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Receptor binding studies were performed on the interleukin-1 (IL-1) sensitive T-helper cell line D10S, a stable line which proliferates to subfemtomolar concentrations of IL-1 in the absence of mitogens. IL-1 binds in a specific and saturable manner and Scatchard analysis at 4 degrees C reveals one class of binding affinity. On D10S cells, the Kd for IL-1 is 227 pM +/- 80, with 11,000 (range 3,300 to 23,800) sites per cell. EL4.6.1 cells, which are less sensitive to IL-1, bind with a single class of high affinity sites (55 pM; 4,000 sites). D10S cells incubated 18 h with IL-1 display reduced IL-1 receptor (IL-1R) numbers and affinities, consistent with reduced (75%, p less than 0.005) proliferation to subsequent IL-1; preincubation with IL-4 increases the number of IL-1R which is associated with increased (200%, p less than 0.001) proliferation to IL-1. The molecular mass of the major (80 kD) IL-1R binding [125I]IL-1 alpha on D10S cells was consistently observed at 73 kD as compared to the 80 kD molecule on the EL4 cells. On the other hand, crosslinking studies with [125I]IL-1 beta on D10S cells revealed a novel 46 kD band on gradient SDS-PAGE corresponding to a binding protein of 29 to 30 kD, which is antigenically distinct from the 80 kD IL-1R. Crosslinking of D10S or EL4 cells at 4 degrees C in the presence of phytohemagglutinin (PHA) and labeled IL-1 enhanced the appearance of the 30 kD IL-1 binding protein. The findings are consistent with a two-chain model for the IL-1R, although Scatchard analysis did not consistently indicate two classes of affinities. IL-1 binding to the 80 kD protein may form a heteroduplex with the 30 kD IL-1R which could account for the presence of the 120 to 130 kD IL-1 crosslinked proteins observed by several investigators.

Most studies have shown that interleukin-1 (IL-1) acts as a helper or co-stimulator in T-lymphocyte activation and proliferation by mitogens or antigens. We describe here a stable subclone (D10S) of the murine D10.G4.1 helper T-cell which proliferates to subfemtomolar (attomolar) concentrations of IL-1 beta or alpha in the absence of mitogens. D10S cells have been maintained in culture for over two years without splenic cell feeder layers nor antigen stimulation. Detection of proliferation can be made by either uptake of tritiated thymidine at 72 h or in 48 h by a colorimetric assay which measures mitochondrial dehydrogenases; the latter assay is rapid and inexpensive. D10S cells are distinct from the parent clone D10.G4., which requires mitogens for IL-1 activity. IL-1-induced proliferation is independent of the elaboration of IL-2, IL-4, or IL-6, although these cells proliferate to these lymphokines at considerably higher concentrations when compared to IL-1. The D10S cells proliferate in direct correlation to the duration of IL-1 presence in the culture. We found no evidence that IL-1 induced more IL-1 in these cells. The subclone is highly specific for IL-1: proliferation was not observed to endotoxin, human or murine interferon-gamma (IFN gamma), tumor necrosis factor (TNF), lymphotoxin, or granulocyte-macrophage colony stimulating factor (GM-CSF). There was no suppressive effect of transforming growth factor (TGF beta). Only at high concentrations (100 ng/ml) did IL-6 induce proliferation. We conclude that this stable, feeder layer-free cell line is highly sensitive to IL-1 which acts as a direct stimulant for these cells; they are also useful for bioassays as well as the study of IL-1 receptors as described in the accompanying paper.