Publications

A recombinant amphotropic retrovirus was used to introduce the protein-coding region of the IL-2 receptor cDNA derived from HUT-102 cells into human CEM leukemic T-cells that lack these receptors. CEM T-cells that contained the virus expressed functional IL-2 receptors that transiently mediated five- to tenfold increases in [3H]thymidine incorporation following the addition of picomolar quantities of IL-2. Although IL-2 responsiveness was subsequently lost, it could be reinduced by cellular activation with the OKT11 monoclonal antibody. This phenotype also proved unstable with progressive time in culture. Despite the loss of IL-2 responsiveness, the infected CEM T-cells continued to express Tac antigen and displayed 50 to 200 high-affinity IL-2 receptors per cell that bound IL-2 with a dissociation constant of 4.3 pM. This affinity is fully equivalent to that detected on activated normal T-cells (2 to 50 pM). The apparent molecular size of the Tac antigen on these cells (55,000 to 60,000 daltons) was comparable to that on normal activated T-cells but 5000 daltons larger than the aberrant IL-2 receptors on HUT-102 cells. These data demonstrate that expression of a human IL-2 receptor cDNA in human T-cells results in high-affinity IL-2 receptor display that transiently imparts an IL-2 responsive state of growth. These results also raise the possibility that the T11 surface receptor may play an important regulatory role in high-affinity IL-2 receptor expression.

High-affinity IL-2-R correspond to a membrane receptor complex composed of two different IL-2-binding proteins, the Tac antigen (alpha chain) and a 70-75 kD beta chain. Using cell lines that express either the alpha or the beta protein, we demonstrate that IL-2 internalization occurs when ligand is bound to the isolated beta chain, but not when it is bound to the isolated alpha chain. The kinetics of IL-2 internalization mediated by the intermediate-affinity beta chain were nearly identical to those of the high-affinity alpha/beta heterodimer (t1/2 of 10-15 min), and each type of receptor targeted the bound IL-2 for intracellular degradation in lysosomes. The beta chain thus appeared to provide the essential element necessary for ligand internalization by both types of IL-2-R.

Interleukin 2 (IL-2) receptors on activated T cells exist in high- and low-affinity configurations, both of which share a ligand-binding component known as the Tac protein. Although almost all binding of IL-2 to such cells was inhibited by an antibody to Tac, the predominant component of binding on the natural killer (NK)-like cell line YT was resistant to this reagent. The ligand-binding component on YT cells also differed from Tac in its affinity constant (Kd approximately 8.2 X 10(-10) M vs. Kd approximately equal to 1.1 X 10(-8) M for low-affinity Tac sites) and in its susceptibility to inhibition by certain antibodies to IL-2. When the YT cells were stimulated in a manner that induced expression of the Tac protein, the IL-2 binding sites were converted to a high-affinity configuration (Kd approximately 1.8 X 10(-11) M). Thus, the original binding component on unstimulated YT cells appeared to combine with Tac and IL-2 to produce a high-affinity receptor complex. Use of bifunctional crosslinking agents following ligand binding to unstimulated YT cells yielded covalent IL-2-receptor complexes of 83 and 90 kDa. These complexes were similar in size to those derived from high-affinity receptors on activated T cells and shared a similar fragmentation pattern upon proteolysis. These results demonstrate the existence of a second IL-2 binding component in addition to the Tac protein and suggest that this component combines with Tac and IL-2 to form high-affinity receptor sites.

Both interleukin 2 (IL-2) and epidermal growth factor (EGF) receptors exist in two forms that differ with respect to affinity for their ligand. Only the high-affinity receptors appear to be responsible for the proliferation signal delivered upon binding of the growth factor. Fibroblasts transfected with IL-2 receptor cDNA generate only low-affinity receptors for IL-2, but fusion of membranes from these fibroblasts with T-cell membranes converts some receptors to high affinity, indicating the involvement of a T cell-specific factor in the generation of high-affinity receptors. We have constructed a chimeric cDNA molecule containing the extracellular IL-2-binding domain of the IL-2 receptor cDNA and the transmembrane and intracellular tyrosine kinase domains of the EGF receptor cDNA. When transfected into fibroblasts, this IL-2-EGF receptor cDNA generated high-affinity receptors for IL-2. Moreover, fibroblasts transfected with the chimeric molecule were morphologically transformed and produced rapidly growing tumors in nude mice.

We evaluated the importance of pH, titratable acidity, and specific chemical composition in acid aerosol-induced bronchoconstriction in 8 asthmatic subjects. We administered aerosols of HCl and H2SO4 at pH 2.0 in an unbuffered state and buffered with glycine. The buffered acids were given in order of increasing titratable acidity (defined as the number of ml of 1 N NaOH required to neutralize 100 ml of acid solution to pH 7.0). Each set of buffered or unbuffered acid aerosols was given on a separate day and each aerosol was inhaled through a mouthpiece during 3 min of tidal breathing. Bronchoconstriction was assessed by measurement of specific airway resistance (SRaw) before and after inhalation of each aerosol. SRaw increased by more than 50% above baseline in 1 of 8 subjects after inhalation of unbuffered HCl and in no subjects after inhalation of unbuffered H2SO4, even at pH 2.0. In contrast, SRaw increased by greater than 50% in all 8 subjects after inhalation of HCl and glycine at pH 2.0 and 7 of 8 subjects after inhalation of H2SO4 and glycine at pH 2.0. The mean titratable acidity required to increase SRaw by 50% above baseline was calculated for each challenge by linear interpolation; these values for H2SO4 and glycine (5.1 ml of 1 N NaOH) and HCl and glycine (2.2 ml of 1 N NaOH) were slightly, but significantly, different (p = 0.01) and were considerably higher than the titratable acidity of the unbuffered acids at pH 2 (1.0 ml of 1 N NaOH).(ABSTRACT TRUNCATED AT 250 WORDS)

The airway edema that develops in guinea pigs after exposure to toluene diisocyanate (TDI) requires the presence of polymorphonuclear leukocytes (PMN). To determine whether this airway edema is mediated by the release of hydrogen peroxide from PMN, we treated animals intravenously with catalase bound to polyethylene glycol and examined the extravasation of Evans blue dye into the tracheal wall after exposure to air or 3 ppm TDI for 1 h. Catalase (25,000, 100,000, and 300,000 IU/kg) caused a dose-dependent inhibition of the TDI-induced increase in dye extravasation. However, treatment with catalase, inactivated at the peroxide binding site with 3-aminotriazole, inhibited dye extravasation after exposure to TDI as effectively as the equimolar 100,000 IU/kg dose of active catalase. Injection of polyethylene glycol alone was without effect. Dose-dependent decreases in extravascular migration of PMN and in circulating PMN also were noted after catalase treatment. These results suggest that the catalase preparations used in these studies inhibited the PMN-dependent airway edema by an effect other than hydrogen peroxide scavenging. Examination of this and other commercially available catalase preparations revealed trace concentrations of endotoxin at levels that could be responsible for the observed effects on PMN function. Treatment of animals with doses of Escherichia coli endotoxin similar to those inadvertantly administered to the catalase-treated groups (0.1 ng/kg to 100 ng/kg, intravenously) inhibited TDI-induced extravasation of Evans blue dye in a dose-dependent manner. These results suggest that contaminating endotoxin may contribute to some of the protective effects of preparations of catalase observed in previous studies of vascular permeability.