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Production and purification of bovine monocyte-derived interleukin 1
1Department of Pathobiological Sciences, University of Wisconsin, Madison 53706.
Insights
Researchers investigated bovine interleukin 1 (IL-1) production by monocytes. Lipopolysaccharide (LPS) and fetal bovine serum (FBS) enhanced IL-1 release, and purification revealed multiple IL-1 molecular species.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Limited understanding of bovine interleukin 1 (IL-1) biological and molecular characteristics.
- Need to elucidate the role of bovine IL-1 in bovine immunology.
Purpose of the Study:
- Investigate parameters of bovine IL-1 production by monocytes in vitro.
- Purify bovine IL-1 to characterize its molecular nature and facilitate future functional studies.
Main Methods:
- Isolation and culture of bovine monocytes.
- Stimulation with Escherichia coli lipopolysaccharide (LPS) and fetal bovine serum (FBS).
- Measurement of IL-1 activity using a murine thymocyte assay.
- Purification of IL-1 using size-exclusion and reverse-phase high-performance liquid chromatography (HPLC).
Main Results:
- Bovine monocytes constitutively released IL-1 activity.
- LPS significantly augmented monocyte IL-1 release.
- FBS enhanced LPS-stimulated IL-1 release.
- Size-exclusion HPLC indicated an IL-1 molecular weight of approximately 18,000 daltons.
- Reverse-phase HPLC revealed at least three distinct molecular species of bovine IL-1.
Conclusions:
- Characterized bovine IL-1 production by monocytes in vitro.
- Developed a purification protocol for bovine IL-1.
- Identified multiple molecular forms of bovine IL-1, paving the way for further functional research.
Abstract:
Few studies have addressed the biological and molecular nature of bovine interleukin 1 (IL-1). In an effort to increase our understanding of the role of bovine IL-1 in bovine immunology, we investigated various parameters of its production by LPS-stimulated monocytes in vitro. Bovine monocytes isolated by our methods constitutively released IL-1 activity, as measured by the murine thymocyte IL-1 assay. Monocyte release of IL-1 activity was further augmented when the cells were incubated with 0.005-10 micrograms per ml of Escherichia coli lipopolysaccharide (LPS). The presence of 1, 5, or 10 percent heat-inactivated fetal bovine serum (FBS) enhanced LPS-stimulated bovine monocyte release of IL-1 activity as compared with monocytes cultured under serum-free conditions. We used a combination of size-exclusion and reverse-phase high-performance liquid chromatography (HPLC) to purify bovine IL-1 from serum-free monocyte culture supernatants. Size-exclusion HPLC resulted in a single peak of biological activity with an approximate molecular weight of 18,000 daltons. Further purification by reverse-phase HPLC demonstrated at least three major molecular species with IL-1 activity. Besides providing information about production of IL-1 by bovine monocytes in vitro, this study also describes a protocol to purify bovine IL-1 for future studies addressing its biological functions.
