometry links variability of multiple markers on single cells to a clinical phenotype. Sci Rep, 7 (1), pp. 5471. | Show Abstract | Read more

Multicolour Flow Cytometry (MFC) produces multidimensional analytical data on the quantitative expression of multiple markers on single cells. This data contains invaluable biomedical information on (1) the marker expressions per cell, (2) the variation in such expression across cells, (3) the variability of cell marker expression across samples that (4) may vary systematically between cells collected from donors and patients. Current conventional and even advanced data analysis methods for MFC data explore only a subset of these levels. The Discriminant Analysis of MultiAspect CYtometry (DAMACY) we present here provides a comprehensive view on health and disease responses by integrating all four levels. We validate DAMACY by using three distinct datasets: in vivo response of neutrophils evoked by systemic endotoxin challenge, the clonal response of leukocytes in bone marrow of acute myeloid leukaemia (AML) patients, and the complex immune response in blood of asthmatics. DAMACY provided good accuracy 91-100% in the discrimination between health and disease, on par with literature values. Additionally, the method provides figures that give insight into the marker expression and cell variability for more in-depth interpretation, that can benefit both physicians and biomedical researchers to better diagnose and monitor diseases that are reflected by changes in blood leukocytes.

Salimi M, Stöger L, Liu W, Go S, Pavord I, Klenerman P, Ogg G, Xue L. 2017. Cysteinyl leukotriene E4 activates human group 2 innate lymphoid cells and enhances the effect of prostaglandin D2 and epithelial cytokines. J Allergy Clin Immunol, 140 (4), pp. 1090-1100.e11. | Show Abstract | Read more

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are a potential innate source of type 2 cytokines in the pathogenesis of allergic conditions. Epithelial cytokines (IL-33, IL-25, and thymic stromal lymphopoietin [TSLP]) and mast cell mediators (prostaglandin D2 [PGD2]) are critical activators of ILC2s. Cysteinyl leukotrienes (cysLTs), including leukotriene (LT) C4, LTD4, and LTE4, are metabolites of arachidonic acid and mediate inflammatory responses. Their role in human ILC2s is still poorly understood. OBJECTIVES: We sought to determine the role of cysLTs and their relationship with other ILC2 stimulators in the activation of human ILC2s. METHODS: For ex vivo studies, fresh blood from patients with atopic dermatitis and healthy control subjects was analyzed with flow cytometry. For in vitro studies, ILC2s were isolated and cultured. The effects of cysLTs, PGD2, IL-33, IL-25, TSLP, and IL-2 alone or in combination on ILC2s were defined by using chemotaxis, apoptosis, ELISA, Luminex, quantitative RT-PCR, and flow cytometric assays. The effect of endogenous cysLTs was assessed by using human mast cell supernatants. RESULTS: Human ILC2s expressed the LT receptor CysLT1, levels of which were increased in atopic subjects. CysLTs, particularly LTE4, induced migration, reduced apoptosis, and promoted cytokine production in human ILC2s in vitro. LTE4 enhanced the effect of PGD2, IL-25, IL-33, and TSLP, resulting in increased production of type 2 and other proinflammatory cytokines. The effect of LTE4 was inhibited by montelukast, a CysLT1 antagonist. Interestingly, addition of IL-2 to LTE4 and epithelial cytokines significantly amplified ILC2 activation and upregulated expression of the receptors for IL-33 and IL-25. CONCLUSION: CysLTs, particularly LTE4, are important contributors to the triggering of human ILC2s in inflammatory responses, particularly when combined with other ILC2 activators.

Hilvering B, Vijverberg SJH, Jansen J, Houben L, Schweizer RC, Go S, Xue L, Pavord ID, Lammers J-WJ, Koenderman L. 2017. Diagnosing eosinophilic asthma using a multivariate prediction model based on blood granulocyte responsiveness. Allergy, 72 (8), pp. 1202-1211. | Show Abstract | Read more

BACKGROUND: The identification of inflammatory asthma phenotypes, using sputum analysis, has proven its value in diagnosis and disease monitoring. However due to technical limitations of sputum analysis, there is a strong need for fast and noninvasive diagnostics. This study included the activation state of eosinophils and neutrophils in peripheral blood to phenotype and monitor asthma. OBJECTIVES: To (i) construct a multivariable model using the activation state of blood granulocytes, (ii) compare its diagnostic value with sputum eosinophilia as gold standard and (iii) validate the model in an independent patient cohort. METHODS: Clinical parameters, activation of blood granulocytes and sputum characteristics were assessed in 115 adult patients with asthma (training cohort/Utrecht) and 34 patients (validation cohort/Oxford). RESULTS: The combination of blood eosinophil count, fractional exhaled nitric oxide, Asthma Control Questionnaire, medication use, nasal polyposis, aspirin sensitivity and neutrophil/eosinophil responsiveness upon stimulation with formyl-methionyl-leucyl phenylalanine was found to identify sputum eosinophilia with 90.5% sensitivity and 91.5% specificity in the training cohort and with 77% sensitivity and 71% specificity in the validation cohort (relatively high percentage on oral corticosteroids [OCS]). CONCLUSIONS: The proposed prediction model identifies eosinophilic asthma without the need for sputum induction. The model forms a noninvasive and externally validated test to assess eosinophilic asthma in patients not on OCS.

Cheung KL, Jarrett R, Subramaniam S, Salimi M, Gutowska-Owsiak D, Chen Y-L, Hardman C, Xue L, Cerundolo V, Ogg G. 2016. Psoriatic T cells recognize neolipid antigens generated by mast cell phospholipase delivered by exosomes and presented by CD1a. J Exp Med, 213 (11), pp. 2399-2412. | Show Abstract | Read more

Psoriasis is a chronic inflammatory skin disease associated with a T helper 17 response. Yet, it has proved challenging to identify relevant peptide-based T cell antigens. Antigen-presenting Langerhans cells show a differential migration phenotype in psoriatic lesions and express constitutively high levels of CD1a, which presents lipid antigens to T cells. In addition, phospholipase A2 (PLA2) is highly expressed in psoriatic lesions and is known to generate neolipid skin antigens for recognition by CD1a-reactive T cells. In this study, we observed expression of a cytoplasmic PLA2 (PLA2G4D) in psoriatic mast cells but, unexpectedly, also found PLA2G4D activity to be extracellular. This was explained by IFN-α-induced mast cell release of exosomes, which transferred cytoplasmic PLA2 activity to neighboring CD1a-expressing cells. This led to the generation of neolipid antigens and subsequent recognition by lipid-specific CD1a-reactive T cells inducing production of IL-22 and IL-17A. Circulating and skin-derived T cells from patients with psoriasis showed elevated PLA2G4D responsiveness compared with healthy controls. Overall, these data present an alternative model of psoriasis pathogenesis in which lipid-specific CD1a-reactive T cells contribute to psoriatic inflammation. The findings suggest that PLA2 inhibition or CD1a blockade may have therapeutic potential for psoriasis.

Salimi M, Xue L, Jolin H, Hardman C, Cousins DJ, McKenzie ANJ, Ogg GS. 2016. Group 2 Innate Lymphoid Cells Express Functional NKp30 Receptor Inducing Type 2 Cytokine Production. J Immunol, 196 (1), pp. 45-54. | Show Abstract | Read more

Group 2 innate lymphoid cells (ILC2) are important in effector functions for eliciting allergic inflammation, parasite defense, epithelial repair, and lipid homeostasis. ILC2 lack rearranged Ag-specific receptors, and although many soluble factors such as cytokines and lipid mediators can influence ILC2, direct interaction of these cells with the microenvironment and other cells has been less explored. Natural cytotoxicity receptors are expressed by subsets of group 1 ILC and group 3 ILC and thought to be important for their effector function, but they have not been shown to be expressed by ILC2. Therefore, we sought to investigate the expression and functional properties of the natural cytotoxicity receptor NKp30 on human ILC2. A subset of ex vivo and cultured ILC2 express NKp30 that upon interaction with its cognate activatory ligand B7-H6 induces rapid production of type 2 cytokines. This interaction can be blocked by NKp30 blocking Ab and an inhibitory ligand, galectin-3. Higher expression of B7-H6 was observed in lesional skin biopsies of patients with atopic dermatitis, and incubation of keratinocytes with proinflammatory and type 2 cytokines upregulated B7-H6, leading to increased ILC2 cytokine production. NKp30-B7-H6 interaction is a novel cell contact mechanism that mediates activation of ILC2 and identifies a potential target for the development of novel therapeutics for atopic dermatitis and other atopic diseases.

Hilvering B, Xue L, Pavord ID. 2015. Evidence for the efficacy and safety of anti-interleukin-5 treatment in the management of refractory eosinophilic asthma. Ther Adv Respir Dis, 9 (4), pp. 135-145. | Show Abstract | Read more

Two recent phase III trials in patients with severe eosinophilic asthma have shown that anti-interleukin 5 (IL-5) therapy with mepolizumab reduces the frequency of asthma attacks, improves symptoms and allows patients to reduce oral glucocorticoid use without loss of control of asthma. An earlier large 616 patient Dose Ranging Efficacy And safety with Mepolizumab in severe asthma (DREAM) study had shown that the only variables associated with treatment efficacy were a prior history of asthma attacks and the peripheral blood eosinophil count. The link between blood eosinophil counts and treatment efficacy is biologically obvious given that IL-5 has a pivotal role in eosinophil production, proliferation and chemotaxis. It is also clinically relevant as the blood eosinophil count is routinely measured and thus readily available in patients with asthma. Recognition of the link between airway or blood eosinophilia and treatment response was also important in the clinical testing of the alternative IL-5 blocker, such as reslizumab, which is currently being evaluated in a phase III randomized controlled trial (RCT) after having shown to improve lung function, improve symptom score and reduce sputum eosinophilia in a smaller phase IIb study. In addition, benralizumab, an IL-5α receptor blocker, has shown good effects in a phase IIb RCT with patients with severe asthma that had sputum eosinophilia and more recently in a phase IIa trial with patients with eosinophilic chronic obstructive pulmonary disease. Therefore anti-IL-5 treatment seems generally effective in eosinophilic asthma, either assessed by blood or airway eosinophilia. This factor together with the impressive clinical efficacy and good safety profile make anti-IL-5 (mepolizumab, reslizumab) and benralizumab (anti-IL-5 receptor α) very promising drugs for the treatment of patients with severe eosinophilic asthma, a subgroup that is in desperate need of better treatments.

Hilvering B, Xue L, Pavord ID. 2015. IL-33-dependent Th2 response after rhinovirus infection in asthma: more information needed. Am J Respir Crit Care Med, 191 (2), pp. 237. | Read more

Cited:

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Scopus

Xue L, Fergusson J, Salimi M, Panse I, Ussher JE, Hegazy AN, Vinall SL, Jackson DG, Hunter MG, Pettipher R et al. 2015. Prostaglandin D<inf>2</inf> and leukotriene E<inf>4</inf> synergize to stimulate diverse T<inf>H</inf>2 functions and T<inf>H</inf>2 cell/neutrophil crosstalk Journal of Allergy and Clinical Immunology, 135 (5), pp. 1358-1366e11. | Show Abstract | Read more

© 2015 American Academy of Allergy, Asthma & Immunology. Background Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. Objective We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. Methods The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. Results PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. Conclusions PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Xue L, Fergusson J, Salimi M, Panse I, Ussher JE, Hegazy AN, Vinall SL, Jackson DG, Hunter MG, Pettipher R et al. 2015. Prostaglandin D2 and leukotriene E4 synergize to stimulate diverse TH2 functions and TH2 cell/neutrophil crosstalk. J Allergy Clin Immunol, 135 (5), pp. 1358-66.e1-11. | Show Abstract | Read more

BACKGROUND: Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. OBJECTIVE: We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. METHODS: The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. RESULTS: PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. CONCLUSIONS: PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Salimi M, Barlow J, Saunders S, Xue L, Gutowska-Owsiak D, Wang X, Huang L-C, Johnson D, Scanlon S, McKenzie A et al. 2014. The role of type 2 innate lymphoid cells in the pathogenesis of atopic dermatitis BRITISH JOURNAL OF DERMATOLOGY, 170 (4), pp. E32-E32.

Xue L, Salimi M, Panse I, Mjösberg JM, McKenzie ANJ, Spits H, Klenerman P, Ogg G. 2014. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells. J Allergy Clin Immunol, 133 (4), pp. 1184-1194. | Show Abstract | Read more

BACKGROUND: Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2), a receptor for prostaglandin D₂ (PGD₂), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear. OBJECTIVES: We sought to determine the role of PGD₂ and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33. METHODS: The effects of PGD₂, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD₂ under physiologic conditions were evaluated by using the supernatant from activated mast cells. RESULTS: PGD₂ binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD₂ on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. CONCLUSIONS: PGD₂ is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s.

Salimi M, Barlow JL, Saunders SP, Xue L, Gutowska-Owsiak D, Wang X, Huang L-C, Johnson D, Scanlon ST, McKenzie ANJ et al. 2013. A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. J Exp Med, 210 (13), pp. 2939-2950. | Show Abstract | Read more

Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease.

Schröder R, Xue L, Konya V, Martini L, Kampitsch N, Whistler JL, Ulven T, Heinemann A, Pettipher R, Kostenis E. 2012. PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor CRTH2/DP2. PLoS One, 7 (3), pp. e33329. | Show Abstract | Read more

Prostaglandin H(1) (PGH(1)) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as "anti-inflammatory". Herein we present evidence that PGH(1) is a potent activator of the pro-inflammatory PGD(2) receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca(2+) flux studies reveal that PGH(1) activates CRTH2 as PGH(2), PGD(2) or PGD(1) do. The PGH(1) precursor DGLA and the other PGH(1) metabolites did not display such effect. PGH(1) specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH(1) is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH(1) mediates migration of and Ca(2+) flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH(1) as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase.

Xue L, Barrow A, Fleming VM, Hunter MG, Ogg G, Klenerman P, Pettipher R. 2012. Leukotriene E4 activates human Th2 cells for exaggerated proinflammatory cytokine production in response to prostaglandin D2. J Immunol, 188 (2), pp. 694-702. | Show Abstract | Read more

PGD(2) exerts a number of proinflammatory responses through a high-affinity interaction with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and has been detected at high concentrations at sites of allergic inflammation. Because cysteinyl leukotrienes (cysLTs) are also produced during the allergic response, we investigated the possibility that cysLTs may modulate the response of human Th2 cells to PGD(2). PGD(2) induced concentration-dependent Th2 cytokine production in the absence of TCR stimulation. Leukotrienes D(4) and E(4) (LTE(4)) also stimulated the cytokine production but were much less active than PGD(2). However, when combined with PGD(2), cysLTs caused a greater than additive enhancement of the response, with LTE(4) being most effective in activating Th2 cells. LTE(4) enhanced calcium mobilization in response to PGD(2) in Th2 cells without affecting endogenous PGD(2) production or CRTH2 receptor expression. The effect of LTE(4) was inhibited by montelukast but not by the P2Y(12) antagonist methylthioadenosine 5'-monophosphate. The enhancing effect was also evident with endogenous cysLTs produced from immunologically activated mast cells because inhibition of cysLT action by montelukast or cysLT synthesis by MK886, an inhibitor of 5-lipoxygenase-activating protein, reduced the response of Th2 cells to the levels produced by PGD(2) alone. These findings reveal that cysLTs, in particular LTE(4), have a significant proinflammatory impact on T cells and demonstrate their effects on Th2 cells are mediated by a montelukast-sensitive receptor.

Pettipher R, Vinall SL, Xue L, Speight G, Townsend ER, Gazi L, Whelan CJ, Armer RE, Payton MA, Hunter MG. 2012. Pharmacologic profile of OC000459, a potent, selective, and orally active D prostanoid receptor 2 antagonist that inhibits mast cell-dependent activation of T helper 2 lymphocytes and eosinophils. J Pharmacol Exp Ther, 340 (2), pp. 473-482. | Show Abstract | Read more

D prostanoid receptor 2 (DP₂) [also known as chemoattractant receptor-homologous molecule expressed on T helper 2 (Th2) cells (CRTH2)] is selectively expressed by Th2 lymphocytes, eosinophils, and basophils and mediates recruitment and activation of these cell types in response to prostaglandin D₂ (PGD₂). (5-Fluoro-2-methyl-3-quinolin-2-ylmethylindo-1-yl)-acetic acid (OC000459) is an indole-acetic acid derivative that potently displaces [³H]PGD₂ from human recombinant DP₂ (K(i) = 0.013 μM), rat recombinant DP₂ (K(i) = 0.003 μM), and human native DP₂ (Th2 cell membranes; K(i) = 0.004 μM) but does not interfere with the ligand binding properties or functional activities of other prostanoid receptors (prostaglandin E₁₋₄ receptors, D prostanoid receptor 1, thromboxane receptor, prostacyclin receptor, and prostaglandin F receptor). OC000459 inhibited chemotaxis (IC₅₀ = 0.028 μM) of human Th2 lymphocytes and cytokine production (IC₅₀ = 0.019 μM) by human Th2 lymphocytes. OC000459 competitively antagonized eosinophil shape change responses induced by PGD₂ in both isolated human leukocytes (pK(B) = 7.9) and human whole blood (pK(B) = 7.5) but did not inhibit responses to eotaxin, 5-oxo-eicosatetraenoic acid, or complement component C5a. OC000459 also inhibited the activation of Th2 cells and eosinophils in response to supernatants from IgE/anti-IgE-activated human mast cells. OC000459 had no significant inhibitory activity on a battery of 69 receptors and 19 enzymes including cyclooxygenase 1 (COX1) and COX2. OC000459 was found to be orally bioavailable in rats and effective in inhibiting blood eosinophilia induced by 13,14-dihydro-15-keto-PGD₂ (DK-PGD₂) in this species (ED₅₀ = 0.04 mg/kg p.o.) and airway eosinophilia in response to an aerosol of DK-PGD₂ in guinea pigs (ED₅₀ = 0.01 mg/kg p.o.). These data indicate that OC000459 is a potent, selective, and orally active DP₂ antagonist that retains activity in human whole blood and inhibits mast cell-dependent activation of both human Th2 lymphocytes and eosinophils.

Pérez-Novo CA, Holtappels G, Vinall SL, Xue L, Zhang N, Bachert C, Pettipher R. 2010. CRTH2 mediates the activation of human Th2 cells in response to PGD(2) released from IgE/anti-IgE treated nasal polyp tissue. Allergy, 65 (3), pp. 304-310. | Show Abstract | Read more

BACKGROUND: Mast cells release mediators upon stimulation that contribute to the pathogenesis of chronic airway disease, including the recruitment and activation of Th2 lymphocytes. The objective was to determine the involvement of prostaglandin D(2) (PGD(2)) and its receptors in the chemotaxis of Th2 cells, using nasal polyp tissue. METHODS: Tissue explants from ten patients with nasal polyposis were incubated with RPMI alone or RPMI containing IgE/anti-IgE for 30 min. Some samples were treated with diclofenac to inhibit the production of PGD(2). Supernatants were assayed for PGD(2) content and for their ability to promote human Th2 cell chemotaxis in the presence and absence of a CRTH2 antagonist. Transcript levels of D protanoid receptor type 1 (DP(1)), chemoattractant receptor-homologous receptor expressed on Th2 cells (CRTH2) and PGD(2) synthase were analysed by real time PCR. RESULTS: Increased release of PGD(2) by nasal polyp tissue treated with IgE/anti-IgE was significantly inhibited by preincubation of the tissue with diclofenac. Transcript levels of PGD(2) synthase, DP(1) and CRTH2 receptors increased after stimulation with IgE/anti-IgE. Supernatants from IgE/anti-IgE-stimulated nasal polyp tissue caused significantly increased chemotaxis of Th2 cells. The levels of PGD(2) produced and the degree of Th2 cell chemotaxis were highly correlated. Diclofenac inhibited the production of Th2 cell chemotactic activity, and the chemotactic effect of the supernatant on Th2 cells was inhibited by the CRTH2 antagonist ramatroban. CONCLUSION: These data suggest that in immunologically activated nasal polyp tissue, PGD(2) produced by mast cells promotes the migration of Th2 cells through a CRTH2 dependent mechanism.

Xue L, Barrow A, Pettipher R. 2009. Novel function of CRTH2 in preventing apoptosis of human Th2 cells through activation of the phosphatidylinositol 3-kinase pathway. J Immunol, 182 (12), pp. 7580-7586. | Show Abstract | Read more

It is now well established that interaction of PGD(2) with chemoattractant receptor- homologous molecule expressed on Th2 cells (CRTH2) promotes chemotaxis and proinflammatory cytokine production by Th2 lymphocytes. In this study we show a novel function of CRTH2 in mediating an inhibitory effect of PGD(2) on the apoptosis of human Th2 cells induced by cytokine deprivation. This effect was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD(2), inhibited by the CRTH2 antagonists ramatroban and TM30089, and not observed in CRTH2-negative T cells. D prostanoid receptor 1 (DP(1)) or the thromboxane-like prostanoid (TP) receptor did not play a role in mediating the effects of PGD(2) on the apoptosis of Th2 cells because neither the DP(1) antagonist BW868C nor the TP antagonist SQ29548 had any effect on the antiapoptotic effect of PGD(2). Apoptosis of Th2 cells induced by Fas ligation was not suppressed by treatment with PGD(2), illustrating that activation of CRTH2 only inhibits apoptosis induced by cytokine deprivation. Treatment with PGD(2) induced phosphorylation of Akt and BAD, prevented release of cytochrome c from mitochondria, and suppressed cleavage of caspase-3 and poly(ADP-ribose) polymerase in Th2 cells deprived of IL-2. The PI3K inhibitor LY294002 blocked the effect of PGD(2) both on the signaling events and on the apoptotic death of Th2 cells. These data suggest that in addition to promoting the recruitment and activation of Th2 cells, PGD(2) may also impede the resolution of allergic inflammation through inhibiting apoptosis of Th2 cells.

Xue L, Barrow A, Pettipher R. 2009. Interaction between prostaglandin D and chemoattractant receptor-homologous molecule expressed on Th2 cells mediates cytokine production by Th2 lymphocytes in response to activated mast cells. Clin Exp Immunol, 156 (1), pp. 126-133. | Show Abstract | Read more

The mechanisms by which immunologically activated mast cells stimulate the production of proinflammatory cytokines by T helper type 2 (Th2) lymphocytes were investigated in a human cell culture system. Supernatants collected from cord blood-derived mast cells after treatment with immunoglobulin E (IgE)/anti-IgE contained an activity that stimulated the production of interleukin (IL)-4, IL-5 and IL-13 (both mRNA and protein) by Th2 lymphocytes. This activity was not detected in supernatants from unactivated mast cells and its production was inhibited by treatment of activated mast cells with the cyclo-oxygenase inhibitor diclofenac. The concentration of diclofenac used inhibited completely the production of prostaglandin D(2) (PGD(2)) but did not inhibit the release of histamine or leukotriene C(4). The effect of supernatants from activated mast cells was mimicked by exogenous PGD(2) at concentrations similar to those detected in the cultures of activated mast cells, and addition of exogenous PGD(2) to supernatants from diclofenac-treated mast cells restored their ability to stimulate Th2 cytokine production. The ability of the mast cell supernatants to stimulate production of Th2 cytokines was not affected by addition of diclofenac to the Th2 cells directly, indicating that the production, but not the action, of the factor was sensitive to diclofenac treatment. Inhibition of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) abolished the effect of the mast cell supernatants on Th2 cytokine production. These data indicate that mast cells have the ability to stimulate Th2 cells to elaborate cytokines independently of T cell receptor activation or co-stimulation and this response is mediated by PGD(2) acting upon CRTH2 expressed by Th2 cells.

Xue L, Gyles SL, Barrow A, Pettipher R. 2007. Inhibition of PI3K and calcineurin suppresses chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)-dependent responses of Th2 lymphocytes to prostaglandin D(2). Biochem Pharmacol, 73 (6), pp. 843-853. | Show Abstract | Read more

Interaction of prostaglandin D2 (PGD2) with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) triggers chemotaxis and pro-inflammatory cytokine production by Th2 lymphocytes. We have investigated the role of inhibitors of various cell-signalling pathways on the responses of human CRTH2+ CD4+ Th2 cells to PGD2. Phosphatidylinositol 3-kinase (PI3K) and Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) pathways were activated by PGD2 in Th2 cells in a CRTH2-dependent manner. Inhibition of the PI3K pathway with LY294002 significantly reduced both PGD2-induced cell migration and cytokine (interleukin-4, interleukin-5 and interleukin-13) production. The inhibitory effect of LY294002 on cell migration is likely to be related to cytoskeleton reorganization as it showed a similar potency on PGD2-induced actin polymerization. The calcineurin inhibitors, tacrolimus (FK506) and cyclosporin A, had no effect on cell migration but completely blocked both cytokine production and the nuclear translocation of NFATc1 suggesting that Ca2+/calcineurin/NFAT is involved in CRTH2-dependent cytokine production but not chemotaxis. The promotion of NFAT nuclear location by PI3K activation may be mediated by negative regulation of glycogen synthase kinase-3beta (GSK3beta), since the PGD2-stimulated increase in phospho-GSK3beta was down-regulated by LY294002, and inhibition of GSK3beta by SB216763 enhanced PGD2-induced Th2 cytokine production and reversed the inhibitory effect of LY294002. These data suggest that PI3K and Ca2+/calcineurin/NFAT signalling pathways are critically involved in pro-inflammatory responses of Th2 cells to PGD2.

Gyles SL, Xue L, Townsend ER, Wettey F, Pettipher R. 2006. A dominant role for chemoattractant receptor-homologous molecule expressed on T helper type 2 (Th2) cells (CRTH2) in mediating chemotaxis of CRTH2+ CD4+ Th2 lymphocytes in response to mast cell supernatants. Immunology, 119 (3), pp. 362-368. | Show Abstract | Read more

Human cultured mast cells, immunologically activated with immunoglobuin E (IgE)/anti-IgE, released a factor(s) that promoted chemotaxis of human CRTH2+ CD4+ T helper type 2 (Th2) lymphocytes. Mast cell supernatants collected at 20 min, 1 hr, 2 hr and 4 hr after activation caused a concentration-dependent increase in the migration of Th2 cells. The effect of submaximal dilutions of mast-cell-conditioned media was inhibited in a dose-dependent manner by ramatroban (IC50 = 96 nm), a dual antagonist of both the thromboxane-like prostanoid (TP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), but not by the selective TP antagonist SQ29548, implicating CRTH2 in mediating the chemotactic response of these Th2 cells. The effect of mast-cell-conditioned media was mimicked by prostaglandin D2 (PGD2) and this eicosanoid was detected in the conditioned media from activated mast cells in concentrations sufficient to account for the activity of the mast cell supernatants. Treatment of the mast cells with the cyclo-oxygenase inhibitor diclofenac (10 microm) inhibited both the production of PGD2 and the CRTH2+ CD4+ Th2-stimulatory activity, while addition of exogenous PGD2 to conditioned media from diclofenac-treated mast cells restored the ability of the supernatants to promote chemotaxis of these Th2 cells. The degree of inhibition caused by diclofenac treatment of the mast cells was concordant with the degree of inhibition of chemotactic responses afforded by CRTH2 blockade. These data suggest that PGD2, or closely related metabolites of arachidonic acid, produced from mast cells may play a central role in the activation of CRTH2+ CD4+ Th2 lymphocytes through a CRTH2-dependent mechanism.

Wettey FR, Xue L, Pettipher R. 2006. Salbutamol inhibits trypsin-mediated production of CXCL8 by keratinocytes. Cytokine, 36 (1-2), pp. 29-34. | Show Abstract | Read more

Treatment of primary keratinocytes (HEKAp) with trypsin led to the production and release of CXCL8. Production of CXCL8 was exquisitely sensitive to inhibition by co-treatment with the beta(2) agonist sabutamol (IC(50)=1.1 nM). The inhibitory effect of salbutamol was beta receptor-mediated since the effect was prevented by the beta antagonist sotalol. Salbutamol also elevated intracellular levels of cAMP (EC(50)=82 nM) but the relationship to the inhibition of CXCL8 secretion was not clear-cut since much higher concentrations of salbutamol were required to elevate total cellular cAMP than inhibit CXCL8 production. However, the effect of salbutamol is likely to be mediated by elevation of cAMP since forskolin, an adenylyl cyclase activator, mimicked the effects of salbutamol while the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine inhibited the effects of salbutamol. Potentiation of cAMP production by co-treatment with the phosphodiesterase type 4 inhibitor rolipram only marginally enhanced the inhibitory effect of salbutamol on CXCL8 production. Taken together, these data suggest that elevation of cAMP production is required for the inhibitory effect of salbutamol on CXCL8 production by keratinocytes and that low threshold levels of cAMP are sufficient to mediate this effect.

Xue L, Gyles SL, Wettey FR, Gazi L, Townsend E, Hunter MG, Pettipher R. 2005. Prostaglandin D2 causes preferential induction of proinflammatory Th2 cytokine production through an action on chemoattractant receptor-like molecule expressed on Th2 cells. J Immunol, 175 (10), pp. 6531-6536. | Show Abstract | Read more

PGD2, produced by mast cells, has been detected in high concentrations at sites of allergic inflammation. It can stimulate vascular and other inflammatory responses by interaction with D prostanoid receptor (DP) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) receptors. A significant role for PGD2 in mediating allergic responses has been suggested based on the observation that enhanced eosinophilic lung inflammation and cytokine production is apparent in the allergen-challenged airways of transgenic mice overexpressing human PGD2 synthase, and PGD2 can enhance Th2 cytokine production in vitro from CD3/CD28-costimulated Th2 cells. In the present study, we investigated whether PGD2 has the ability to stimulate Th2 cytokine production in the absence of costimulation. At concentrations found at sites of allergic inflammation, PGD2 preferentially elicited the production of IL-4, IL-5, and IL-13 by human Th2 cells in a dose-dependent manner without affecting the level of the anti-inflammatory cytokine IL-10. Gene transcription peaked within 2 h, and protein release peaked approximately 8 h after stimulation. The effect of PGD2 was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD2 but not by the selective DP agonist BW245C, suggesting that the stimulation is mediated by CRTH2 and not DP. Ramatroban, a dual CRTH2/thromboxane-like prostanoid receptor antagonist, markedly inhibited Th2 cytokine production induced by PGD2, while the selective thromboxane-like prostanoid receptor antagonist SQ29548 was without effect. These data suggest that PGD2 preferentially up-regulates proinflammatory cytokine production in human Th2 cells through a CRTH2-dependent mechanism in the absence of any other costimulation and highlight the potential utility of CRTH2 antagonists in the treatment of allergic diseases.

Armer RE, Ashton MR, Boyd EA, Brennan CJ, Brookfield FA, Gazi L, Gyles SL, Hay PA, Hunter MG, Middlemiss D et al. 2005. Indole-3-acetic acid antagonists of the prostaglandin D2 receptor CRTH2. J Med Chem, 48 (20), pp. 6174-6177. | Show Abstract | Read more

Prostaglandin D2 (PGD2) acting at the CRTH2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells) has been linked with a variety of allergic and other inflammatory diseases. We describe a family of indole-1-sulfonyl-3-acetic acids that are potent and selective CRTH2 antagonists that possess good oral bioavailability. The compounds may serve as novel starting points for the development of treatments of inflammatory disease such as asthma, allergic rhinitis, and atopic dermatitis.

Gazi L, Gyles S, Rose J, Lees S, Allan C, Xue L, Jassal R, Speight G, Gamble V, Pettipher R. 2005. Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes. Prostaglandins Other Lipid Mediat, 75 (1-4), pp. 153-167. | Show Abstract | Read more

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of delta12-PGD2, a product of PGD2 isomerization by albumin. Delta12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. delta12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, delta12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. delta12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that delta12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.

Ke X, Yang Y, Yin W-Y, Xue L. 2004. Effects of low pH environment on the collembolan Onychiurus yaodai Pedobiologia, 48 (5-6), pp. 545-550. | Show Abstract | Read more

The effects of pH on taxis, mortality and activation of MAP-kinase pathways in the collembolan Onychiurus yaodai were studied. The animals preferred to stay at pH 8. They were particularly stressed by a reduction in pH but were more tolerant of elevated pH. A short period of exposure to low pH led to phosphorylation of Drosophila homolog of extracellular signal-regulated kinase (ERKa) and the c-Jun N-terminal kinase (DJNK) in the animals, indicating activation of these signal transduction pathways. Our observations suggest that reduced environmental pH, even for a limited period, is potentially harmful to O. yaodai. © 2004 Elsevier GmbH. All rights reserved.

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Allen M, Heinzmann A, Noguchi E, Abecasis G, Broxholme J, Ponting CP, Bhattacharyya S, Tinsley J, Zhang YM, Holt R et al. 2003. Positional cloning of a novel gene influencing asthma from Chromosome 2q14 NATURE GENETICS, 35 (3), pp. 258-263. | Read more

Allen M, Heinzmann A, Noguchi E, Abecasis G, Broxholme J, Ponting CP, Bhattacharyya S, Tinsley J, Zhang Y, Holt R et al. 2003. Positional cloning of a novel gene influencing asthma from chromosome 2q14. Nat Genet, 35 (3), pp. 258-263. | Show Abstract | Read more

Asthma is a common disease in children and young adults. Four separate reports have linked asthma and related phenotypes to an ill-defined interval between 2q14 and 2q32 (refs. 1-4), and two mouse genome screens have linked bronchial hyper-responsiveness to the region homologous to 2q14 (refs. 5,6). We found and replicated association between asthma and the D2S308 microsatellite, 800 kb distal to the IL1 cluster on 2q14. We sequenced the surrounding region and constructed a comprehensive, high-density, single-nucleotide polymorphism (SNP) linkage disequilibrium (LD) map. SNP association was limited to the initial exons of a solitary gene of 3.6 kb (DPP10), which extends over 1 Mb of genomic DNA. DPP10 encodes a homolog of dipeptidyl peptidases (DPPs) that cleave terminal dipeptides from cytokines and chemokines, and it presents a potential new target for asthma therapy.

Yung HW, Xue L, Tolkovsky AM. 2002. Apoptosis-specific protein (ASP 45 kDa) is distinct from human Apg5, the homologue of the yeast autophagic gene apg5. FEBS Lett, 531 (2), pp. 168-172. | Show Abstract | Read more

We have examined whether the apoptosis-specific protein p45ASP and human Apg5 are identical proteins. Like p45ASP, myc-hApg5 cross-reacted with a c-Jun antibody and approximately 50% of myc-hApg5 was bound to a Triton X-100-insoluble fraction in HeLa cells. However, soluble myc-hApg5 was degraded during apoptosis induced by staurosporine or TNFalpha/cycloheximide whilst expression of soluble p45ASP was stabilised. Furthermore, myc-hApg5 degradation was blocked by the caspase inhibitor Boc-Asp(OMe)FMK whilst p45ASP expression was eliminated. Moreover, myc-hApg5 ( approximately 32 kDa) never assumed the size of p45ASP (45 kDa). It is therefore likely that p45ASP and human Apg5 are distinct proteins although they do share some common characteristics.

Xue L, Borutaite V, Tolkovsky AM. 2002. Inhibition of mitochondrial permeability transition and release of cytochrome c by anti-apoptotic nucleoside analogues. Biochem Pharmacol, 64 (3), pp. 441-449. | Show Abstract | Read more

We have investigated whether nucleoside drugs that induce or protect neurones against apoptosis might directly activate or inhibit mitochondrial permeability transition (mPT) since opening of the mPT pore can promote release of cytochrome c and apoptosis, while its closure can prevent these changes. We found that the pro-apoptotic pyrimidine analogues cytosine beta-D-arabinofuranoside and cytosine beta-D-arabinofuranoside 5'-triphosphate, which activated apoptosis in post-mitotic neurones without incorporation into nuclear DNA, induced rapid calcium-dependent mitochondrial swelling of isolated liver mitochondria in a dose-dependent manner. Induction of up to 50 and 80%, respectively, of maximal swelling induced by high calcium was obtained at 1mM concentrations, which also promoted a 17-fold increase in the release of cytochrome c. Both activities were inhibited by cyclosporine A to unstimulated levels; dCTP had no effect. In contrast, the anti-apoptotic adenine analogues, 3-methyladenine (3-MA) and olomoucine (but not iso-olomoucine), inhibited swelling induced by calcium or phenylarsine oxide in a dose-dependent manner at concentrations that protect neurones from apoptosis. Both compounds also inhibited the release of cytochrome c (by 82%, 20 mM 3-MA and 95%, 0.9 mM olomoucine), similar to the inhibition obtained with cyclosporine A, and 5mM ADP or ATP. Similar inhibitory effects with olomoucine and 3-MA were found in isolated heart mitochondria. These studies identify the mPT as an important target for hitherto untested pro- and anti-apoptotic nucleoside-based drugs and suggest that screening for mPT modulation is an important component in the validation of a drug's mechanism of action.

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Tolkovsky AM, Xue L, Fletcher GC, Borutaite V. 2002. Mitochondrial disappearance from cells: a clue to the role of autophagy in programmed cell death and disease? Biochimie, 84 (2-3), pp. 233-240. | Show Abstract | Read more

When cells are induced to undergo apoptosis in the presence of general caspase inhibitors and then returned to their normal growth environment, there follows an extended period of life during which the entire cohort of mitochondria (including mitochondrial DNA) disappears from the cells. This phenomenon is widespread; it occurs in NGF-deprived sympathetic neurons, in NGF-maintained neurons treated with cytosine arabinoside, and in diverse cell lines treated with staurosporine, including HeLa, CHO, 3T3 and Rat 1 cells. Mitochondrial removal is highly selective since the structure of all other organelles remains unperturbed. Since Bcl2 overexpression blocks the removal of mitochondria without preventing death-inducing signals, it appears that the mitochondria are responsible for initiating their own demise. Degradation of mitochondria is not in itself a rare event. It occurs in large part by autophagy during normal cell house-keeping, during ecdysis in insects, as well as after induction of apoptosis. However, the complete and selective removal of an entire cohort of mitochondria in otherwise living mammalian cells has not been described previously. These findings raise several questions. What are the mechanisms which remove mitochondria in such a 'clean' fashion? What are the signals that target mitochondria for such selective degradation? How are cells that have lost their mitochondria different from rho0 cells (which retain mitochondria but lack mitochondrial DNA, and cannot carry out oxidative phosphorylation)? Are the cells which have lost mitochondria absolutely committed to die or might they be repaired by mitochondrial therapy? The answers will be especially relevant when considering treatment of diseases affecting long-lived and non-renewable organs such as the nervous system.

Xue L, Fletcher GC, Tolkovsky AM. 2001. Mitochondria are selectively eliminated from eukaryotic cells after blockade of caspases during apoptosis. Curr Biol, 11 (5), pp. 361-365. | Show Abstract | Read more

Pan caspase inhibitors are potentially powerful cell-protective agents that block apoptosis in response to a wide variety of insults that cause tissue degeneration. In many conditions, however, the blockade of apoptosis by caspase inhibitors does not permit long-term cell survival, but the reasons are not entirely clear. Here we show that the blockade of apoptosis by Boc.Aspartyl(O-methyl)CH2F can result in the highly selective elimination of the entire cohort of mitochondria, including mitochondrial DNA, from both neurons and HeLa cells, irrespective of the stimulus used to trigger apoptosis. In cells that lose their mitochondria, the nuclear DNA, Golgi apparatus, endoplasmic reticulum, centrioles, and plasma membrane remain undamaged. The capacity to remove mitochondria is both specific and regulated since mitochondrial loss in neurons is completely prevented by the expression of the antiapoptotic protein Bcl-2 and partially suppressed by the autolysosomal inhibitor bafilomycin. Cells without mitochondria are more tolerant to an anaerobic environment but are essentially irreversibly committed to death. Prevention of mitochondrial loss may be crucial for the long-term regeneration of tissues emerging from an apoptotic episode in which death was prevented by caspase blockade.

Harding TC, Xue L, Bienemann A, Haywood D, Dickens M, Tolkovsky AM, Uney JB. 2001. Inhibition of JNK by overexpression of the JNL binding domain of JIP-1 prevents apoptosis in sympathetic neurons. J Biol Chem, 276 (7), pp. 4531-4534. | Show Abstract | Read more

Studies in non-neuronal cells show that c-Jun N-terminal kinases (JNK) play a key role in apoptotic cell death. In some neurons JNK is also thought to initiate cell death by the activation of c-Jun. JNK inhibition has been achieved pharmacologically by inhibiting upstream kinases, but there has been no direct demonstration that inhibition of JNK can prevent neuronal death. We have therefore examined whether the JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1, a scaffold protein and specific inhibitor of JNK) can inhibit c-Jun phosphorylation and support the survival of sympathetic neurons deprived of NGF. We show that expression of the JBD in >80% of neurons was sufficient to prevent the phosphorylation of c-Jun and its nuclear accumulation as well as abrogate neuronal cell death induced by NGF deprivation. JBD expression also preserved the capacity of mitochondria to reduce MTT. Interestingly, although the PTB domain of JIP was reported to interact with rhoGEF, expression of the JBD domain was sufficient to localize the protein to the membrane cortex and growth cones. Hence, JNK activation is a key event in apoptotic death induced by NGF withdrawal, where its point of action lies upstream of mitochondrial dysfunction.

Fletcher GC, Xue L, Passingham SK, Tolkovsky AM. 2000. Death commitment point is advanced by axotomy in sympathetic neurons. J Cell Biol, 150 (4), pp. 741-754. | Show Abstract | Read more

Axotomized neurons have several characteristics that are different from intact neurons. Here we show that, unlike established cultures, the axotomized sympathetic neurons deprived of NGF become committed to die before caspase activation, since the same proportion of NGF-deprived neurons are rescued by NGF regardless of whether caspases are inhibited by the pan-caspase inhibitor Boc-Asp(O-methyl)-CH(2)F (BAF). Despite prolonged Akt and ERK signaling induced by NGF after BAF treatment has prevented death, the neurons fail to increase protein synthesis, recover ATP levels, or grow. Within 3 d, all the mitochondria disappear without apparent removal of any other organelles or loss of membrane integrity. Although NGF does rescue intact BAF-treated 6-d cultures after NGF deprivation, rescue by NGF fails when these neurons are axotomized before NGF deprivation and BAF treatment. Moreover, cytosolic cytochrome c rapidly kills axotomized neurons. We propose that axotomy induces signals that make sympathetic neurons competent to die prematurely. NGF cannot repair these NGF-deprived, BAF-treated neurons because receptor signaling (which is normal) is uncoupled from protein renewal, and the mitochondria (which are damaged) go on to be eliminated. Hence, the order of steps underlying neuronal death commitment is mutable and open to regulation.

Xue L, Murray JH, Tolkovsky AM. 2000. The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons. J Biol Chem, 275 (12), pp. 8817-8824. | Show Abstract | Read more

Ras promotes robust survival of many cell systems by activating the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, but little is understood about the survival functions of the Ras/ERK pathway. We have used three different effector-loop mutant forms of Ras, each of which activates a single downstream effector pathway, to dissect their individual contributions to survival of nerve growth factor (NGF)-dependent sympathetic neurons. The PI3-kinase pathway-selective protein Ras(Val-12)Y40C was as powerful as oncogenic Ras(Val-12) in preventing apoptosis induced by NGF deprivation but conferred no protection against apoptosis induced by cytosine arabinoside. Identical results were obtained with transfected Akt. In contrast, the ERK pathway-selective protein Ras(Val-12)T35S had no protective effects on NGF-deprived neurons but was almost as strongly protective as Ras(Val-12) against cytosine arabinoside-induced apoptosis. The protective effects of Ras(Val-12)T35S against cytosine arabinoside were completely abolished by the ERK pathway inhibitor PD98059. Ras(Val-12)E37G, an activator of RalGDS, had no survival effect on either death pathway, similar to RasS17N, the full survival antagonist. Thus, Ras provides two independent survival pathways each of which inhibits a distinct apoptotic mechanism. Our study presents one of the few clear-cut cases where only the Ras/ERK, but not the Ras/PI3K/Akt pathway, plays a dominant survival signaling role.

Xue L, Fletcher GC, Tolkovsky AM. 1999. Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution. Mol Cell Neurosci, 14 (3), pp. 180-198. | Show Abstract | Read more

Autophagy is a mechanism whereby cells digest themselves from within and so may be used in lieu of apoptosis to execute cell death. Little is known about its role in neurons. In newly isolated sympathetic neurons, two independent apoptotic stimuli, NGF-deprivation or cytosine arabinoside added in the presence of NGF, caused a 30-fold increase in autophagic particle numbers, many autophagosomes appearing before any signs of DNA-fragmentation. The anti-autophagic drug 3-methyladenine also delayed apoptosis, its neuroprotection correlating with inhibition of cytochrome c release from mitochondria and prevention of caspase activation. In contrast, autophagic activity remained elevated in neurons treated with the pan-caspase inhibitor Boc-Asp(OMe)fmk, which inhibited morphological apoptosis but did not inhibit cytochrome c release nor prevent cell death. We propose that the same apoptotic signals that cause mitochondrial dysfunction also activate autophagy. Once activated, autophagy may mediate caspase-independent neuronal death.

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Virdee K, Xue L, Hemmings BA, Goemans C, Heumann R, Tolkovsky AM. 1999. Nerve growth factor-induced PKB/Akt activity is sustained by phosphoinositide 3-kinase dependent and independent signals in sympathetic neurons. Brain Res, 837 (1-2), pp. 127-142. | Show Abstract | Read more

Phosphoinositide 3-kinase and its downstream effector kinase PKB/Akt have been suggested to have crucial roles in suppressing apoptosis in several classes of neurons. However, few studies have conducted a long-term investigation of either kinase activity, many studies relying instead on use of the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. When we added LY294002 or wortmannin to sympathetic neurons, apoptosis in the presence of nerve growth factor (NGF) was very slow compared to that obtained by NGF deprivation. However, expression of a kinase-inactive mutant of PKB/Akt in the presence of NGF induced apoptosis in a significant proportion of the neurons. To understand this discrepancy, we investigated more closely the regulation of PKB/Akt activity by NGF. NGF stimulation induced a rapid increase in PKB/Akt activity which was sustained at approximately 6-fold up to 24 h. Phosphoinositide 3-kinase was also rapidly activated by NGF. However, concentrations of wortmannin which completely blocked phosphoinositide 3-kinase activity in the neurons inhibited no more than 50-70% of cellular PKB/Akt activity. Similarly, approximately 50% of maximal NGF-stimulated PKB/Akt activity remained elevated at concentrations of LY294002 which completely blocked neurite outgrowth, a process known to be phosphoinositide 3-kinase dependent. We suggest that a proportion of the sustained PKB/Akt activity induced by NGF is mediated by phosphoinositide 3-kinase-independent pathways. These results raise a cautionary note as to the usefulness of LY294002 or wortmannin as tools to dissect the role of PKB/Akt in neuronal survival.

Anderson CNG, Sampson CBP, Virdee K, Xue L, Tolkovsky AM. 1999. Use of mutlitple signalling pathways is crucial for suppressing neuronal apoptosis. JOURNAL OF NEUROCHEMISTRY, 73 pp. S108-S108.

Xue L, Lucocq J. 1998. ERK2 signalling from internalised epidermal growth factor receptor in broken A431 cells. Cell Signal, 10 (5), pp. 339-348. | Show Abstract | Read more

Activation of extracellular signal-regulated kinase 2 (ERK2) signalling from epidermal growth factor receptors (EGFRs) is widely assumed to originate in the plasma membrane. Using an in vitro assay, we investigated whether EGF/EGFR complexes internalised by endocytosis in A431 cells can initiate signalling in the ERK2 pathway. At 0 degrees C, binding of EGF induced tyrosine phosphorlyation of EGFR and, when the cells were subsequently broken by scraping and warmed in the presence of exogenous cytosol, activation of ERK2 occurred. At 0 degrees C, washes with pH 4.5 media reversed EGF binding, tyrosine phosphorylation and ERK-2 activation in exogenous cytosol, providing conditions in which signalling from the cell surface and internalised EGFRs could be distinguished. When cells containing internalised EGF/EGFR complexes were first washed in low pH media at 0 degrees C and then broken and incubated in exogenous cytosol, substantial activation of ERK2 occurred. This activation reached a maximum after a 5-min internalisation and was almost completely prevented by incubation in 0.45 M sucrose, a known inhibitor of receptor-mediated endocytosis. These data are consistent with activation of the ERK2 signalling pathway by internalised EGRFs situated in endosomal compartments. Our observation that EGFR tyrosine dephosphorylation is incomplete above pH 5.5 suggests that signalling is initiated in early endosomes.

Xue L, Lucocq JM. 1997. Low extracellular pH induces activation of ERK 2, JNK, and p38 in A431 and Swiss 3T3 cells. Biochem Biophys Res Commun, 241 (2), pp. 236-242. | Show Abstract | Read more

The mechanism by which mammalian cells respond to low environmental pH is unclear. A wide range of environmental stresses are known to induce activation of MAP kinases ERK 2, JNK and p38 and recent work has shown that low pH can activate the p38 homologue in yeast HOG1. In this study we show that ERK2 MAP kinase is activated in human A431 cells exposed to low pH media. Activation is sustained throughout low pH treatment, is reversible, and occurs maximally at pH 4 or 5. Stimulation is not accompanied by tyrosine phosphorylation of the EGF receptor or Raf-1 activation, indicating that acid conditions act via pathways independendent of those required for EGF mediated MAPK stimulation. The MAP kinase homologue JNK and MAPKAP kinase-2 reactivating kinase (p38) were also activated in A431 cells by low pH and so low pH induces parallel activation of multiple MAP kinase pathways. Strong activation of p42, and p44 ERKs as well as p38 and JNK was also found in mouse Swiss 3T3 cells treated at pH 5. These results indicate that MAP kinases may be important markers of the acid induced cellular stress that occurs in human disease.

Christie KN, Thomson C, Xue L, Lucocq JM, Hopwood D. 1997. Carbonic anhydrase isoenzymes I, II, III, and IV are present in human esophageal epithelium. J Histochem Cytochem, 45 (1), pp. 35-40. | Show Abstract | Read more

Carbonic anhydrase (CA) isoenzymes have been widely studied in the gastrointestinal tract, where they mediate membrane transport events and pH regulation. However, the esophagus has generally received scant attention. In an immunohistochemical study confirmed by Western blotting, we have detected for CA isoenzymes (CAI, II, III, and IV) in the epithelium of human esophagus. Isoenzymes I, III, and sometimes IV (< 10%) were present in the cytoplasm of basal cells and II and IV in the cytoplasm and cell surface membranes, respectively, of suprabasal cells (prickle cells). The localization of CAIV to the plasma membranes was confirmed by electron microscopic immunocytochemistry. CA was effectively divided at the basal-suprabasal interface between low-activity CAI and III (basal) and high-activity CAII and IV (suprabasal). Carbonic anhydrase in esophageal epithelial cells may have several functions: elimination of CO2 and metabolites, participation in membrane transport events during active cell growth, and pH regulation as a protective mechanism against acidic gastric reflux.

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Odorizzi CG, Trowbridge IS, Xue L, Hopkins CR, Davis CD, Collawn JF. 1994. Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment. J Cell Biol, 126 (2), pp. 317-330. | Show Abstract | Read more

Targeting of MHC class II molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain (Ii). By analysis of Ii-transferrin receptor (TR) chimera trafficking, we have identified sorting signals in the Ii cytoplasmic tail and transmembrane region that mediate this process. Two non-tyrosine-based sorting signals in the Ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrin-coated pits and promote rapid endocytosis. Leu7 and Ile8 were required for the activity of the signal most distal to the cell membrane whereas Pro15 Met16 Leu17 were important for the membrane-proximal signal. The same or overlapping non-tyrosine-based sorting signals are essential for delivery of Ii-TR chimeras, either by an intracellular route or via the plasma membrane, to an endocytic compartment where they are rapidly degraded. The Ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the Ii cytoplasmic tail. More than 80% of the Ii-TR chimera containing the Ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the Ii sorting signals are efficiently recognized by sorting machinery located in the trans-Golgi.

XUE L, DALLAI R, YIN WY. 1993. TRACHEAL ULTRASTRUCTURE OF PROTURA (INSECTA, APTERYGOTA) INTERNATIONAL JOURNAL OF INSECT MORPHOLOGY & EMBRYOLOGY, 22 (1), pp. 13-23. | Show Abstract | Read more

The tracheal systems of Sinentomon and Eosentomon (Apterygota : Protura) were examined in thin sections and compared with the tracheae of collembolan, Allacma. The tracheal system of Protura consists of spiracles and tracheae. The spiracle is a simple, concave cuticular cavity known as an atrium. A globular chamber is present between the atrium and trachea. The atrium of Eosentomon is decorated with ridges and has 2 small openings to tracheal recesses beside the central tracheal opening. The tracheae of Protura are characterized by a high frequency of taenidia and the absence of intima folds and intertaenidial spaces. The taenidia of Sinentomon have a rectangular section and those of Eosentomon are gable-shaped. The results also suggest that the tracheal recess of Eosentomon is a kind of stigmatic gland. The tracheal structure of Protura was compared with that of collembolans, insects, and other arthropods, and discussed in terms of phylogeny. © 1993.

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WOS

DALLAI R, XUE L, YIN W. 1992. FLAGELLATE SPERMATOZOA OF PROTURA (INSECTA, APTERYGOTA) ARE MOTILE INTERNATIONAL JOURNAL OF INSECT MORPHOLOGY & EMBRYOLOGY, 21 (2), pp. 137-148. | Show Abstract | Read more

A new model of sperm axoneme with 16 + 0 doublets is described. The spermatozoon of Acerentulus confinis (Apterygota : Protura) has a short conical acrosome, a long helicoidal nucleus, well-developed centriolar adjunct material, and a long flagellum. Using fixation with a glutaraldehyde-tannic acid mixture, without osmium post-fixation, doublet protofilaments, inner dynein arms, radial spokes, nexin bridges, and Y-links of the sperm axoneme of A. confinis and Acerentomon italicum were clearly observed. Optical observation shows that the proturan flagellate spermatozoa are motile cells. The process involving the transformation of the spermatozoa from a coiled to an elongated swimming form was studied by scanning electron microscope. The findings confirmed that flagellar motility is due to the presence of a single dynein arm on doublets in spite of the unusual axonemal pattern. © 1992.

Xué L, Romano D. 1992. Cell junctions in the gut of Protura. Tissue Cell, 24 (1), pp. 51-59. | Show Abstract | Read more

The main cell junctions in the intestinal tract of a small group of apterygotan insects, Protura, were examined in conventional thin sections, tracer-infiltrated sections and freeze-fracture replicas. The smooth septate junctions in the midgut of collembolan Tomocerus minor were also studied for comparison. Pleated septate junctions are found in foregut, hindgut and Malpighian papillae. They exhibit regular septa crossing the intercellular clefts in thin sections; and the septa with a pronounced zig-zag appearance run parallel to form a honeycomb structure in tracer-impregnated sections. After freeze-fracture undulating rows of intramembranous particles (IMPs) are visible on the P face. Smooth septate junctions are observed in the midgut. The intercellular septa often run in pairs for long tracts and exhibit a wavy course in lanthanum impregnated sections. The IMPs exhibited on the E face are usually separated one from another. Twin arrangement of particle rows is also apparent on the replicas. Gap junctions are frequent in both the midgut and hindgut and possess the conventional characteristics of 'inverted gap junction' with E face connexons. These results provide further evidence relating Protura closely to Collembola as well as to primitive arthropods.

XUE L, DALLAI R, YIN WY. 1990. FINE-STRUCTURE OF THE MIDGUT AND HINDGUT IN LEPIDOCAMPA-WEBERI (INSECTA, DIPLURA) ACTA ZOOLOGICA, 71 (4), pp. 201-209. | Show Abstract | Read more

The fine structure of the alimentary canal, especially the midgut and hindgut of Lepidocampa weberi (Diplura: Campodeidae) is described. The general organization of the canal is similar to that of Campodea. The midgut epithelium is composed of columnar apical microvillated cells. Each nucleus contains a single intranuclear crystal. Close to the pyloric region, the posterior midgut cells are devoid of microvilli and intranuclear crystals. There is no special pyloric chamber as in Protura or pyloric cuticular ring as in Collembola but a morphological transformation from midgut to hindgut cells. Eight globular Malpighian papillae, consisting of distal microvillated cells and flat proximal cells, open into the gut lumen via ducts formed by hindgut cells. The structure of the hindgut is complicated and can be divided into three segments. The anterior hindgut cells have an irregular shape and compact cytoplasm. A striking interdigitation between the large bottle‐shaped epithelial cells and longitudinal muscle cells occurs in the middle segment of the hindgut. The thick cuticle gives rise to long spikes projecting into the gut lumen. The posterior hindgut cells possess the morphological features for water reabsorption. Some hypotheses are advanced about the function of the different regions of the gut. 1990 The Royal Swedish Academy of Sciences

DALLAI R, XUE L, YIN WY. 1990. AFLAGELLATED SPERMATOZOA OF HUHENTOMON AND ACERELLA (PROTURA, APTERYGOTA) INTERNATIONAL JOURNAL OF INSECT MORPHOLOGY & EMBRYOLOGY, 19 (3-4), pp. 211-217. | Show Abstract | Read more

The spermatozoa of Huhentomon plicatunguis (Protura : Hesperentomidae) and Acerella tiarnea (Protura : Acerellidae) are aflagellate and of simple structure. An ovoidal nucleus with condensed chromatin is located at the end of the cell; the cytoplasm is devoid of organelles, except for mitochondria, and contains compact material derived from centriolar adjunct-like material. Centriole and mitochondrial cristae degenerate during spermiogenesis. These results show that the evolutionary trends of proturan spermatozoa tend towards aflagellate state. © 1990.

DALLAI R, YIN WY, XUE L, FRANCOIS J. 1989. THE GUT STRUCTURE OF SINENTOMON-ERYTHANUM YIN (PROTURA, SINENTOMIDAE) INTERNATIONAL JOURNAL OF INSECT MORPHOLOGY & EMBRYOLOGY, 18 (2-3), pp. 173-184. | Show Abstract | Read more

The fine structure of the midgut, pyloric region, Malpighian papillae, and hindgut of Sinentomon erythranum (Protura : Sinentomidae) is described. Midgut cells are rich in mineral concretions and are presumably involved in excretory activity; the pyloric chamber, a cavity in the proturan intestine behind the midgut, is formed by cells with microvilli pointing anteriorly; the secretion from 6 Malpighian papillae flows into this cavity. The hindgut consists of 2 regions; the anterior of the 2 has a series of specializations typical of cells engaged in active water reabsorption. Long infoldings of the apical plasma membrane reach deep into the cells. The findings are compared with the gut organization of other genera of Protura examined to date. © 1989.

Yin W-Y, Yang Y, Xue L, Dallai R. 1985. A “13+0” axonemal pattern in the spermatozoon ofNeocondeellum dolichotarsum (Insecta, Protura) Journal of Ultrastructure Research, 93 (3), pp. 179-185. | Show Abstract | Read more

The new 13+0 axonemal model has been described in the proturan Neocondeellum. All the doublets are devoid of dynein arms and the spermatozoa are immotile. A protruded acrosome is evident in the mature coiled spermatozoa. On the basis of the spermatological results Protentomidae seem closely related to Acerentomidae. © 1985.

Hilvering B, Hinks TSC, Stöger L, Marchi E, Salimi M, Shrimanker R, Liu W, Chen W, Luo J, Go S et al. 2018. Synergistic activation of pro-inflammatory type-2 CD8+ T lymphocytes by lipid mediators in severe eosinophilic asthma. Mucosal Immunol, 11 (5), pp. 1408-1419. | Show Abstract | Read more

Human type-2 CD8+ T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia-a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

Salimi M, Stöger L, Liu W, Go S, Pavord I, Klenerman P, Ogg G, Xue L. 2017. Cysteinyl leukotriene E4 activates human group 2 innate lymphoid cells and enhances the effect of prostaglandin D2 and epithelial cytokines. J Allergy Clin Immunol, 140 (4), pp. 1090-1100.e11. | Show Abstract | Read more

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are a potential innate source of type 2 cytokines in the pathogenesis of allergic conditions. Epithelial cytokines (IL-33, IL-25, and thymic stromal lymphopoietin [TSLP]) and mast cell mediators (prostaglandin D2 [PGD2]) are critical activators of ILC2s. Cysteinyl leukotrienes (cysLTs), including leukotriene (LT) C4, LTD4, and LTE4, are metabolites of arachidonic acid and mediate inflammatory responses. Their role in human ILC2s is still poorly understood. OBJECTIVES: We sought to determine the role of cysLTs and their relationship with other ILC2 stimulators in the activation of human ILC2s. METHODS: For ex vivo studies, fresh blood from patients with atopic dermatitis and healthy control subjects was analyzed with flow cytometry. For in vitro studies, ILC2s were isolated and cultured. The effects of cysLTs, PGD2, IL-33, IL-25, TSLP, and IL-2 alone or in combination on ILC2s were defined by using chemotaxis, apoptosis, ELISA, Luminex, quantitative RT-PCR, and flow cytometric assays. The effect of endogenous cysLTs was assessed by using human mast cell supernatants. RESULTS: Human ILC2s expressed the LT receptor CysLT1, levels of which were increased in atopic subjects. CysLTs, particularly LTE4, induced migration, reduced apoptosis, and promoted cytokine production in human ILC2s in vitro. LTE4 enhanced the effect of PGD2, IL-25, IL-33, and TSLP, resulting in increased production of type 2 and other proinflammatory cytokines. The effect of LTE4 was inhibited by montelukast, a CysLT1 antagonist. Interestingly, addition of IL-2 to LTE4 and epithelial cytokines significantly amplified ILC2 activation and upregulated expression of the receptors for IL-33 and IL-25. CONCLUSION: CysLTs, particularly LTE4, are important contributors to the triggering of human ILC2s in inflammatory responses, particularly when combined with other ILC2 activators.

Cheung KL, Jarrett R, Subramaniam S, Salimi M, Gutowska-Owsiak D, Chen Y-L, Hardman C, Xue L, Cerundolo V, Ogg G. 2016. Psoriatic T cells recognize neolipid antigens generated by mast cell phospholipase delivered by exosomes and presented by CD1a. J Exp Med, 213 (11), pp. 2399-2412. | Show Abstract | Read more

Psoriasis is a chronic inflammatory skin disease associated with a T helper 17 response. Yet, it has proved challenging to identify relevant peptide-based T cell antigens. Antigen-presenting Langerhans cells show a differential migration phenotype in psoriatic lesions and express constitutively high levels of CD1a, which presents lipid antigens to T cells. In addition, phospholipase A2 (PLA2) is highly expressed in psoriatic lesions and is known to generate neolipid skin antigens for recognition by CD1a-reactive T cells. In this study, we observed expression of a cytoplasmic PLA2 (PLA2G4D) in psoriatic mast cells but, unexpectedly, also found PLA2G4D activity to be extracellular. This was explained by IFN-α-induced mast cell release of exosomes, which transferred cytoplasmic PLA2 activity to neighboring CD1a-expressing cells. This led to the generation of neolipid antigens and subsequent recognition by lipid-specific CD1a-reactive T cells inducing production of IL-22 and IL-17A. Circulating and skin-derived T cells from patients with psoriasis showed elevated PLA2G4D responsiveness compared with healthy controls. Overall, these data present an alternative model of psoriasis pathogenesis in which lipid-specific CD1a-reactive T cells contribute to psoriatic inflammation. The findings suggest that PLA2 inhibition or CD1a blockade may have therapeutic potential for psoriasis.

Hilvering B, Xue L, Pavord ID. 2015. IL-33-dependent Th2 response after rhinovirus infection in asthma: more information needed. Am J Respir Crit Care Med, 191 (2), pp. 237. | Read more

Cited:

20

Scopus

Xue L, Fergusson J, Salimi M, Panse I, Ussher JE, Hegazy AN, Vinall SL, Jackson DG, Hunter MG, Pettipher R et al. 2015. Prostaglandin D<inf>2</inf> and leukotriene E<inf>4</inf> synergize to stimulate diverse T<inf>H</inf>2 functions and T<inf>H</inf>2 cell/neutrophil crosstalk Journal of Allergy and Clinical Immunology, 135 (5), pp. 1358-1366e11. | Show Abstract | Read more

© 2015 American Academy of Allergy, Asthma & Immunology. Background Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. Objective We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. Methods The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. Results PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. Conclusions PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Xue L, Fergusson J, Salimi M, Panse I, Ussher JE, Hegazy AN, Vinall SL, Jackson DG, Hunter MG, Pettipher R et al. 2015. Prostaglandin D2 and leukotriene E4 synergize to stimulate diverse TH2 functions and TH2 cell/neutrophil crosstalk. J Allergy Clin Immunol, 135 (5), pp. 1358-66.e1-11. | Show Abstract | Read more

BACKGROUND: Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. OBJECTIVE: We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. METHODS: The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. RESULTS: PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. CONCLUSIONS: PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Salimi M, Barlow J, Saunders S, Xue L, Gutowska-Owsiak D, Wang X, Huang L-C, Johnson D, Scanlon S, McKenzie A et al. 2014. The role of type 2 innate lymphoid cells in the pathogenesis of atopic dermatitis BRITISH JOURNAL OF DERMATOLOGY, 170 (4), pp. E32-E32.

Xue L, Salimi M, Panse I, Mjösberg JM, McKenzie ANJ, Spits H, Klenerman P, Ogg G. 2014. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells. J Allergy Clin Immunol, 133 (4), pp. 1184-1194. | Show Abstract | Read more

BACKGROUND: Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2), a receptor for prostaglandin D₂ (PGD₂), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear. OBJECTIVES: We sought to determine the role of PGD₂ and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33. METHODS: The effects of PGD₂, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD₂ under physiologic conditions were evaluated by using the supernatant from activated mast cells. RESULTS: PGD₂ binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD₂ on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. CONCLUSIONS: PGD₂ is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s.

Salimi M, Barlow JL, Saunders SP, Xue L, Gutowska-Owsiak D, Wang X, Huang L-C, Johnson D, Scanlon ST, McKenzie ANJ et al. 2013. A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. J Exp Med, 210 (13), pp. 2939-2950. | Show Abstract | Read more

Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease.

Schröder R, Xue L, Konya V, Martini L, Kampitsch N, Whistler JL, Ulven T, Heinemann A, Pettipher R, Kostenis E. 2012. PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor CRTH2/DP2. PLoS One, 7 (3), pp. e33329. | Show Abstract | Read more

Prostaglandin H(1) (PGH(1)) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as "anti-inflammatory". Herein we present evidence that PGH(1) is a potent activator of the pro-inflammatory PGD(2) receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca(2+) flux studies reveal that PGH(1) activates CRTH2 as PGH(2), PGD(2) or PGD(1) do. The PGH(1) precursor DGLA and the other PGH(1) metabolites did not display such effect. PGH(1) specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH(1) is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH(1) mediates migration of and Ca(2+) flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH(1) as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase.

Xue L, Barrow A, Fleming VM, Hunter MG, Ogg G, Klenerman P, Pettipher R. 2012. Leukotriene E4 activates human Th2 cells for exaggerated proinflammatory cytokine production in response to prostaglandin D2. J Immunol, 188 (2), pp. 694-702. | Show Abstract | Read more

PGD(2) exerts a number of proinflammatory responses through a high-affinity interaction with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and has been detected at high concentrations at sites of allergic inflammation. Because cysteinyl leukotrienes (cysLTs) are also produced during the allergic response, we investigated the possibility that cysLTs may modulate the response of human Th2 cells to PGD(2). PGD(2) induced concentration-dependent Th2 cytokine production in the absence of TCR stimulation. Leukotrienes D(4) and E(4) (LTE(4)) also stimulated the cytokine production but were much less active than PGD(2). However, when combined with PGD(2), cysLTs caused a greater than additive enhancement of the response, with LTE(4) being most effective in activating Th2 cells. LTE(4) enhanced calcium mobilization in response to PGD(2) in Th2 cells without affecting endogenous PGD(2) production or CRTH2 receptor expression. The effect of LTE(4) was inhibited by montelukast but not by the P2Y(12) antagonist methylthioadenosine 5'-monophosphate. The enhancing effect was also evident with endogenous cysLTs produced from immunologically activated mast cells because inhibition of cysLT action by montelukast or cysLT synthesis by MK886, an inhibitor of 5-lipoxygenase-activating protein, reduced the response of Th2 cells to the levels produced by PGD(2) alone. These findings reveal that cysLTs, in particular LTE(4), have a significant proinflammatory impact on T cells and demonstrate their effects on Th2 cells are mediated by a montelukast-sensitive receptor.

Pettipher R, Vinall SL, Xue L, Speight G, Townsend ER, Gazi L, Whelan CJ, Armer RE, Payton MA, Hunter MG. 2012. Pharmacologic profile of OC000459, a potent, selective, and orally active D prostanoid receptor 2 antagonist that inhibits mast cell-dependent activation of T helper 2 lymphocytes and eosinophils. J Pharmacol Exp Ther, 340 (2), pp. 473-482. | Show Abstract | Read more

D prostanoid receptor 2 (DP₂) [also known as chemoattractant receptor-homologous molecule expressed on T helper 2 (Th2) cells (CRTH2)] is selectively expressed by Th2 lymphocytes, eosinophils, and basophils and mediates recruitment and activation of these cell types in response to prostaglandin D₂ (PGD₂). (5-Fluoro-2-methyl-3-quinolin-2-ylmethylindo-1-yl)-acetic acid (OC000459) is an indole-acetic acid derivative that potently displaces [³H]PGD₂ from human recombinant DP₂ (K(i) = 0.013 μM), rat recombinant DP₂ (K(i) = 0.003 μM), and human native DP₂ (Th2 cell membranes; K(i) = 0.004 μM) but does not interfere with the ligand binding properties or functional activities of other prostanoid receptors (prostaglandin E₁₋₄ receptors, D prostanoid receptor 1, thromboxane receptor, prostacyclin receptor, and prostaglandin F receptor). OC000459 inhibited chemotaxis (IC₅₀ = 0.028 μM) of human Th2 lymphocytes and cytokine production (IC₅₀ = 0.019 μM) by human Th2 lymphocytes. OC000459 competitively antagonized eosinophil shape change responses induced by PGD₂ in both isolated human leukocytes (pK(B) = 7.9) and human whole blood (pK(B) = 7.5) but did not inhibit responses to eotaxin, 5-oxo-eicosatetraenoic acid, or complement component C5a. OC000459 also inhibited the activation of Th2 cells and eosinophils in response to supernatants from IgE/anti-IgE-activated human mast cells. OC000459 had no significant inhibitory activity on a battery of 69 receptors and 19 enzymes including cyclooxygenase 1 (COX1) and COX2. OC000459 was found to be orally bioavailable in rats and effective in inhibiting blood eosinophilia induced by 13,14-dihydro-15-keto-PGD₂ (DK-PGD₂) in this species (ED₅₀ = 0.04 mg/kg p.o.) and airway eosinophilia in response to an aerosol of DK-PGD₂ in guinea pigs (ED₅₀ = 0.01 mg/kg p.o.). These data indicate that OC000459 is a potent, selective, and orally active DP₂ antagonist that retains activity in human whole blood and inhibits mast cell-dependent activation of both human Th2 lymphocytes and eosinophils.

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