2554 S. Mark Duffy et al. Eur. J. Immunol. 2008. 38: 2548–2556 other systems such as smooth muscle relaxation has been chal- lenged [25]. Opening of KCa3.1 enhances IgE-dependent Ca21 inﬂux and degranulation to a submaximal stimulus [8], and its blockade by charybdotoxin attenuates this [7]. Thus, while cAMP plays some role in the inhibition of mast cell mediator release, the demonstration that PGE2 also closes KCa3.1 supports the view that cAMP-independent, KCa3.1-dependent mechanisms also contribute in part to EP2-dependent inhibition of HLMC mediator release. In many diseases the recruitment of mast cells to key tissue structures appears critical for their pathophysiological effects [4, 26, 27]. For example, the contribution of mast cells to the disordered airway physiology of asthma is undoubtedly facilitated by their migration into the airway epithelium [3], submucosal glands [5] and ASM [4]. Inhibition of their migration and subsequent microlocalisation within these structures might therefore offer a novel approach to therapy. Blockade of KCa3.1 markedly inhibits HLMC migration in response to a number of diverse chemotactic stimuli including conditioned medium from activated asthmatic ASM [6]. The ability of PGE2 to close KCa3.1 suggested that it should also inhibit HLMC migration. However, its ability to inhibit HLMC migration in response to ASM-conditioned medium was variable and did not reach statistical signiﬁcance. In contrast, the selective EP2 receptor agonist butaprost, which was more potent at closing KCa3.1 than PGE2, produced a marked and consistent inhibition of HLMC migration. PGE2 was also chemotactic at high concen- trations when used alone, but was more potent as a chemoat- tractant when EP2 receptors were blocked. This indicates that there are competing pro-migratory and anti-migratory signals when PGE2 is present, mediated by the EP2 receptor (inhibitory) and most probably the EP3 receptor (pro-migratory). These ﬁndings are in marked contrast to those in mouse bone marrow- derived mast cells in which PGE2 in isolation is a potent chemoattractant. This chemotactic activity is mediated through the EP3 receptor, and the lack of inhibition can be attributed to the absence of EP2 receptors in mouse bone marrow-derived mast cells [13]. In human cord blood-derived mast cells, which express both EP2 and EP3 receptors, PGE2 is not a chemoattractant [13], compatible with an inhibitory role for the EP2 receptor. In human lung therefore, PGE2 is not likely to act as a HLMC chemoat- tractant, in keeping with its anti-inﬂammatory activities in this tissue [18]. In humans PGE2 inhibits the early and late asthmatic airway response to allergen challenge [18]. The ability of PGE2 to close KCa3.1 provides a mechanism through which it is able to achieve these effects. It will be of great interest to investigate whether PGE2 has similar effects on lung T-cell KCa3.1 function, cytokine secretion and migration, and whether it can inhibit KCa3.1- dependent ASM proliferation [28]. Drugs that block KCa3.1 are also in development as anti-inﬂammatory treatments [29]. Our demonstration that the EP2 receptor closes KCa3.1 in HLMC provides further support for the development of speciﬁc EP2 receptor agonists for the treatment of mast cell-mediated pulmonary disease. & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Materials and methods Reagents We used the following reagents: SCF, IL-6 and IL-10 (R&D, Abingdon, UK); goat polyclonal anti-human IgE, PGE2, AH6809, butaprost, PAF, LPA, UTP (Sigma, Poole, Dorset, UK); 17-phenyl trinor PGE2 (Cayman Chemical Company, Ann Arbor, Michigan, US); mouse IgG1 mAb YB5.B8 (anti-CD117) (Cambridge Bioscience, Cambridge, UK); sheep anti-mouse IgG1 Dynabeads (Dynal, Wirral, UK); Dulbecco’s Modiﬁed Essential Medium (DMEM)/glutamax/HEPES, antibiotic/antimycotic solution, MEM non-essential aminoacids, and fetal calf serum (Life Technologies, Paisley, Scotland, UK). Human mast cell puriﬁcation and culture All human subjects gave written informed consent, and the study was approved by the Leicestershire Research Ethics Committee. HLMC were dispersed and puriﬁed from macro- scopically normal lung (n 5 14 donors) obtained within 1 h of resection for lung cancer using immunomagnetic afﬁnity selec- tion as described previously [30]. Final mast cell purity determined by Kimura stain was 499% and viability determined by Trypan blue was 497%. HLMC were cultured in DMEM/ glutamax/HEPES containing antibiotic/antimycotic solution, non-essential amino acids, 10% fetal calf serum, 100 ng/mL SCF, 50 ng/mL IL-6, and 10 ng/mL IL-10 for up to 10 wk as described previously [8, 31]. Electrophysiology The whole-cell variant of the patch-clamp technique was used [7,32]. Patch pipettes were made from borosilicate ﬁbre-contain- ing glass (Clark Electromedical Instruments, Reading, UK), and their tips were heat polished, typically resulting in resistances of 4–6 MO. The standard pipette solution contained (in mM) KCl, 140; MgCl2, 2; HEPES, 10; Na1-ATP, 2; GTP, 0.1 (pH 7.3). The standard external solution contained (in mM) NaCl, 140; KCl, 5, CaCl2, 2; MgCl2,1; HEPES, 10 (pH 7.3). For recording, mast cells were placed in 35-mm dishes containing standard external solution. Whole-cell currents were recorded using an Axoclamp 200A ampliﬁer (Axon Instruments, Foster City, CA, USA), and currents were evoked by applying voltage commands to a range of potentials in 10 mV steps from a holding potential of 20 mV. The currents were digitised (sampled at a frequency of 10 kHz), stored on computer, and subsequently analysed using pClamp software (Axon Instruments). Capacitance transients were mini- mised using the capacitance neutralisation circuits on the ampliﬁer. Correction for series resistance was not routinely applied. Experiments were performed at 271C, and the tempera- ture controlled by a Peltier device. Experiments were performed www.eji-journal.eu