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High-affinity pan-specific monoclonal antibodies that target cysteinyl leukotrienes and show efficacy in an acute model of colitis.

ABSTRACT: Cysteinyl leukotrienes (CysLTs) are a small family of biological signaling lipids produced by active leukocytes that contribute to diverse inflammatory disease states as a consequence of their engagement with dedicated G protein-coupled receptors. Immunization of mice with a CysLT-modified hapten carrier protein yielded novel monoclonal antibodies that display variable binding affinity to CysLTs. Solution binding assays indicated differing specificities among the antibodies tested, with antibody 10G4 displaying a preference for leukotriene C4 (LTC4). X-ray crystallography of a humanized 10G4 Fab fragment in complex with LTC4 revealed that binding induces a hook-like conformation within the hydrocarbon tail of the lipid arachidonic acid moiety. Specific hydrogen bonding to the LTC4 carboxylate groups further stabilized the complex, while a water molecule mediated a hydrogen bond network that connected the N-terminal arm of l-glutathione to both the arachidonyl carboxylate of LTC4 and the antibody heavy chain. Prophylactic administration of two anti-CysLT antibodies in mice followed by challenge with LTC4 demonstrated their in vivo efficacy against acute inflammation in a vascular permeability model. 10G4 ameliorated the effects of acute dextran sulfate sodium-induced colitis, suggesting that anti-CysLT antibodies could provide a therapeutic benefit in the treatment of inflammatory diseases.

SUBMITTER: King AN

PROVIDER: S-EPMC5496036 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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High-affinity pan-specific monoclonal antibodies that target cysteinyl leukotrienes and show efficacy in an acute model of colitis.

King Ashlee N AN Fleming Jonathan K JK Knapik Stephanie S SS Visentin Barbara B Wojciak Jonathan M JM Huxford Tom T

Journal of lipid research 20170515 7

Cysteinyl leukotrienes (CysLTs) are a small family of biological signaling lipids produced by active leukocytes that contribute to diverse inflammatory disease states as a consequence of their engagement with dedicated G protein-coupled receptors. Immunization of mice with a CysLT-modified hapten carrier protein yielded novel monoclonal antibodies that display variable binding affinity to CysLTs. Solution binding assays indicated differing specificities among the antibodies tested, with antibody ...[more]

PMID: 28507038

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Project description:BackgroundCysteinyl leukotrienes (CysLTs) are potent prophlogistic mediators in asthmatic patients; however, inhibition of CysLT receptor 1 is not a consistently effective treatment, suggesting additional regulatory mechanisms. Other cysteinyl-containing lipid mediators (LMs) derived from docosahexaenoic acid, namely maresin conjugates in tissue regeneration (MCTRs), were recently discovered. Therefore their production and actions in the lung are of considerable interest.ObjectiveWe sought to determine MCTR production, bioactions, and mechanisms in the human lung and in patients with experimental allergic airway inflammation.MethodsLM metabololipidomic profiling of the lung was performed by using liquid chromatography with tandem mass spectrometry. Donor-derived human precision-cut lung slices were exposed to leukotriene (LT) D4, MCTRs, or both before determination of airway contraction. The actions of exogenous MCTRs on murine allergic host responses were determined in the setting of ovalbumin- and house dust mite-induced lung inflammation.ResultsLipidomic profiling showed that the most abundant cysteinyl LMs in healthy human lungs were MCTRs, whereas CysLTs were most prevalent in patients with disease. MCTRs blocked LTD4-initiated airway contraction in human precision-cut lung slices. In mouse allergic lung inflammation MCTRs were present with temporally regulated production. With ovalbumin-induced inflammation, MCTR1 was most potent for promoting resolution of eosinophils, and MCTR3 potently decreased airway hyperreactivity to methacholine, bronchoalveolar lavage fluid albumin, and serum IgE levels. MCTR1 and MCTR3 inhibited lung eosinophilia after house dust mite-induced inflammation.ConclusionThese results identified lung MCTRs that blocked human LTD4-induced airway contraction and promoted resolution of murine allergic airway responses when added exogenously. Together, these findings uncover proresolving mechanisms for lung responses that can be disrupted in patients with disease.

Project description:BackgroundPhospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined.ObjectivesTo examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils.MethodsPeripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT.ResultsEosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the expression of PLA2G10 by eosinophils.ConclusionsThese results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.

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High-affinity pan-specific monoclonal antibodies that target cysteinyl leukotrienes and show efficacy in an acute model of colitis.

Publications

High-affinity pan-specific monoclonal antibodies that target cysteinyl leukotrienes and show efficacy in an acute model of colitis.

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