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Human tumour necrosis factor alpha (TNF‐alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF‐alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF‐alpha can be dissociated. The TNFR55‐selective mutants (R32W, E146K and R32W‐S86T) which bind poorly to TNFR75 displayed similar potency to wild‐type TNF in causing cytotoxicity of a human laryngeal carcinoma‐derived cell line (HEp‐2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55‐selective mutants exhibited lower proinflammatory activity than wild‐type TNF. Specifically, TNF‐alpha's priming of human neutrophils for superoxide production and antibody‐dependent cell‐mediated cytotoxicity, platelet‐activating factor synthesis and adhesion to endothelium were reduced by up to 170‐fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E‐selectin expression, neutrophil transmigration and IL‐8 secretion were also reduced by up to 280‐fold. On the other hand, D143F, a TNFR75‐selective mutant tested either alone or in combination with TNFR55‐selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75‐transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32‐‐‐‐Trp, Leu36‐‐‐‐Phe, Ser86‐‐‐‐Phe and Ala84‐‐‐‐Val) behaved similar to the wild‐type in various physico‐chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84‐‐‐‐Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non‐conservative alterations were introduced (Leu29‐‐‐‐Ser and Glu146‐‐‐‐Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val‐‐‐‐Ala) caused a significant drop (500‐fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor‐interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.

The receptor for interleukin‐5 (IL‐5) is composed of two different subunits. The IL‐5 receptor alpha (IL‐5R alpha) is required for ligand‐specific binding while association with the beta‐chain results in increased binding affinity. Murine IL‐5 (mIL‐5) has similar activity on human and murine cells, whereas human IL‐5 (hIL‐5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL‐5 by their respective murine counterpart yields a molecule which is as potent as mIL‐5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL‐5R alpha subunit, we have used chimeric IL‐5R alpha molecules to define regions of hIL‐5R alpha involved in species‐specific hIL‐5 ligand binding. We found that this property is largely determined by the NH2‐terminal module of hIL‐5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2‐terminal module and the membrane proximal (WSXWS‐containing) module of hIL‐5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta‐strands on the proposed tertiary structure of the NH2‐terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM‐CSF), interleukin‐3 (IL‐3) and interleukin‐5 (IL‐5) is essential for high affinity ligand‐binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM‐CSF, IL‐3 and IL‐5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL‐2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site‐directed mutagenesis and binding assays with radiolabelled GM‐CSF, IL‐3 and IL‐5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365‐Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM‐CSF and IL‐5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL‐3 binding to high affinity by beta c. To identify likely contact points in GM‐CSF involved in binding to the 365‐368 beta c region we used the GM‐CSF mutant eco E21R which is unable to interact with wild‐type beta c whilst retaining full GM‐CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild‐type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM‐CSF and IL‐5 can be dissociated from IL‐3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM‐CSF.

The mouse interleukin‐5 receptor (mIL‐5R) consists of two components one of which, the mIL‐5R alpha‐chain, binds mIL‐5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL‐5R beta‐chain, immunoprecipitate a p130–140 protein doublet which corresponds to the mIL‐3R and the mIL‐3R‐like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL‐5R can be reconstituted on COS1 cells by co‐expression of the mIL‐5R alpha‐chain with the mIL‐3R‐like protein (beta‐chain). Cross‐linking of 125I‐labeled mIL‐5 to the COS1 cells co‐transfected with both cDNAs revealed the same pattern as in B13 cells, i.e. two proteins of 60 and 130 kd which correspond to the low affinity mIL‐5R alpha‐chain and the mIL‐3R‐like protein, respectively. The dissociation rate of mIL‐5 from this reconstituted high affinity site was lower than that of the low affinity site, whereas the association rate was unchanged. Nonetheless, the apparent dissociation constant (Kd) for this reconstituted receptor was still 10‐fold higher than the Kd observed for B13 cells. Although the mIL‐3R is greater than 90% homologous to the mIL‐3R‐like protein, no increase in affinity for mIL‐5 was detected on COS1 cells co‐transfected with the cDNAs for the mIL‐5R alpha‐chain and the mIL‐3R protein.

Southern hybridization using 32P‐labelled human interleukin 2 (IL2) cDNA probes revealed the existence of a single human IL2 gene. Five clones containing the human IL2 chromosomal gene were isolated from two different human DNA libraries cloned in either lambda Charon 4A or L47 phages. Analysis of the clones showed that they contained different, overlapping portions of human DNA which were derived from the same chromosomal segment. Restriction fragments which hybridized with labelled IL2 cDNA probes were subcloned into plasmid pUR250 and the sequence and organization of the IL2 gene was determined. It contains three introns, 90 bp, +/‐ 2400 bp and +/‐ 1900 bp in length, respectively. The organization of the genomic clone resembles that of another lymphokine, interferon‐gamma, but no clear homology was found by comparing either the coding sequence or the 5′‐ and 3′‐flanking sequences of the two genes.

Two clones containing the human immune interferon‐gamma (IFN‐gamma) chromosomal gene were isolated from a human DNA library present in lambda Charon4A phage. DNA from these clones specified biologically active interferon upon injection into the nuclei of Xenopus laevis oocytes. Analysis of the clones revealed that they were derived from the same chromosomal segment. Restriction fragments that hybridized with 32P‐labeled cDNA probes were subcloned into plasmids and the complete sequence of the IFN‐gamma gene was determined. Unlike IFNs‐alpha and ‐beta, IFN‐gamma does contain introns. Their presence was also revealed by electron microscopy. It is intriguing that the smallest of the three introns is located just in the middle of the Glu‐Glu sequence which is conserved among all three forms of interferon at approximately the same position. The promoter region was found to contain a prototype TATA box, many palindromic structures and several repeating sequences and two symmetrical structures. Particularly interesting was the existence of two sequences homologous to those present in the chicken albumin and the human IFN‐beta gene promoter region. A sequence GTGTTG common to several other genes was found in the region approximately 10 nucleotides downstream from the polyadenylation site.