Kinetics of receptor-mediated uptake and processing of interferon-α₂a and tumor necrosis factor-α by human tumor cells

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.