Uptake and destruction of ¹²⁵I‐CSF‐1 by peritoneal exudate macrophages

The binding and uptake of the colony‐stimulating factor CSF‐1 by peritoneal exudate macrophages (PEM) from lipopolysaccharide insensitive C3H/HeJ mice was examined at 2°C, and at 37°C. At 2°C, ¹²⁵I‐CSF‐1 was bound irreversibly to the cell surface. At 37°C, 90% of the cell surface associated ¹²⁵I‐CSF‐1 was rapidly internalized and subsequently degraded and the remaining 10% dissociated as intact ¹²⁵I‐CSF‐1. Thus classical equilibrium or steady state methods could not be used to quantitatively analyze ligand–cell interactions at either temperature, and alternative approaches were developed. At 2°C, the equilibrium constant (K_d ⩽ 10⁻¹³M) was derived from estimates of the rate constants for the binding (k_on ≃ 8 × 10⁵M⁻¹s⁻¹) and dissociation (k_off ⩽ 2 × 10⁻⁷s⁻¹) reactions. At 37°C, the processes of dissociation and internalization of bound ligand were kinetically competitive, and the data was formally treated as a system of competing first order reactions, yielding first order rate constants for dissociation, k_off = 0.7 min⁻¹ (t_1/2 = 10 min) and internalization, k_in = 0.07 min⁻¹ (t_1/2 = 1 min). Approximately 15 min after internalization, low‐molecular weight ¹²⁵I‐labeled degradation products began to appear in the medium. Release of this degraded ¹²⁵I‐CSF‐1 was kinetically first order over three half‐lives (K_d = 4.3 × 10⁻² min⁻¹, t_1/2 = 16 min). Thus CSF‐1 binds to a single class of receptors on PEM, is internalized with a single rate limiting step, and is rapidly destroyed without segregation into more slowly degrading intracellular compartments.