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A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor
Published on: June 27, 2011
Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo
F Sayani1, F A Montero-Julian, V Ranchin
1Alberta Bone Marrow/Stem Cell Transplant Program and Division of Hematology, Departments of Medicine and Oncology, University of Calgary, Calgary, Alberta, Canada.
Abstract:
On the basis of the finding of alternatively spliced mRNAs, the alpha-subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMRalpha) and a soluble form (solGMRalpha). However, only limited data has been available to support that the solGMRalpha protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMRalpha would have the potential to also produce solGMRalpha. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMRalpha (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMRalpha-specific band by Western blot, whereas a tmGMRalpha-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMRalpha. The solGMRalpha protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMRalpha. A human solGMRalpha ELISA was developed that confirmed the presence of solGMRalpha in supernatant conditioned by the tmGMRalpha-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMRalpha in normal human plasma (36 +/- 17 pmol) and provided data suggesting that plasma solGMRalpha levels can be elevated in acute myeloid leukemias. (Blood. 2000;95:461-469)
Insights
The soluble form of the GM-CSF receptor alpha-subunit (solGMRalpha) is present in vivo, produced by hematopoietic cells expressing the membrane-bound form (tmGMRalpha). This finding has implications for understanding acute myeloid leukemias.
Area of Science:
- Hematology
- Molecular Biology
- Immunology
Background:
- The alpha-subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to exist in membrane-spanning (tmGMRalpha) and soluble (solGMRalpha) forms, based on mRNA splicing.
- Limited in vivo evidence existed for the solGMRalpha protein product.
Purpose of the Study:
- To investigate whether hematopoietic cells expressing tmGMRalpha also produce solGMRalpha in vivo.
- To characterize the presence and properties of solGMRalpha in biological samples.
Main Methods:
- Analysis of conditioned media from human leukemic cell lines (HL60, U937, THP1, K562) using functional assays, Western blotting, and immunologic means.
- Examination of leukapheresis products and freshly isolated human neutrophils.
- Development and application of a human solGMRalpha ELISA.
- Determination of dissociation constant (Kd) for in vivo solGMRalpha.
Main Results:
- Leukemic cell lines expressing tmGMRalpha secreted GM-CSF binding activity and a solGMRalpha-specific band, unlike the tmGMRalpha-negative cell line.
- solGMRalpha was detected in leukapheresis products and neutrophil-conditioned media.
- In vivo solGMRalpha exhibited a Kd similar to recombinant solGMRalpha.
- ELISA confirmed solGMRalpha presence in various hematopoietic cell supernatants and detected steady-state levels in normal human plasma.
- Elevated plasma solGMRalpha levels were suggested in acute myeloid leukemias.
Conclusions:
- Hematopoietic cells expressing tmGMRalpha are capable of producing solGMRalpha in vivo.
- solGMRalpha is detectable in various hematopoietic cell types and human plasma.
- Plasma solGMRalpha may serve as a biomarker for acute myeloid leukemias.
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