Generation of adenosine A3 receptor functionally humanized mice for the evaluation of the human antagonists.

Although the adenosine A(3) receptor (A3AR), which is a G(i/o) protein-coupled receptor, has attracted considerable interest as a potential target for drugs against asthma or inflammation, the in vivo evaluation of the antagonists using rodents in the first step of drug development has been hampered by the lack of highly potent antagonists for the rodent A3AR. To evaluate the pharmacological effects of human A3AR antagonists in mice, we previously generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. However, the human A3AR did not lead to the phosphoinositide 3-kinase (PI3K) gamma-signaling pathway such as IgE/antigen-dependent mast cell degranulation, probably due to the uncoupling of the mouse G(i/o) protein(s). To overcome the uncoupling, we here generated A3AR functionally humanized mice by replacing the mouse A3AR gene with a human/mouse chimeric A3AR sequence in which whole intracellular regions of the human A3AR were substituted for the corresponding regions of the mouse A3AR. The chimeric A3AR led to intracellular Ca(2+) elevation and activation of the PI3Kgamma-signaling pathway, which are equivalent to the actions induced by A3AR in wild-type mice. The human A3AR antagonist had the same binding affinities for the chimeric A3AR as the human A3AR and completely antagonized this potentiation. This is the first direct evidence that the uncoupling of mouse G protein(s) to the human A3AR is due to a sequence difference in the intracellular regions of A3AR. The A3AR functionally humanized mice can be widely employed for pharmacological evaluations of the human A3AR antagonists.

Human adenosine A(3) receptor leads to intracellular Ca(2+) mobilization but is insufficient to activate the signaling pathway via phosphoinositide 3-kinase gamma in mice.

Selective antagonists for the adenosine A(3) receptor (A3AR), a member of the G protein-coupled receptors, have been indicated as potential drugs for anti-asthma or anti-inflammation. However, potent antagonists for the rodent A3AR have not been identified. To evaluate the pharmacological effects of human A3AR antagonists in mice, we here generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. The expression levels of human A3AR in the A3AR-humanized mice were equivalent to those of mouse A3AR in wild-type mice. Elevation of the intracellular Ca(2+) concentration induced by an A3AR agonist was observed in bone marrow-derived mast cells from the A3AR-humanized mice and this Ca(2+) mobilization was completely antagonized by a human A3AR antagonist. However, antigen-dependent degranulation was not potentiated by the A3AR agonist in the mast cells from A3AR-humanized mice. The agonist-stimulated human A3AR did not lead to the phosphorylation of either extracellular signal-regulated kinase 1/2 or protein kinase B in A3AR-humanized mice. The rate of human A3AR internalization in the mast cells was also markedly decreased compared with that of mouse A3AR in the mast cells. These results demonstrate that the human A3AR is insufficient to activate phosphoinositide 3-kinase gamma-dependent signaling pathways in mice, probably due to the uncoupling of member(s) of the G proteins, which are capable of activating phosphoinositide 3-kinase gamma, to the human A3AR, despite the mouse G protein(s) responsible for the Ca(2+) elevation are coupled with the human A3AR.

Functional analysis of PU.1 domains in monocyte-specific gene regulation.

The Ets family transcription factor PU.1 is required for the development of various lymphoid and myeloid cell lineages, and regulates the expression of several genes in a cell type-specific manner. Recently we found that overproduction of PU.1 in mouse bone marrow-derived mast cell progenitors induced the expression of monocyte-specific genes. This prompted us to analyze the functions of each domain of PU.1 in monocyte-specific gene expression, using transfection of mast cell progenitors with a series of retrovirus vectors for overexpression of various truncation mutants. Both the acidic region and the Ets domain of PU.1 were required for expression of monocyte-specific genes, and for enhanced interleukin-6 production in response to lipopolysaccharide. The Gln-rich region was suggested to be involved in expression of both MHC class II and F4/80. On the other hand, when PU.1 protein lacking the PEST domain was produced in the progenitor cells, expression of monocyte-specific genes was substantially enhanced, suggesting that the PEST domain plays a negative role in monocyte-specific gene expression.

Differential regulation of IL-4 expression and degranulation by anti-allergic olopatadine in rat basophilic leukemia (RBL-2H3) cells.

Olopatadine hydrochloride (olopatadine) is an anti-allergic drug that functions as a histamine H(1) antagonist and inhibits both mast cell degranulation and the release of arachidonic acid metabolites in various types of cells. In this study, we examined the ability of olopatadine to inhibit the expression of cytokine genes in vitro via high-affinity receptors for immunoglobulin E in mast cells, using a rat basophilic leukemia (RBL-2H3) cell line and an in vivo mouse model. Levels of gene expression in RBL-2H3 cells were determined by semi-quantitative RT-PCR, and serum interleukin-4 (IL-4) level in mice was quantified by ELISA. Olopatadine inhibited significantly the induction of IL-4 expression by mast cells both in vivo and in vitro. Olopatadine inhibited Ca(2+) influx through receptor-operated channels (ROC) without affecting Ca(2+) release from intracellular stores. Comparative analysis of olopatadine with other anti-allergic drugs and the ROC blocker SKF-96365 demonstrated that the potency of inhibition of Ca(2+) influx correlated with the degree of suppression of degranulation and arachidonic acid release. Inhibition of Ca(2+) influx decreased phosphorylation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase, which participate in regulation of cytokine (e.g. IL-4) gene expression. However, the rank order of inhibition of Ca(2+) influx did not correspond to reduction of IL-4 expression, suggesting that an unknown mechanism(s) of action, in addition to inhibition of Ca(2+) influx, is involved in the expression of cytokines in mast cells.

Effect of olopatadine and other histamine H1 receptor antagonists on the skin inflammation induced by repeated topical application of oxazolone in mice.

Histamine H1 receptor antagonists have long been prescribed for atopic dermatitis as an adjuvant therapy with topical therapy by local applied steroids. Olopatadine is one of the second-generation histamine H1 receptor antagonists that are treated for allergic disorders. We investigated that the effect of olopatadine on oxazolone-induced chronic contact hypersensitivity response in BALB/c mice compared with other histamine H1 receptor antagonists loratadine, cetirizine and fexofenadine. The chronic contact hypersensitivity induced by repeated application of oxazolone was treated with olopatadine and other histamine H1 receptor antagonists at the effective doses on histamine-induced paw edema in mice. The effects of these drugs in the oxazolone-induced model were quantified by measurements of ear swelling, and levels of cytokines in the lesioned ear. Olopatadine significantly inhibited the ear swelling and the increased production of IL-4, IL-1beta, IL-6, GM-CSF and NGF in the lesioned ear. On the other hand, the other histamine H1 receptor antagonists did not significantly suppress the increase in ear thickness. Moreover, they did not affect the production of cytokines in the lesioned ear. These results indicate that olopatadine appears to exert additional biological effects besides its blockade of the histamine H1 receptor.

GATA-1 is required for expression of Fc{epsilon}RI on mast cells: analysis of mast cells derived from GATA-1 knockdown mouse bone marrow.

The high-affinity receptor for IgE (FcepsilonRI) that is expressed on the surface of mast cells plays an important role in antigen/IgE-mediated allergic reactions. We have previously found that critical elements in the promoter of the FcepsilonRI alpha- and beta-chain genes are recognized by the transcription factor GATA-1 in electrophoretic mobility shift assays coupled with a transient expression system for the alpha- and beta-chain promoters. To confirm that GATA-1 is involved in the expression of FcepsilonRI definitively, we generated bone marrow-derived mast cells from GATA-1 knockdown (KD) heterozygous mice. FACS analysis showed that the frequency of FcepsilonRI-positive cells was significantly decreased in mast cells derived from bone marrow of GATA-1 KD mice. Reverse transcription-PCR analysis showed that the level of transcripts not only for GATA-1 but also for both the alpha- and beta-chains was significantly lower in KD mast cells, whereas that of the FcepsilonRI gamma-chain was not affected. Degranulation caused by cross-linking of FcepsilonRI on mast cells prepared from KD mice was markedly repressed in comparison with that of wild-type mast cells. We concluded that the transcription factor GATA-1 positively regulates FcepsilonRI alpha- and beta-chain expression and therefore is involved in mast cell development.

Overproduction of PU.1 in mast cell progenitors: its effect on monocyte- and mast cell-specific gene expression.

The Ets family transcription factor PU.1 is required for development of various lymphoid and myeloid cell lineages, and regulates the expression of several genes in a cell type-specific manner. Mouse bone marrow-derived hematopoietic progenitor cells are programmed to differentiate into mast cells, when the cells are maintained in the presence of pokeweed mitogen-stimulated spleen-conditioned medium. However, by retroviral introduction of PU.1 cDNA, the progenitor cells expressed MHC class II, CD11b, CD11c, and F4/80, and acquired the ability to stimulate T cells. Furthermore, PU.1-overproducing cells exhibited the morphology, in part, similar to that of monocyte. These results indicate that the mast cell progenitors still have the ability to express monocyte-specific genes by increased expression of PU.1.

Properties of olopatadine hydrochloride, a new antiallergic/antihistaminic drug.

Olopatadine hydrochloride (CAS 140462-76-6, KW-4679, AL-4943A; hereinafter referred to as olopatadine) is a novel antiallergic drug that is a selective histamine H1 receptor antagonist possessing inhibitory effects on the release of inflammatory lipid mediators such as leukotriene and thromboxane from human polymorphonuclear leukocytes and eosinophils. Olopatadine also inhibits the tachykininergic contractions in guinea pig bronchi by prejunctional inhibition of peripheral sensory nerves. Oral administration of olopatadine at doses of 0.03 mg/kg or higher reduces the symptoms of experimental allergic cutaneous responses and rhinoconjunctivitis in sensitized animals. Preclinical and clinical evaluations have demonstrated that olopatadine is a safe drug. After oral administration to healthy volunteers, olopatadine was rapidly and extensively absorbed. Unlike most other antiallergic drugs which are eliminated via hepatic metabolism, olopatadine is mainly excreted into urine. Olopatadine did not affect cytochrome P450 activities in human liver microsomes and consequently drug-drug metabolic interactions are unlikely. In double-masked clinical trials, olopatadine was shown to be effective at alleviating symptoms of allergic diseases. The drug (Allelock) was approved in Japan for the treatment of allergic rhinitis, chronic urticaria, eczema dermatitis, prurigo, cutaneous pruritus, psoriasis vulgaris and erythema exsudativum multiforme in December, 2000. An ophthalmic solution of olopatadine is also useful for the treatment of allergic conjunctivitis: this formulation (Patanol) was approved in the USA and the European Union for the treatment of seasonal and perennial allergic conjunctivitis in 1996 and 2002, respectively.