Introduction Protease activated receptors (PARs) are members of a newly discovered family of G-protein coupled receptors whose acti- vation is induced by proteolytic cleavage of an extracellular N-terminal domain, exposing a tethered ligand. So far, four receptors have been characterised (PAR-1, -2, -3, -4) which are differentially activated by various physiological factors such as thrombin (PAR-1, -3, -4), mast cell-derived tryptase (PAR-2) and trypsin (PAR-2 and PAR-4). It is thought that PARs may play a role in allergic inflammation since they reg- ulate airway smooth muscle mitogenesis and neuronal activ- ity, thus contributing to increased airway reactivity in asthma [1, 2]. Additionally, PARs may also be involved in the innate conditioning of the immune system, favouring a Th2-type response, which is central to the production of IgE antibod- ies and subsequent allergy. The house dust-mite allergens Der p1, Der p3 and Der p9 as well as various parasitic extracts have all been shown to activate PARs leading to Th2 responses and subsequent specific IgE production. More- over, basophils and KU812 basophilic cell lines release mediators to hookworm (Necator americanus) secretions, which are rich in proteases, as well as to Der p1, suggesting a role for PARs in modulating basophil activity [3]. Human basophils are an important early source of IL-4, which criti- cally augments the development of Th2 cells [4]. Given this, we asked whether basophils express PARs and release IL-4, as well as other inflammatory mediators such as histamine and IL-13, upon stimulation with various PAR agonists. Materials and methods Human basophils were obtained from buffy coats which were freshly prepared from healthy donors undergoing routine blood donation. Basophils were purified by Ficoll-density centrifugation followed by elutriation and negative selection using magnetic cell sorting (MACS) [5]. Purities ranged between 93% and 99%, as determined by alcian blue Inflamm. res. 54, Supplement 1 (2005) S 13– S14 1023-3830/05/01S13-02 DOI 10.1007/s00011-004-0405-y staining. Basophils were incubated at 37°C for 15 min before stimula- tion with various synthetic PAR agonists (TRAP-6 for PAR-1, H-Ser- Leu-Ile-Gly-Lys-Val-NH 2 for PAR-2 and H-Gly-Tyr-Pro-Gly-Lys-Phe- OH for PAR-4), anti-IgE (positive control) or buffer alone (negative control). Histamine secretions were assayed after 30 min stimulation by spectrofuorometric autoanalysis, cytokine release after 4 h stimulation using ELISA. For PAR expression at mRNA level, primers were designed using the Primer3 web utility (http://frodo.wi.mit.edu/cgi-bin/primer3/ primer3_www.cgi). All PAR primers are intron-spanning and specific for their target gene. Total RNA was extracted from 200,000 to 500,000 basophils from three blood donors with an average basophil purity of 98%. RNA was purified using the GenElute Mammalian total RNA Mini prep kit (Sigma), with an additional DNAse digestion step (20 min at RT). Success of DNAse treatment was monitored using primers spe- cific for intronic regions of the target genes. Reverse transcription was performed using Oligo-dT primers with the GeneAmp RT-PCR kit as directed. PCR was performed using ABgenes’ ReddyMix containin Taq polymerase on a MJ Research PTC200 thermal cycler using low profile PCR tubes. Human mononuclear cells and neutrophils served as posi- tive controls for expression of PAR-receptors and were purified as pre- viously described [6]. Results and discussion Functional assays revealed that none of the synthetic agonists of PAR-1, PAR-2 and PAR-4 employed significantly induced histamine, IL-4 or IL-13 release from basophils (Table 1), even though the cells were highly responsive to anti-IgE stimulation (used as a positive control). The actions of PAR- 3 activation could not be examined since there are no specif- ic agonists for this receptor, which by itself does not directly induce signalling but potentiates the activities of other PARs. In agreement to the functional assays, we failed to observe any mRNA expression for PARs in basophils, which was in stark contrast to mononuclear cells and neutrophils (Fig. 1). Our results clearly show that human basophils do not express PARs, at least under normal conditions, and this is consistent with previous work showing that human basophils are unresponsive to trypsin and thrombin [7]. We can also conclude that the reported mediator release from basophils caused by hookworm secretions and Der p1 antigen in unsen- sitized individuals does not occur via PAR activation. © Birkhäuser Verlag, Basel, 2005 Inflammation Research Lack of protease activated receptor (PAR) expression in purified human basophils F. H. Falcone 1 , S. Morroll 1 and B. F. Gibbs 2 1 The School of Pharmacy, University of Nottingham, UK 2 Department of Dermatology, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany, Fax: ++49 451 500 2981, e-mail: bfgibbs@gmx.de Correspondence to: B. F. Gibbs