Mitchell Dukovich

Novel interleukin 2 (IL-2) receptor appears to mediate IL-2-induced activation of natural killer cells.

A novel IL-2 receptor, distinct from the Tac protein, has been identified on the surface of purified human natural killer (NK) cells by chemical cross-linking of 125I-IL-2. This protein is approximately 70,000 D in size (p70) and appears to be identical to the recently recognized second subunit of the human high affinity IL-2 receptor complex. Scatchard analysis of 125I-IL-2 binding to purified NK cells revealed approximately 2,300 p70 binding sites per cell with an apparent dissociation constant of 200 pM, a value intermediate between the previously recognized high and low affinity forms of the human IL-2 receptor. The monoclonal anti-Tac antibody did not inhibit the cross-linking of 125I-IL-2 to the p70 binding sites present on NK cells. Functionally, the addition of high concentrations of recombinant IL-2 to the enriched NK cells promoted a rapid augmentation of cytolytic activity and a more delayed increase in cellular proliferation. Anti-Tac effectively blocked the IL-2-induced proliferative response in these cells, but failed to alter the enhancement of cytotoxicity. Analysis of NK cytoplasmic RNA isolated at various time points after IL-2 stimulation revealed the rapid induction of c-myb and Tac gene expression that was also not inhibited by the anti-Tac antibody. These findings suggest that IL-2 binding to the p70 receptor constitutively expressed on the surface of NK cells may mediate both the development of increased cytolytic activity and rapid changes in gene expression. The activation of the Tac gene may in turn permit the formation of the high affinity IL-2 receptor complex (comprised of at least the Tac and p70 proteins) that appears to transduce the requisite signals involved in NK cell proliferation.

[37] Murine interleukin 1

Publisher Summary This chapter describes the basic protocols for the production and purification of IL-1 from the P388D 1 macrophage cell line, along with assays used to quantitate IL-1 activity. A basic protocol for generating goat antibodies to IL-1 is also explained. Interleukin I (IL-1) 1 is a polypeptide of 12,000 to 17,000 molecular weight that functions as a major communication signal between the macrophage and other cell types that are involved in immune and inflammatory responses. This polypeptide is produced by peripheral blood, peritoneal, alveolar, and placental macrophages. In addition, a number of IL-l-producing glioma and keratinocyte tumor cell lines are described. Murine IL-1 was initially purified to homogeneity as a result of (1) the use of the P388D1 murine macrophage cell line and (2) the use of a superinduction protocol for producing relatively high levels of IL- 1. The chapter further discusses the purification of murine interleukin 1.

New Insights into the Structure of High-Affinity Interleukin-2 Receptors

Publisher Summary Interleukin-2 (IL-2) is a 15,500-Da lymphokine produced by antigen- or mitogen-activated τ cells that functions as an autocrine or paracrine growth factor for τ cells and possibly select populations of β cells. IL-2 exerts its biological effects by binding to specific cell surface receptors that transduce intracellular signals leading to τ-cell proliferation and expansion of the antigen reactive population of τ cells. IL-2 receptors are not constitutively expressed on the surface of resting τ cells, but they are induced in the process of τ-cell activation. Recent studies have led to the identification of both high- and low-affinity forms of the IL-2 receptor. The high-affinity receptors appear to mediate the growth response to IL-2 while no clear function has yet been attributed to the low-affinity receptors. Insights into the structural difference in high- and low-affinity receptors have emerged using I-labeled IL-2 for affinity cross-linking. These approaches have led to the detection of a second human IL-2 binding protein approximately 70,000–75,000 Da in size that assembles with the 55,000-Da Tac antigen, thus forming a high-affinity receptor complex. The p70 protein alone binds IL-2 with only intermediate affinity, lacks reactivity with the anti-Tac monoclonal antibody, appears to interact with IL-2 at an epitope distinct from Tac, and is capable of transducing intracellular signals and mediating endocytosis of bound ligand. This chapter discusses the biochemical and molecular properties of these two subunits of the high-affinity IL-2 receptor.

The Human High Affinity Interleukin-2 Receptor

A novel human interleukin-2 (IL-2) receptor has been identified on the surface of resting and activated human T cells, B cells, and large granular lymphocytes (natural killer cells) by chemical crosslinking of 125I-IL-2. This receptor has an estimated size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and binds IL-2 with an affinity that is intermediate between the previously recognized high and low affinity forms of the IL-2 receptor. High affinity crosslinking of 125I-IL-2 results in the labeling of both the p70 protein and the Tac antigen, a well-characterized membrane IL-2 receptor, and the crosslinking to both proteins is inhibited by the anti-Tac antibody. Transfection of MLA-144 T cells, which display only intermediate affinity p70 IL-2 receptors (Kd of 0.6–1.2 nM), with the Tac cDNA reconstituted high affinity (Kd of 30–75 pM) IL-2 receptor expression in these cells. IL-2 binding to these reconstituted high affinity receptors was blocked by anti-Tac; however, this antibody did not alter IL-2 binding to the intermediate affinity p70 receptors. Similarly, high affinity IL-2 receptors were reconstituted on the “natural killer-like” YT cells which display 8,000–10,000 p70 receptors and little or no Tac by activation of Tac gene expression with forskolin or IL-1. Together, these data suggest that the high affinity IL-2 receptor corresponds to a membrane receptor complex composed of two different IL-2 binding proteins.