Differential regulation of interleukin‐6 receptors by interleukin‐6 and interferons in multiple myeloma cell lines

Abstract

Interleukin-6 (IL-6) mediates pleiotropic functions through specific receptors (IL-6R) composed of an 80-kDa binding protein, associated with a non-ligand binding protein (gp130) which transduces the signal. Because IL-6 is the major tumor growth factor in multiple myeloma, we investigated the regulation of IL-6R in two human multiple myeloma cell lines. Binding experiments with 125I-labeled IL-6 showed that IL-6R were expressed at a high density on RPMI-8226 cells (15 000 receptors/cell), but no specific binding was detected on XG-1 cells, whose growth depends on the presence of exogenous IL-6. However, when IL-6 was removed from the culture medium, high-affinity IL-6R appeared on the surface of XG-1 cells (5300 sites/cell). Treatment of RPMI-8226 cells with IL-6 reduced the number of IL-6R without changing their affinity. This reduction was dose dependent and was not affected by acid treatment which dissociates ligand-receptor complexes. Cross-linking experiments showed that the formation of one IL-6/receptor complex of 160 kDa markedly decreased upon IL-6 treatment, while the other complex of 190 kDa became undetectable. These data provide evidence for ligand-induced down-regulation of membrane IL-6R expression in myeloma cells. Treatment of RPMI-8226 cells with interferon-alpha (IFN-alpha), which inhibits the growth of these cells, stimulated IL-6R expression and increased the formation of the 160-kDa IL-6/receptor complex. This stimulation was specific for IFN-alpha, since IFN-gamma reduced the number of IL-6R. These data indicate that, in myeloma cells, IL-6R are differentially regulated by IL-6 and IFN-alpha.

Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-6-dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains.

“…4 B) and myeloid U937 cells ( Fig. 6 C); similar observations were made by Lasfar et al (49). Importantly, these investigators also reported IFN-a-mediated gp8O down-regulation on another IL-6-dependent myeloma cell line (XG-l) and, similarly, IFN-a-mediated gp8O up-regulation on another IL-6-independent myeloma cells (RPMI-8226).…”

Establishment and characterization of a human stroma-dependent myeloma cell line (MM5.1) and its stroma-independent variant (MM5.2)

“…Despite the apparent involvement of IL-6 in the growth of MM5.1 cells, we failed to demonstrate a significant expression of the IL-6 receptor (gp80) on the surface of the cells when they were cocultured with bone marrow stroma. This observation is consistent with the finding of Lasfar et al 33 who found a down-regulation of membrane IL-6 receptors on the myeloma cell line XG1 in the presence of IL-6, although this line is completely IL-6 dependent for its growth. This may indicate that a low number of surface gp80 molecules (even under the detection limit in FACS analysis) might be sufficient to mediate IL-6 signal transduction.…”

Engineering human interleukin-6 to obtain variants with strongly enhanced bioactivity.

“…The bioactivity was more enhanced when hIL-6 and its analogs were acting through soluble rather than membrane-bound forms of hIL-6Ra, and when the shIL-6Ra concentration was kept below or around the physiological levels present in human sera. Indeed, hIL-6 has been shown to down-regulate the surface expression of hIL-6Ra in A375 (Schwabe et al, 1994) and XG-1 (Lasfar et al, 1994) cells, probably as a consequence of a ligand-dependent increase in the internalization rate (Dittrich et al, 1994). Although we cannot exclude that selected high affinity mutants bind the soluble form of hIL-6Ra more tightly than the cellular form, we rather favor the idea that receptor clustering on the surface of cytokine-stimulated cells increases the local concentration of hIL-6Ra to levels well above the KD of the superbinders.…”

“…Finally, it cannot be excluded that the maximum bioactivity attainable is limited by the faster internalization rate of the membrane-bound versus the soluble hIL-6Rc. Indeed, hIL-6 has been shown to down-regulate the surface expression of hIL-6Ra in A375 (Schwabe et al, 1994) and XG-1 (Lasfar et al, 1994) cells, probably as a consequence of a ligand-dependent increase in the internalization rate (Dittrich et al, 1994). Potential advantages of hlL-6 high affinity variants Several studies have proposed hIL-6 as a thrombopoietic, as well as an anticancer agent, due to well defined effects on hIL-6Ra-bearing cells (Akira et al, 1993).…”