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). A pharmacodynamic model injecting male C57/BL6 mice with IL-15 or IL-15/IL-15Rα, with or without DISC0280, and assessing changes in lymphocytic cell populations and serum cytokines was utilized. KEY RESULTS: DISC0280 inhibited the binding of IL-15 to IL-15Rα and also potently inhibits IL-15 dependent proliferation of cells expressing IL-15Rα, shared interleukin 2/ interleukin 15 receptor ß chain (IL-15Rß) and common gamma chain (γ(c) ). DISC0280 also inhibited the IL-15 dependent proliferation of M-07e cells that only express IL-15Rß/γ(c) subunits. Human IL-15 injected into mice caused an increase in NK1.1(+) and CD3(+) cells in the spleen and peripheral blood and these effects were unexpectedly potentiated by giving DISC0280 with human IL-15. This increase in cells caused by DISC0280/IL-15 co-administration was greater than that observed when IL-15 was administered complexed with soluble IL-15Rα. CONCLUSIONS AND IMPLICATIONS: The ability of DISC0280 to bind to the IL-15Rα-binding site on IL-15 allows trans-presentation of IL-15 by DISC0280 in vivo, similar to the trans-presentation by soluble IL-15Rα. DISC0280 may be therefore suitable as a clinical substitute for IL-15.

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. 2. ATP caused release of IL-1beta from LPS primed THP-1 cells in both a time- and concentration-dependent manner, with a minimal effective ATP concentration of 1 mM. Stimulation of cells with 5 mM ATP resulted in detectable concentrations of IL-1beta in cell supernatants within 30 min. 3. The ATP analogue benzoylbenzoyl ATP (DBATP), a P2X7 receptor agonist, was approximately 10 fold more potent than ATP at eliciting IL-1beta release. 4. KN-62 (1 micro M), PPADS (100 microM) or oxidized ATP (100 uM) significantly inhibited 5 mM ATP-induced IL-1beta release by 81, 90 and 66% respectively, but failed to significantly inhibit LPS-induced IL-1beta release in both THP-1 cells and in freshly isolated human monocytes. 5. In both THP-1 cells and freshly isolated human monocytes, addition of the ATP degrading enzyme apyrase (0.4 U ml(-1)) to cell supernatants prior to LPS activation failed to significantly inhibit the LPS-induced IL-1beta release. In addition there was no correlation between extracellular ATP concentrations and IL-1beta release in THP-1 cells when studied over a 6 h time period. 6. In conclusion our data confirm the involvement of P2X7 receptors in ATP-induced IL-1beta release in human monocytes. However no evidence was obtained which would support the involvement of either endogenous ATP release or P2X7 receptor activation as the mechanism by which LPS-induces IL-1beta release in either the THP-1 cell line or in freshly isolated human monocytes.

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. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

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. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC50 = 191 microM) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells. ATP-stimulated 45Ca2+ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated 45Ca2+ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency:- guanidinium (EC50 = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose). A number of P2 purinoceptor antagonists inhibited ATP-stimulated 45Ca2+ influx. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3-300 microM), pyridoxal 5-phosphate (3-300 microM) and d-tubocurarine (30-300 microM) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC50. Suramin (100-300 microM) and cibacron blue (30-300 microM) produced a surmountable antagonism while DIDS (4,4'-diisothiocyanatostilbene-2,2'disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 microM. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X2 purinoceptors. These findings suggest that ATP-stimulated 45Ca2+ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.

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 presence of these two purinoceptor transcripts in the brain was confirmed by polymerase chain reaction. Two partial cDNAs, identical to sections of the PC12 or vas deferens P2X purinoceptor coding sequences, were amplified from neonatal brain and cerebellum poly(A)+ RNA, respectively. These findings are in broad agreement with earlier Northern blot studies on the PC12 P2X purinoceptor mRNA but differ from those for the vas deferens P2X purinoceptor mRNA, which had not previously been detected in adult brain. This difference is attributed to the low levels seen in the adult compared with the neonate and to the greater sensitivity of the methods used in the present study. The neonate medial habenula had low levels of transcripts for the PC12 but none for the vas deferens P2X purinoceptor. Because pharmacologically the recombinant PC12 P2X purinoceptor differs from the functional purinoceptor in the medial habenula, these results suggest the existence of other, unidentified, P2X purinoceptors in the rat nervous system.

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 10.8 microM. The depolarising effects of alpha beta meATP were not attenuated by beta gamma me-L-ATP (100 microM). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 microM), but not beta gamma me-L-ATP (1-300 microM), evoked rapid (< 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, beta gamma me-D-ATP and alpha beta meATP competed with high affinity for [3H]alpha beta meATP binding sites, with mean pIC50 values of 7.7 and 8.3, respectively. However, beta gamma me-L-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 microM (mean pIC50 value 4.1). In prostatic segments of the rat vas deferens, beta gamma me-L-ATP (1-100 microM) and alpha beta meATP (0.3-100 microM) each produced concentration-related contractile responses with mean EC50 values of 17.1 and 3.6 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)