The histamine H 4 receptor (H 4 R) is involved in the chemotaxis of leukocytes and mast cells to sites of inflammation and is suggested to be a potential drug target for asthma and allergy. So far, selective H 4 R agonists have not been identified. In the present study, we therefore evaluated the human H 4 R (hH 4 R) for its interaction with various known histaminergic ligands. Almost all of the tested H 1 R and H 2 R antagonists, including several important therapeutics, displaced less than 30% of specific [3 H]histamine binding to the hH 4 R at concentrations up to 10 M. Most of the tested H 2 R agonists and imidazolebased H 3 R ligands show micromolar-to-nanomolar range hH 4 R affinity, and these ligands exert different intrinsic hH 4 R activities, ranging from full agonists to inverse agonists. Interestingly, we identified 4-methylhistamine as a high-affinity H 4 R ligand (K i ϭ 50 nM) that has a Ͼ100-fold selectivity for the hH 4 R over the other histamine receptor subtypes. Moreover, 4-methylhistamine potently activated the hH 4 R (pEC 50 ϭ 7.4 Ϯ 0

Interleukin-12 (IL-12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide-bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL-12 (moIL-12) p40-transfected COS cells could inhibit IL-12-dependent responses in vitro (Mattner, F., et al., Eur. J. Immunol. 1993. 23: 2202). We have further characterized the nature of the inhibitory substance. Purified mouse p40 produced in a baculovirus expression system was found to consist of two species: the p40 monomer and a disulfide-linked p40 dimer [(p40)2]. The (p40)2 was 25- to 50-fold more active than the p40 monomer in causing specific, dose-dependent inhibition of IL-12-induced mouse concanavalin A (Con A) blast proliferation and could also inhibit IL-12-induced interferon-gamma (IFN-gamma) secretion by mouse splenocytes and IL-12-dependent activation of mouse NK cells. Competitive binding studies on mouse Con A blasts showed that (p40)2 was equally effective as moIL-12 in competing with 125I-labeled moIL-12 ([125I]moIL-12) for binding to mouse Con A blasts. However, in contrast to moIL-12, mouse (p40)2 displayed little ability to compete with 125I-labeled human IL-12 (huIL-12) for binding to high-affinity IL-12 receptors (IL-12R) on human phytohemagglutinin (PHA) blasts and caused little or no inhibition of huIL-12-induced human PHA blast proliferation. Nonetheless, mouse (p40)2 was equally effective as moIL-12 in competing with [125I] huIL-12 for binding to COS cells transfected with the human IL-12R beta subunit and expressing low-affinity IL-12 binding sites. These results suggest that (i) the majority of the structural determinants required for binding of IL-12 to its receptor are contained within the p40 subunit, but p35 is required for signaling, (ii) the p40 subunit of IL-12 interacts with the beta subunit of IL-12R, and (iii) (p40)2 may be a suitable IL-12 antagonist for studying the role of IL-12 in various immune responses in vivo as well as in vitro. Further studies are required to determine whether or not (p40)2 is produced by normal lymphoid cells and is a physiologic regulator of IL-12 activity.

PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2.

1 During mast cell degranulation, histamine is released in large quantities. Human eosinophils were found to express histamine H 4 but not H 3 receptors. The possible effects of histamine on eosinophils and the receptor mediating these effects were investigated in our studies. 2 Histamine (0.01-30 mM) induced a rapid and transient cell shape change in human eosinophils, but had no effects on neutrophils. The maximal shape change was at 0.3 mM histamine with EC 50 at 19 nM. After 60 min incubation with 1 mM histamine, eosinophils were desensitized and were refractory to shape change response upon histamine restimulation. Histamine (0.01-1 mM) also enhanced the eosinophil shape change induced by other chemokines. 3 Histamine-induced eosinophil shape change was mediated by the H 4 receptor. This effect was completely inhibited by H 4 receptor-specific antagonist JNJ 7777120 (IC 50 0.3 mM) and H 3 /H 4 receptor antagonist thioperamide (IC 50 1.4 mM), but not by selective H 1 , H 2 or H 3 receptor antagonists. H 4 receptor agonists imetit (EC 50 25 nM) and clobenpropit (EC 50 72 nM) could mimic histamine effect in inducing eosinophil shape change. 4 Histamine (0.01-100 mM) induced upregulation of adhesion molecules CD11b/CD18 (Mac-1) and CD54 (ICAM-1) on eosinophils. This effect was mediated by the H 4 receptor and could be blocked by H 4 receptor antagonists JNJ 7777120 and thioperamide. 5 Histamine (0.01-10 mM) induced eosinophil chemotaxis with an EC 50 of 83 nM. This effect was mediated by the H 4 receptor and could be blocked by H 4 receptor antagonists JNJ 7777120 (IC 50 86 nM) and thioperamide (IC 50 519 nM). Histamine (0.5 mM) also enhanced the eosinophil shape change induced by other chemokines. 6In conclusion, we have demonstrated a new mechanism of eosinophil recruitment driven by mast cells via the release of histamine. Using specific histamine receptor ligands, we have provided a definitive proof that the H 4 receptor mediates eosinophil chemotaxis, cell shape change and upregulation of adhesion molecules. The effect of H 4 receptor antagonists in blocking eosinophil infiltration could be valuable for the treatment of allergic diseases. The histamine-induced shape change and upregulation of adhesion molecules on eosinophils can serve as biomarkers for clinical studies of H 4 receptor antagonists.

Three series of H(4) receptor ligands, derived from indoly-2-yl-(4-methyl-piperazin-1-yl)-methanones, have been synthesized and their structure-activity relationships evaluated for activity at the H(4) receptor in competitive binding and functional assays. In all cases, substitution of small lipophilic groups in the 4 and 5-positions led to increased activity in a [(3)H]histamine radiolabeled ligand competitive binding assay. In vitro metabolism and initial pharmacokinetic studies were performed on selected compounds leading to the identification of indole 8 and benzimidazole 40 as potent H(4) antagonists with the potential for further development. In addition, both 8 and 40 demonstrated efficacy in in vitro mast cell and eosinophil chemotaxis assays.