A murine model of allergen-induced airway inflammation and epithelial phenotypic change, and the time-courses of these events, are described. Mice were sensitized to ovalbumin using an adjuvant-free protocol, and challenged by multiple intratracheal instillations of ovalbumin by a non-surgical technique. Many of the characteristic features of human atopic asthma were seen in the mice. A marked eosinophilic infiltration of lung tissue and airways followed allergen challenge, and its severity increased with each challenge, as did the number of eosinophils in the blood. Lymphocytes, neutrophils, and monocytes also invaded the lungs. Airway macrophages showed signs of activation, their appearance resembling those recovered from antigen-challenged human asthmatic airways. The airway epithelium was thickened and displayed a marked goblet cell hyperplasia in terminal bronchioles and larger airways. After repeated challenges, the reticular layer beneath the basement membrane of the airway epithelium showed fibrosis, reproducing a commonly observed histologic feature of human asthma. Goblet cell hyperplasia began to appear before eosinophils or lymphocytes had migrated across the airway epithelium, and persisted for at least 11 days after the third intratracheal challenge with ovalbumin, despite the number of inflammatory cells in the lungs and airways having decreased to near-normal levels by 4 days. Plugs of mucus occluded some of the airways. These results indicate that some of the phenotypic changes in airway epithelium that follow an allergic response in the lung can be initiated before the migration of eosinophils or lymphocytes across the epithelial layer.

Interleukin-5 (IL-5) is a cytokine that plays a major role in the differentiation and activation of eosinophils. In order to identify which charged residues of human IL-5 are important in binding to its receptor and subsequent cellular activation, we have systematically replaced all of the clusters of charged amino acids with alanine residues. The mutants have been expressed in Escherichia coli, renatured, and purified. They were assayed for ability to cause proliferation of the erythroleukaemic cell line TF-1 and the up-regulation of eosinophil adhesion to ICAM-1. In addition, we studied receptor binding using either immobilized recombinant IL-5 receptor alpha-chain or the alpha/beta-receptor complex expressed on TF-1 cells. The key charged residue involved in binding to the beta-chain of the receptor is Glu-12. This residue is in an identical position to those previously identified in IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in binding to the receptor beta-chain. The alpha-chain binding site is shown to involve the side chains Arg-90 and Glu-109, located in the second beta sheet and after the end of the fourth helix, respectively. It is unique to IL-5 and does not occur in IL-3 or GM-CSF. Understanding the topology of the interaction of IL-5 with its receptor chains will help in the search for rationally designed antagonists of IL-5 function.

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heatinducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly ca-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines. INTRODUCTIONInterleukin-5 (IL-5) is a specific haematopoietic growth factor responsible for eosinophil differentiation. Murine IL-5 has also been shown to have B-cell-growth-factor [1] and T-cell-replacingfactor activities [2]. These activities were originally thought to be derived from separate proteins, namely eosinophil differentiating factor (EDF), B-cell growth factor (BCGF-II) and T-cell replacing factor (TRF). The availability of recombinant-derived murine protein showed that these activities were derived from a single protein, namely IL-5.Natural IL-5 has been isolated only from murine T-cell supernatants and shown to be a glycoprotein which exists as a homodimer (Mr 45 000) [2,3]. The cDNA sequence of the murine species predicts an Mr of 13 000 for the polypeptide monomer [5].Human genes have been cloned using the murine IL-5 cDNA [4] as a probe. The murine and human proteins show 70 % sequence similarity and have species cross-reactivity in EDF activity. The natural hIL-5 has not been purified and characterized, but recombinant hIL-5 has been expressed in several eukaryotic systems [7,8] and shown to be a glycosylated, disulphide-linked homodimer of Mr

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Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5–dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5–dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.