Characterization of Potential Antagonists of Human Interleukin 5 Demonstrates Their Cross‐Reactivity with Receptors for Interleukin 3 and Granulocyte‐Macrophage Colony‐Stimulating Factor

Abstract

The ligand-binding a-chain of the human interleukin 5 (IL-5) receptor was expressed in its soluble form, lacking the transmembrane and cytoplasmic domains, from recombinant baculovirus. The soluble receptor was used in a scintillation proximity assay to identify two chemical compounds that inhibit binding of human IL-5 to the soluble receptor a chain with IC,, of 8 pM and 11 pM, These compounds also inhibited the interaction of human IL-5 with its membrane-bound receptor, composed of the ligandbinding a chain and signal-transducing p chain, and prevented signaling through the receptor. Analysis by surface plasmon resonance and matrix-assisted laser-desorptiodionization mass spectrometry showed that the identified compounds bound irreversibly to the receptor at a 1 : 1 (mol/mol) ratio, suggesting a covalent interaction with the a chain of the human IL-5 receptor. Both compounds also inhibited the interaction of the receptors for interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are involved in hematopoietic differentiation and activation of immune cells, thus eliminating them as potential therapeutic agents. The inhibition of the strucuturally closely related receptors for IL-5, IL-3 and GM-CSF by both compounds, while binding of interleukin-4 to its receptor was not affected, suggests that a similar reactive site exists in the ligand-binding domains of the receptors for IL-5, IL-3 and GM-CSF. Keywords : human interleukin 5 ; interleukin-5 receptor; interleukin-3 receptor; granulocyte-macrophagecolony-stimulating-factor receptor; receptor antagonist.Human interleukin (IL-5) is a disulfide-linked homodimeric cytokine produced from T cells that promotes proliferation and differentiation of eosinophils [l, 21. The observed effect of IL-5 on eosinophils suggests that IL-5 has a role in the pathogenic inflammatory responses of hypersensitivity and other diseases in which eosinophil infiltration has been observed. Antagonists of IL-5 might serve as drugs for the treatment of allergic diseases such as bronchial asthma and other hypereosinophilic syndromes.Human IL-5 transduces its signal by binding to its specific receptor expressed on eosinophils [3]. The human IL-5 receptor has been identified as a heterodimer consisting of the ligandbinding a chain of 60 kDa and a signal-transducing /l chain of 120 kDa [4]. Association of the a chain with the p chain is required for the induction of signal-transduction pathways [4, 51 involving Jak2 and STAT1 (signal transducer and activator of transcription 1) or Lyn and the mitogen-activated protein kinases [6-81.The IL-5 receptor and the receptors for interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM- CSF) form a subfamily of cytokine receptors based on shared structural and functional features. Similarly to the a chain of the IL-5 receptor, the a chains of the GM-CSF and IL-3 receptors bind specifically their cognate ligand and associate with the common p chain shared by all three receptors for signal transductio...

Recombinant‐protein production in insect cells utilizing a hollow‐fibre bioreactor

“…We obtained similar results for all samples, showing a molecular mass of 42 kDa (Figure 4). This is consistent with published results [5,48].…”

“…The expression of shIL‐5Rα has been carried out before in insect cells, with both S2 and Sf9 cells, in shaker flasks. It has been shown that insect cells are capable of producing functional shIL‐5Rα [5,48]. Production in prokaryotic cells is not an option because prokaryotic cells are not able to perform the post‐translational modifications necessary to produce functional shIL‐5Rα, and it has been shown that shIL‐5Rα loses activity when deglycosylated [5].…”

New anti-asthma therapies: suppression of the effect of interleukin (IL)‐4 and IL‐5

“…Although this has resulted in the identification of a number of promising molecules [76], some of them have proven to be too toxic for therapeutic applications or have been shown to also block IL-3 and GM-CSF mediated effects, again eliminating them as potential therapeutic agents [77,78]. These various points illustrate the difficulty in properly antagonizing IL-5 receptor activation.…”

Effect of a novel interleukin-5 receptor antagonist, YM-90709 (2,3-dimethoxy-6,6-dimethyl-5,6-dihydrobenzo[7,8]indolizino[2,3-b]quinoxaline), on antigen-induced airway inflammation in BN rats

“…Several low molecular mass compounds which interfere with the binding of IL-5 to IL-5R have been reported [24,25]. However, they are neither potent nor selective enough to suppress IL-5 activity in vitro.…”