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Identification of soluble interleukin-1 binding protein in cell-free supernatants. Evidence for soluble interleukin-1
J G Giri1, R C Newton, R Horuk
1Medical Products Division, E. I. Du Pont De Nemours & Co., Glenolden Laboratory, Pennsylvania 19036.
Insights
Researchers identified a soluble interleukin-1 (IL-1) binding protein from Raji B-lymphoma cells. This protein, similar to the IL-1 receptor
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Interleukin-1 (IL-1) is a key cytokine in immune responses.
- Membrane-bound IL-1 receptors mediate cellular responses to IL-1.
- The existence and nature of soluble IL-1 binding proteins were previously unclear.
Purpose of the Study:
- To identify and characterize a soluble IL-1 binding protein in conditioned media from Raji human B-lymphoma cells.
- To determine the relationship between this soluble protein and the membrane-bound IL-1 receptor.
- To assess the functional properties and specificity of the soluble IL-1 binding protein.
Main Methods:
- IL-1 affinity chromatography for isolation of the binding protein.
- Trypsin treatment to confirm protein nature.
- Specificity assays using IL-1 alpha and IL-1 beta.
- Inhibition assays of IL-1 binding to Raji cells.
- Scatchard analysis for binding kinetics (KD).
- Gel chromatography for molecular mass determination.
Main Results:
- A soluble IL-1 binding protein was isolated from Raji cell conditioned media.
- The protein's binding to IL-1 was sensitive to trypsin, confirming its protein nature.
- The soluble protein exhibited specificity for IL-1, discriminating between IL-1 alpha and IL-1 beta.
- It inhibited IL-1 binding to Raji cells in a dose-dependent manner.
- Scatchard analysis revealed a KD of 5.1 nM for IL-1 beta binding.
- Gel chromatography indicated a molecular mass of 35-45 kDa.
Conclusions:
- The identified soluble IL-1 binding protein is likely the extracellular binding domain of the membrane-bound Raji IL-1 receptor.
- This soluble protein possesses specific IL-1 binding characteristics analogous to its membrane-bound counterpart.
- The findings contribute to understanding IL-1 receptor regulation and signaling.
Abstract:
This study describes the identification and characterization of a soluble interleukin-1 (IL-1) binding protein in the conditioned media from Raji human B-lymphoma cells. The soluble IL-1 binding material was isolated by IL-1 affinity chromatography, and treatment with trypsin decreased its ability to bind to IL-1 demonstrating its protein nature. The soluble IL-1 binding protein was specific for IL-1 and was able to discriminate between Il-1 alpha and IL-1 beta in a manner analogous to the membrane-bound Raji IL-1 receptor. The specificity of the IL-1 binding protein was further established in two ways. 1) Cell-free supernatants from Raji "receptor-negative" cells did not contain any IL-1 binding protein, thus ruling out nonspecific interactions between IL-1 and a serum or other protein present in the conditioned medium; and 2) the soluble binding protein inhibited IL-1 binding to Raji cells in a dose-dependent manner. Scatchard analysis of IL-1 beta binding showed the dissociation constant (KD) to be 5.1 nM for the soluble IL-1 binding protein compared with 0.8 nM for the membrane-bound IL-1 receptor. Gel chromatography of the soluble binding protein yielded a major peak of IL-1 binding activity with a molecular mass of 35-45 kDa. The characteristics of the soluble IL-1 binding protein described above are consistent with those of the extracellular binding domain of the membrane-bound Raji IL-1 receptor.
