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Interleukin 5 (IL-5) is a T-cell derived cytokine that induces eosinophil growth and differentiation in both mouse and human bone marrow cultures. Elevated levels of IL-5 as well as eosinophils have been detected in the sputum and Bronchoalveolar lavage (BAL) fluids of asthmatics. Since the recruitment of inflammatory cells to tissues requires the participation of adhesion molecules, we have developed a rapid and sensitive assay to examine the effect of IL-5 and other activation stimuli on eosinophil adhesion to recombinant intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Human recombinant IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), tumour necrosis factor alpha (TNF-alpha), RANTES, MCP-3, C5a, PAF, fMLP, PMA and ConA all induced adhesion of purified eosinophils obtained from normal donors to ICAM-1 and VCAM-1 in a dose and time dependent manner. Adhesion was rapid, within 15 minutes of culture at 37 degrees C, and plateaued within 30 minutes. Activated eosinophils also adhered rapidly to immobilized IgG via the type II Fc gamma receptor (CD32). Analysis of the effect of IL-5 on surface molecule expression by FACS analysis revealed increased expression of CD11b molecules and decreased expression of L-selectin, but no change in the expression of CD11a, CD18, CD29, CD49d and CD32. We also show that Mac-i plays an important role in the regulation of eosinophil activation, since antibodies to CD11b can block IL-5 induced adhesion to IgG and IL-5 induced degranulation.

In this study we have investigated the effect that interleukin 1 (IL-1) has on cell surface IL-1 receptor expression in the murine thymoma cell line, EL4 6.1. These cells express IL-1 receptors with both high affinity (Kd = 65 pM, 986 receptors/cell) and low affinity (Kd = 14.5 nM, 10,417 receptors/cell). The high- and low-affinity receptors are indistinguishable by crosslinking studies performed at both high and low ligand concentrations. However, the two affinity states could be functionally distinguished on the basis of their internalization of ligand. Receptor-mediated endocytosis was dependent upon the concentration of ligand bound to the cells. In the presence of low IL-1 concentrations receptor-mediated endocytosis was slow, whereas at high IL-1 concentrations, endocytosis was more rapid. Furthermore, receptor-mediated endocytosis of IL-1 did not result in downregulation of surface IL-1 receptors. Indeed, both kinetic and equilibrium binding studies revealed that pre-incubation of cells with IL-1 alpha resulted in an acute upregulation of 125IL-1 alpha binding to high affinity surface receptors in a time and energy dependent manner. Examination of the association kinetics suggested that increased binding was not attributable to positive co-operativity of the high affinity IL-1 receptor, but was due to increasing IL-1 receptor number. This observation was confirmed by equilibrium binding studies. Moreover, receptor numbers were not enhanced by de novo synthesis, nor release of receptors from an intracellular pool. The observed increases in surface ligand binding were most probably due to conversion of the surface pool of low affinity receptors into high affinity receptors.

By screening a human genomic library with an interleukin-1 receptor antagonist (IL-1ra) cDNA probe, we have isolated a 15 kb clone which contains the entire coding region of the gene as expressed in monocytes, and includes 6 kb of 5'-upstream sequence. The gene contains four exons which code for the secreted form of the IL-1ra, however, our clone does not contain the alternative first exon used to generate an intracellular form of the protein as the protein as found in epithelial cells. Analysis of the sequence reveals a consensus TATA box, and three Alu repeats, two of which are in the upstream region and one in intron 3. The sequence also reveals an 86 bp motif tandomly repeated four times within intron 2, and may reflect the polymorphism known to exist in this region of the gene. By in-situ fluorescence hybridization we have shown that the IL-1ra gene is found on the long arm of chromosome 2 and maps to 2q13-14.1. Previous studies have revealed that IL-1 alpha, and IL-1 beta and both type I and type II forms of the IL-1 receptor all map close to this region of chromosome 2.

In this study we have examined the effect of agents known to perturb certain signal transduction pathways on the biological responses of target cells to stimulation with interleukin-1 (IL-1). In the murine thymoma cell line EL4, IL-1 stimulation results in the secretion of interleukin-2 (IL-2), which was subsequently measured by proliferation of an IL-2-dependent cell line. Agents that elevated intracellular cAMP blocked or partially blocked IL-1 induction of IL-2 secretion, whereas agents that activated protein kinase C (PKC) resulted in a synergistic enhancement. Both pertussis and cholera toxins also inhibited IL-1-induced IL-2 secretion, although probably by acting at different levels. IL-1 simulation of human and murine fibroblasts resulted in release of prostaglandin E2. This response was inhibitable by pertussis toxin but not by cholera toxin, whereas co-stimulation of the fibroblasts with IL-1 and phorbol ester resulted in a synergistic response. Murine fibroblasts could also be stimulated to proliferate by IL-1, and this response was also inhibitable by pertussis toxin. These findings are consistent with coupling of the IL-1 receptor to a signalling pathway via a pertussis toxin substrate.

Murine Th1 and Th2 T cell lines differ in their responses to interleukin 1 (IL 1). Therefore, we examined two T-cell lines, D10.G4.1 (Th2) and MTg12B (Th1) in an attempt to correlate IL 1 receptor (IL 1R) expression with their IL 1 responsiveness. D10.G4.1 cells, which respond to IL 1, expressed two forms of the IL 1R, with molecular masses of approximately 80 kDa and approximately 60 kDa. In contrast, MTg12B cells failed to respond to IL 1 and only expressed the approximately 60 kDa receptor form. This suggests that the approximately 80 kDa receptor is essential for signaling. Expression of both IL 1R forms on D10.G4.1 cells could be inhibited by the anti-IL 4 antibody, 11B11. Antigen presentation reversibly upregulated both forms of the IL 1R, whereas stimulation with concanavalin A (ConA) and anti-CD3 only upregulated the approximately 60 kDa moiety. Upregulation of the approximately 80-kDa IL 1R by repeated antigenic stimulation resulted in a marked increase in sensitivity of D10.G4.1 cells to IL 1.

Using affinity crosslinking techniques, we have biochemically characterized the interleukin-1 (IL1) receptor and investigated its distribution on a range of murine and human cell lines. We show that two forms of IL1 receptor can be identified on the basis of specific crosslinking with 125I-IL1 alpha and 125I-IL1 beta. The two receptor forms have an approximate molecular mass of approximately 80 and approximately 60 kDa, and were found on both murine and human cells. Their relative distribution shows no clear cell lineage restriction and does not correlate with preferential binding of IL1 alpha or IL1 beta. Some cells, such as the T helper cell line D10.G4.1, express both forms of the receptor. Iodine 125-IL1 was crosslinked to the two receptor forms and a partial peptide map analysis of the two receptor/ligand complexes was performed. Comigration of the major partial peptide fragments suggests that the approximately 80 and approximately 60 kDa forms of the receptor may be differentially processed forms of the same protein. Treatment of the approximately 60 kDa IL1 receptor on Raji cells with N-glycanase reduced its molecular mass by 12 kDa, showing that this lower molecular mass form is a glycoprotein; glycosylation differences alone probably do not account for the difference in mass between the two forms.