Purification and characterization of recombinant human interleukin 4. Biological activities, receptor binding and the generation of monoclonal antibodies

Abstract

A synthetic gene coding for human interleukin 4 (IL-4) was cloned and expressed in Saccharomyces cerevisiae (baker's yeast) as a C-terminal fusion protein with the yeast prepro alpha-mating factor sequence, resulting in secretion of mature IL-4 into the culture medium (0.6-0.8 micrograms/ml). A protocol was developed for purification of this protein. Crude cell-free conditioned medium was passed over a concanavalin A-Sepharose affinity column; bound proteins were eluted and further purified by S-Sepharose Fast Flow cation exchange and C18 reverse-phase h.p.l.c. Highly purified IL-4 was obtained by this method (0.3-0.4 mg per litre of culture) with a recovery of 51%. Thermospray liquid chromatography-mass spectrometry showed the C-terminal N-glycosylation site to be largely unmodified, and also showed that the N-terminus of the purified recombinant IL-4 (rIL-4) was authentic. Thiol titration revealed no free cysteine residues, implying that there are three disulphide groups, the positions of which remain to be determined. We have characterized the biological activities of the purified rIL-4. This material is active in B-cell co-stimulator assays, T-cell proliferation assays and in the induction of cell-surface expression of CD23 (the low-affinity receptor for IgE) on tonsillar B-cells. Half-maximal biological activity of the rIL-4 was achieved at a concentration of 120 pM. We have radioiodinated rIL-4 without loss of biological activity and performed equilibrium binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding site with high affinity (Kd = 100 pM) and revealed 1100 receptors per cell. Receptor-ligand cross-linking studies demonstrated a single cell-surface receptor with an apparent molecular mass of 124 kDa. Two monoclonal antibodies have been raised to the human rIL-4, one of which blocks both the biological activity of rIL-4 and binding to its receptor.

Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement.

“…Partial agonism of IL-4 variants in B-cell differentiation Compared with the T-cell proliferation assay, much lower concentrations of IL-4 were sufficient to stimulate B-cells for the induction of CD23 (FceRII), the low affinity IgE receptor (Kikutani et al, 1986;DeFrance et al, 1987). After stimulation, the number of CD23 positive cells increased with EC50 of -0.4 pM for IL4 ( Figure 2A) (see also Solari et al, 1989). In this very sensitive assay, mutant protein Y124D behaved as a partial agonist.…”

“…T-cell proliferation assay (see Yokota et al, 1986;Solari et al, 1989) Peripheral blood mononuclear cells obtained from healthy donors were purified by Ficoll-Hypaque centrifugation (Pharmacia) and stored in aliquots at -80°C. The thawed cells were cultured for 7 days with 9 Ag/ml phytohemagglutinine (PHA; Wellcome) and consisted at this stage to a high percentage of activated T-cells (PHA-blasts).…”

Receptor binding properties of four‐helix‐bundle growth factors deduced from electrostatic analysis

“…The IL-4 has a net charge of +7e (measured PI > 9; Solari et al, 1989) and is mainly surrounded by positive potential, which complements the negative potential of IL-4R at the interface (Figs. 4, 9).…”

T cells from atopic individuals produce IgE‐inducing activity incompletely blocked by anti‐interleukin‐4 antibody

“…Using this system, we have now investigated peripheral blood B and T lymphocytes from patients with atopic diseases or normal controls with respect to (a) amounts of IgE secreted by B cells in response to IL-4, (b) generation of IgE-inducing activity by stimulated total T cells and (c) susceptibility of such activity to inhibition by anti-IL-4 antibody. The EL4-B cell assays were performed in 200 pl with 50 000 irradiated mutant EL4 thymoma cells (clone 6.1.5.5) [21], 300 B cells and T-SN as indicated below, with or without human rIL-4, human rIFN-y or anti-IL-4 monoclonal antibody ( [24]; all three reagents kindly provided by Dr. J. Y. Bonnefoy, Glaxo Institute for Molecular Biology, Geneva, Switzerland). All cultures were performed in RPMI 1640 medium with 10 YO FCS, 10" M 2-ME, 25 mM Hepes buffer and penicillin/streptomycin as described [23].…”