Elizabeth A. Mellor

Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells

The cysteinyl leukotrienes (cys-LTs) LTC4, LTD4, and LTE4 are a class of peptide-conjugated lipids formed from arachidonic acid and released during activation of mast cells (MCs). We now report that human cord-blood-derived MCs (hMCs) express the CysLT1 receptor, which responds not only to inflammation-derived cys-LTs, but also to a pyrimidinergic ligand, UDP. hMCs express both CysLT1 protein and transcript, and respond to LTC4, LTD4, and UDP with concentration-dependent calcium fluxes, each of which is blocked by a competitive CysLT1 receptor antagonist, MK571. Stably transfected Chinese hamster ovary cells expressing the CysLT1 receptor also exhibit MK571-sensitive calcium flux to all three agonists. Both hMCs and CysLT1 transfectants stimulated with UDP are desensitized to LTC4, but only partially to LTD4. Priming of hMCs with IL-4 for 5 days enhances their sensitivity to each agonist, but preferentially lowers their threshold for activation by LTC4 and UDP (≈3 log10-fold shifts in dose-response for each agonist) over LTD4 (1.3 log10-fold shift), without altering CysLT1 receptor mRNA or surface protein expression, implying the likely induction of a second receptor with CysLT1-like dual ligand specificity. hMCs thus express the CysLT1 receptor, and possibly a closely related IL-4-inducible receptor, which mediate dual activation responses to cys-LTs and UDP, providing an apparent intersection linking the inflammatory and neurogenic elements of bronchial asthma.

Cysteinyl Leukotrienes and Uridine Diphosphate Induce Cytokine Generation by Human Mast Cells Through an Interleukin 4–regulated Pathway that Is Inhibited by Leukotriene Receptor Antagonists

We previously reported that interleukin (IL)-4 upregulates the expression of leukotriene C4 synthase (LTC4S) by human cord blood–derived mast cells (hMCs), augments their high-affinity Fc receptor for IgE (FcεRI)-dependent generation of eicosanoids and cytokines, and induces a calcium flux in response to cysteinyl leukotrienes (cys-LTs) and uridine diphosphate (UDP) that is blocked by cys-LT receptor antagonists. We speculated that this IL-4–dependent, receptor-mediated response to the cys-LTs and UDP might induce cytokine generation by hMCs without concomitant exocytosis. Unlike hMCs maintained in cytoprotective stem cell factor (SCF) alone, hMCs primed for 5 d with IL-4 responded to UDP (1 μM), LTC4 (100 nM), and LTD4 (100 nM) by producing IL-5, tumor necrosis factor (TNF)-α, and especially large quantities of macrophage inflammatory protein (MIP)-1β de novo at 6 h, preceded by the induced expression of the corresponding mRNAs. Cys-LT– and UDP-mediated cytokine production by the primed hMCs occurred without histamine release or PGD2 generation and was inhibited by the CysLT1 receptor antagonist MK571. Additionally, pretreatment of hMCs with MK571 or with the cys-LT biosynthetic inhibitor MK886 decreased IL-5 and TNF-α production in response to IgE receptor cross-linkage, implying a positive feedback by endogenously produced cys-LTs. Cys-LTs and UDP thus orchestrate a novel, IL-4–regulated, non-IgE–dependent hMC activation for cytokine gene induction that could be initiated by microbes, cellular injury, or neurogenic or inflammatory signals; and this pathobiologic event would not be recognized in tissue studies where hMC activation is classically defined by exocytosis.

Expression of the type 2 receptor for cysteinyl leukotrienes (CysLT2R) by human mast cells: Functional distinction from CysLT1R

Cysteinyl leukotrienes (cysLTs) mediate vascular leakage and bronchoconstriction through the smooth muscle-associated CysLT type 1 receptor (CysLT1R), one of at least two loosely homologous cysLT-binding G protein-coupled receptors. We previously reported that CysLT1R is expressed by cultured human mast cells (hMCs), and that priming these cells with IL-4 enhances their sensitivity to calcium flux and cytokine generation in response to cys-LTs and the nucleotide ligand, uridine diphosphate (UDP), without increasing their surface expression of CysLT1R. We now report that hMCs express the type 2 receptor for cysLTs (CysLT2R) as well, and that the amount of surface CysLT2R protein increases in response to priming with IL-4. The selective function of CysLT2R was evident based on uninhibited IL-8 secretion by IL-4-primed hMCs stimulated with cys-LTs or UDP in the presence of the selective CysLT1R antagonist MK571. MK571 did inhibit IL-5 generation, calcium flux, and phosphorylation of extracellular signal-regulated kinase. IL-8 secretion was inhibited by pertussis toxin and a selective p38 kinase inhibitor, SB203580. The CysLT2 response may permit the cys-LTs and nucleotides generated in infection and tissue injury to elicit IL-8 generation by hMCs, potentially leading to neutrophilic infiltration, a characteristic of aerosol challenge-induced late-phase responses and of sudden death associated with asthma.

Biochemical and functional characterization of human transmembrane tryptase (TMT)/tryptase γ: TMT is an exocytosed mast cell protease that induces airway hyperresponsiveness in vivo via an interleukin-13/interleukin-4 receptor α/signal transducer and activator of transcription (STAT) 6-dependent pathway

Transmembrane tryptase (TMT)/tryptase γ is a membrane-bound serine protease stored in the secretory granules of human and mouse lung mast cells (MCs). We now show that TMT reaches the external face of the plasma membrane when MCs are induced to degranulate. Analysis of purified recombinant TMT revealed that it is a two-chain neutral protease. Thus, TMT is the only MC protease identified so far which retains its 18-residue propeptide when proteolytically activated. The genes that encode TMT and tryptase βI reside on human chromosome 16p13.3. However, substrate specificity studies revealed that TMT and tryptase βI are functionally distinct even though they are ∼50% identical. Although TMT is rapidly inactivated by the human plasma serpin α1-antitrypsin in vitro, administration of recombinant TMT (but not recombinant tryptase βI) into the trachea of mice leads to airway hyperresponsiveness (AHR) and increased expression of interleukin (IL) 13. T cells also increase their expression of IL-13 mRNA when exposed to TMT in vitro. TMT is therefore a novel exocytosed surface mediator that can stimulate those cell types that are in close proximity. TMT induces AHR in normal mice but not in transgenic mice that lack signal transducer and activator of transcription (STAT) 6 or the α-chain of the cytokine receptor that recognizes both IL-4 and IL-13. Based on these data, we conclude that TMT is an exocytosed MC neutral protease that induces AHR in lungs primarily by activating an IL-13/IL-4Rα/STAT6-dependent pathway.

Biochemical and functional characterization of human transmembrane tryptase (TMT)/tryptase γ. TMT is an exocytosed mast cell protease that induces airway hyperresponsiveness in vivo via an IL-13/IL-4Rα/STAT6-dependent pathway

Transmembrane tryptase (TMT)/tryptase γ is a membrane-bound serine protease stored in the secretory granules of human and mouse lung mast cells (MCs). We now show that TMT reaches the external face of the plasma membrane when MCs are induced to degranulate. Analysis of purified recombinant TMT revealed that it is a two-chain neutral protease. Thus, TMT is the only MC protease identified so far which retains its 18-residue propeptide when proteolytically activated. The genes that encode TMT and tryptase βI reside on human chromosome 16p13.3. However, substrate specificity studies revealed that TMT and tryptase βI are functionally distinct even though they are ∼50% identical. Although TMT is rapidly inactivated by the human plasma serpin α1-antitrypsin in vitro, administration of recombinant TMT (but not recombinant tryptase βI) into the trachea of mice leads to airway hyperresponsiveness (AHR) and increased expression of interleukin (IL) 13. T cells also increase their expression of IL-13 mRNA when exposed to TMT in vitro. TMT is therefore a novel exocytosed surface mediator that can stimulate those cell types that are in close proximity. TMT induces AHR in normal mice but not in transgenic mice that lack signal transducer and activator of transcription (STAT) 6 or the α-chain of the cytokine receptor that recognizes both IL-4 and IL-13. Based on these data, we conclude that TMT is an exocytosed MC neutral protease that induces AHR in lungs primarily by activating an IL-13/IL-4Rα/STAT6-dependent pathway.

Biochemical and Functional Characterization of Human Transmembrane Tryptase(TMT)Ttryptase g.

Transmembrane tryptase (TMT)/tryptase γ is a membrane-bound serine protease stored in the secretory granules of human and mouse lung mast cells (MCs). We now show that TMT reaches the external face of the plasma membrane when MCs are induced to degranulate. Analysis of purified recombinant TMT revealed that it is a two-chain neutral protease. Thus, TMT is the only MC protease identified so far which retains its 18-residue propeptide when proteolytically activated. The genes that encode TMT and tryptase βI reside on human chromosome 16p13.3. However, substrate specificity studies revealed that TMT and tryptase βI are functionally distinct even though they are ∼50% identical. Although TMT is rapidly inactivated by the human plasma serpin α1-antitrypsin in vitro, administration of recombinant TMT (but not recombinant tryptase βI) into the trachea of mice leads to airway hyperresponsiveness (AHR) and increased expression of interleukin (IL) 13. T cells also increase their expression of IL-13 mRNA when exposed to TMT in vitro. TMT is therefore a novel exocytosed surface mediator that can stimulate those cell types that are in close proximity. TMT induces AHR in normal mice but not in transgenic mice that lack signal transducer and activator of transcription (STAT) 6 or the α-chain of the cytokine receptor that recognizes both IL-4 and IL-13. Based on these data, we conclude that TMT is an exocytosed MC neutral protease that induces AHR in lungs primarily by activating an IL-13/IL-4Rα/STAT6-dependent pathway.