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Biological and structural properties of MIP-1 alpha expressed in yeast
J M Clements1, S Craig, A J Gearing
1British Bio-technology, Cowley, Oxford.
Abstract:
The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).
Insights
Macrophage inflammatory protein-1 alpha (MIP-1 alpha) inhibits hematopoietic stem cell growth and protects stem cells in vivo. This study characterized MIP-1 alpha
Area of Science:
- Immunology
- Hematology
- Molecular Biology
Background:
- Macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are related cytokines.
- MIP-1 alpha is known to inhibit hematopoietic stem cell growth.
- The specific properties of MIP-1 alpha require further investigation.
Purpose of the Study:
- To express and purify MIP-1 alpha.
- To characterize the structure and function of MIP-1 alpha.
- To confirm its in vitro and in vivo activity on stem cells.
Main Methods:
- Expression of MIP-1 alpha in yeast and purification.
- Structural analysis using circular dichroism and fluorescence spectroscopy.
- In vitro assays using murine progenitor cells (FACS-1, FDCP-mix A4, CFU-S).
- In vivo studies involving bolus injection in mice and assessment of stem cell protection and proliferation.
Main Results:
- Purified MIP-1 alpha exhibits similar secondary and tertiary structures to platelet factor 4 and interleukin 8.
- MIP-1 alpha confirmed to inhibit in vitro stem cell growth at concentrations of 0.15 µg/ml.
- In vivo administration of MIP-1 alpha protected stem cells from tritiated thymidine-induced killing.
- MIP-1 alpha enhanced the proliferation of granulocyte-macrophage progenitor cells (GM-CFC).
Conclusions:
- MIP-1 alpha is a structurally conserved cytokine with potent in vitro and in vivo effects on hematopoietic stem cells.
- MIP-1 alpha demonstrates both inhibitory effects on stem cell growth and stimulatory effects on progenitor cell proliferation.
- These findings highlight MIP-1 alpha's complex role in hematopoiesis and immune regulation.