Identification of a potent anti-IL-15 antibody with opposing mechanisms of action in vitro and in vivo.

Background And Purpose: Interleukin-15 (IL-15) is important in the activation and proliferation of lymphocytic cell populations and is implicated in inflammatory disease. We report the characterization of a novel monoclonal antibody DISC0280 which is specific for human IL-15.

Experimental Approach: DISC0280 was characterized in a direct binding assay of IL-15 with IL-15 receptor α (IL-15Rα) and by its ability to alter IL-15 mediated proliferation of a range of cell lines (cytotoxic T lymphocyte line-2, M-07e, KIT225).

E. coli expression and purification of human and cynomolgus IL-15.

The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)(6)-tag was expressed in Escherichia coli as an insoluble protein.

Identification of a putative intracellular allosteric antagonist binding-site in the CXC chemokine receptors 1 and 2.

The chemokine receptors CXCR1 and CXCR2 are G-protein-coupled receptors (GPCRs) implicated in mediating cellular functions associated with the inflammatory response. Potent CXCR2 receptor antagonists have been discovered, some of which have recently entered clinical development. The aim of this study was to identify key amino acid residue differences between CXCR1 and CXCR2 that influence the relative antagonism by two compounds that have markedly different chemical structures.

Evaluation of a series of bicyclic CXCR2 antagonists.

The CXCR2 SAR of a series of bicyclic antagonists such as the 2-aminothiazolo[4,5-d]pyrimidine 3b was investigated by systematic variation of the fused pyrimidine-based heterocyclic cores. Replacement of the aminothiazole ring with a 2-thiazolone alternative led to a series of thiazolo[4,5-d]pyrimidine-2(3H)-one antagonists with markedly improved biological and pharmacokinetic properties, which are suitable pharmacological tools to probe the in vivo effects of CXCR2 antagonism combined with the associated CCR2 activity.

Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain.

The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1.

A truncated form of CKbeta8-1 is a potent agonist for human formyl peptide-receptor-like 1 receptor.

1. Human formyl peptide-receptor-like-1 (FPRL-1) is a promiscuous G protein-coupled receptor (GPCR), and belongs to a chemoattractant receptor family protein. This receptor has been reported to interact with various host-derived peptides and lipids involved in inflammatory responses.

Pharmacological characterization of ATP- and LPS-induced IL-1beta release in human monocytes.

1. We have utilized the human monocytic cell line, THP-1, and freshly isolated adherent human monocytes with the compounds pyridoxalphosphate-6-azophenyl-2',4'-disuphonic acid (PPADS), oxidized ATP, and 1-(N, O-bis[5-isoquinolinesufonyll]-N-methyl-L-tyrosyl)-4-phenylpiper azi ne (KN-62) to pharmacologically characterize the P2 receptor involved in ATP-induced release of interleukin 1beta (IL-1beta). We have also investigated the involvement of P2 receptors in lipopolysaccharide (LPS)-induced IL-1beta release from both cell types.

Blockade of human P2X7 receptor function with a monoclonal antibody.

A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections.

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx.

The aim of this study was to determine whether 45Ca2+ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC50 = 45 microM), ATP gamma S (EC50 = 50 microM) and 2-meSATP (EC50 = 81 microM) but not alpha beta meATP (1 mM) stimulated 45Ca2+ influx 2-5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADP beta S did not produce any significant effect.

Localization of P2X purinoceptor transcripts in the rat nervous system.

We used transcript-specific oligonucleotides to examine the localization in the rat nervous system of the corresponding mRNAs for the two P2X purinoceptor genes cloned recently from the rat vas deferens and PC12 cells. PC12 P2X purinoceptor mRNA was labeled in the olfactory tubercle, striatum, hypothalamus, hippocampus, dentate gyrus, amygdala, cortex, and cerebellum, whereas the vas deferens P2X purinoceptor-specific probes labeled the cerebellum and, at lower levels of expression, the striatum, hippocampus, and cortex. Both types of P2X purinoceptor transcript were found on cell bodies in the nodose and superior cervical ganglia.

The selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate, discriminates between smooth muscle and neuronal P2X purinoceptors.

The effects of the putative selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate (beta gamma me-L-ATP), were determined at rat neuronal and smooth muscle P2X purinoceptors. beta gamma Me-L-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 microM. In contrast, the archetypal P2X purinoceptor agonist, alpha,beta-methylene ATP (alpha beta meATP; 1-100 microM), produced concentration-related depolarisation responses with a mean EC50 value of 10.