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Kinetics of receptor-mediated uptake and processing of interferon-alpha 2a and tumor necrosis factor-alpha by human
S L Dunne1, Z Bajzer, S Vuk-Pavlović
1Division of Developmental Oncology Research, Mayo Foundation, Rochester, Minnesota 55905.
Abstract:
The kinetics of uptake and processing of recombinant human interferon-alpha 2a (IFN) and recombinant human tumor necrosis factor-alpha (TNF) were studied in human epithelial tumor 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 degrees C were measured as a function of time. Cells incubated with [125I]IFN exhibited transient maxima of surface-associated and internalized [125I]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 endocytotic rate constant (ke,IFN) varied among cell lines from 2.4 x 10(-4) to 7.8 x 10(-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 ke,TNF ranged among cell lines from 8.4 x 10(-4) to 2.5 x 10(-3) sec-1. For every cell line, the value of ke,TNF was larger than the value of ke,IFN. We tested the significance of these differences by substituting ke,IFN for ke,TNF 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.
Insights
Recombinant human interferon-alpha 2a (IFN) and tumor necrosis factor-alpha (TNF) uptake kinetics differ significantly. TNF is internalized faster and cleared slower than IFN, with TNF receptor recycling being essential, unlike IFN receptor kinetics.
Area of Science:
- Cellular and Molecular Biology
- Immunology
- Pharmacology
Background:
- Recombinant human interferon-alpha 2a (IFN) and tumor necrosis factor-alpha (TNF) are critical cytokines with distinct biological roles.
- Understanding their cellular uptake and processing kinetics is crucial for elucidating their therapeutic mechanisms and potential side effects.
- Human epithelial tumor cell lines exhibit varying sensitivities to IFN and TNF, suggesting differences in receptor-mediated cellular responses.
Purpose of the Study:
- To investigate and compare the kinetic differences in cellular uptake and processing between recombinant human interferon-alpha 2a (IFN) and tumor necrosis factor-alpha (TNF).
- To analyze the role of receptor recycling in the internalization and processing of IFN and TNF in human epithelial tumor cells.
- To quantify the rate constants for endocytosis and other kinetic parameters for both cytokines in different cell lines.
Main Methods:
- Utilized radiolabeled [125I]IFN and [125I]TNF to quantify cell surface-bound and internalized cytokines over time in confluent human epithelial tumor cell monolayers at 37°C.
- Employed a compartmental model incorporating free surface receptors, ligand-receptor complexes, internalized ligand, and receptor recycling to analyze time-dependent cytokine concentrations.
- Determined rate constants for concentration changes by fitting model functions to experimental data, including endocytotic rate constants (ke,IFN and ke,TNF).
Main Results:
- IFN exhibited transient surface and internalized maxima within 1-2 hours, reaching a steady-state after approximately 6 hours, with best fits achieved without receptor recycling.
- TNF showed a plasma membrane association maximum within 20 minutes, with increasing internalized radioactivity over time and no steady-state, requiring receptor recycling for model fit.
- Endocytotic rate constants for TNF (ke,TNF) were consistently higher than for IFN (ke,IFN) across all cell lines, with statistically significant differences observed.
Conclusions:
- Receptor recycling is insignificant for the kinetics of the Type-I IFN receptor but is necessary to explain the kinetics of the TNF receptor.
- TNF is internalized more rapidly and eliminated more slowly from cells compared to IFN, indicating distinct cellular processing pathways.
- The observed kinetic differences in uptake and processing highlight distinct receptor-mediated mechanisms for IFN and TNF signaling in tumor cells.