Kinetics of Receptor-Mediated Uptake and Processing of Interferon-α₂a and Tumor Necrosis Factor-α by Human Tumor Cells

Abstract

The kinetics of uptake and processing of recombinant human interferon-a₂a (IFN) and recombinant human tumor necrosis factor-a (TNF) were studied in human epithelial rumor cell lines differing in sensitivity to growth inhibition by IFN and TNF. Concentrations of [¹²⁵I]IFN or [¹²⁵I]TNF at the cell surface and internalized by confluent cell monolayers incubated at 37°C were measured as a function of time. Cells incubated with [¹²⁵I]IFN exhibited transient maxima of surface-associated and internalized [^I25I]IFN after 1-2 hr of incubation followed by a steady-state attained after approximately 6 hr of exposure to [¹²⁵I]IFN. Concentration of [¹²⁵I]TNF associated with the plasma membrane displayed a maximum within 20 min of incubation. Internalized radioactivity increased with time and did not achieve a steady state. We analyzed the time-dependent concentrations of membrane-associated and internalized cytokines by use of a compartmental model. This model describes changes in concentrations of free surface receptors, of ligand-receptor complex at the membrane and of internalized ligand and contains a term for receptor recycling. The rate constants for concentration changes were evaluated by fitting model functions to data. The best fits for IFN were obtained without receptor recycling. The best-fit values for the endo-cytotic rate constant (k_tlFN) varied among cell lines from 2.4x10⁻⁴ to 7.8 x10⁻⁴ sec⁻¹. To obtain fits to time-dependent concentrations of surface-associated and internalized [¹²⁵I]TNF, introduction of a term for receptor recycling was required. Best-fit values for k, _TNF ranged among cell lines from 8.4x10⁻⁴ to 2.5x10⁻³ sec⁻¹. For every cell line, the value of k_lTNF was larger than the value of k_clFN. We tested the significance of these differences by substituting K.IFN ^tor K.TNT ^as fixed parameters in fits to data for TNF and v.v., respectively. Under these conditions, fits were significantly worse. Our data indicate that recycling is insignificant in kinetics of Type-I IFN receptor; recycling is necessary to explain the kinetics of TNF receptor. Upon binding, TNF is taken up by cells faster than IFN and is eliminated from cells more slowly than IFN.

Key Words:

• receptor-mediated endocytosis
• interferon Type-I receptor
• tumor necrosis factor receptor
• kinetic analysis
• compartmental modeling