Recombinant human GM-CSF protein

Summary

• High purity human protein (Uniprot number: P04141)
• 14.6 kDa (monomer)
• >98%, by SDS-PAGE quantitative densitometry
• Expressed in E. coli
• Animal origin-free (AOF) and carrier protein-free
• Manufactured in Qkine's Cambridge, UK laboratories
• Lyophilized from acetonitrile, TFA
• Resuspend in sterile-filtered water at >50 µg/ml, add carrier protein if desired, prepare single use aliquots and store frozen at -20 °C (short-term) or -80 °C (long-term).

Featured applications

• Asymmetric stem cell self-renewal in human keratinocytes
• Differentiation of peripheral blood monocytes to myeloid lineages
• Differentiation of stem cells into erythrocytes and megakaryocytes
• Generation of immature dendritic cells
• Generation of iPSC-derived granulocytes
• Polarization of macrophages

Bioactivity

Purity

Recombinant GM-CSF migrates as a major band at approximately 14.6 kDa in non-reducing (NR) ad reduced (R) conditions. No contaminating protein bands are present. The purified recombinant protein (3 µg) was resolved using 15% w/v SDS-PAGE in reduced (+β-mercaptoethanol, R) and non-reduced conditions and stained with Coomassie Brilliant Blue R250. Data from Qk076 batch #204525.

Qkine GM-CSF is as biologically active as the comparable alternative supplier protein. Stimulation of proliferation of TF-1 cells with Qkine GM-CSF (Qk076, green) and alternative supplier GM-CSF (Supplier B, black). Cells were treated in triplicate with a serial dilution of GM-CSF for 48 hours and proliferation measured using the CellTiter-Glo (Promega) luminescence assay.

Protein background

Granulocyte-macrophage colony-stimulating factor (GM-CSF), or colony-stimulating factor 2 (CSF-2), is a secreted glycoprotein belonging to the hematopoietic family of growth factors. GM-CSF induces the development of myeloid, erythroid, and megakaryocytic cell lineages[1-4]. It plays a crucial role in the survival and recruitment of myeloid cells to inflammation sites and is also involved in regulating T-cell function [1,3,5-7]. Its role in the differentiation and proliferation of myeloid progenitors towards macrophages and dendritic cells was first identified, followed by its role as a cytokine during inflammation [1,3,5,6]. Other growth factors involved in monocyte/macrophage regulation include M-CSF and IFN-γ [8,9].

GM-CSF is a monomer composed of 144 amino acids with a molecular weight of approximately 14.6 kDa [10]. It is produced by various cells including activated T cells, B cells, macrophages, monocytes, mast cells, vascular endothelial cells, and fibroblasts [1,11]. GM-CSF binds to the GM-CSF receptor (GM-CSFR or CSF2R) expressed by myeloid cells and some non-hematopoietic cells [1,12]. This receptor is not present in lymphoid cells. GM-CSFR is composed of an α chain and β chain. The ternary complex of GM-CSF to GM-CSFR assembles into a dodecamer or higher-order complex and activates the downstream signaling pathways: JAK2/ STAT5, Ras/ ERK, NF-κB, and PI3K pathways [1,7].

GM-CSF is commonly used in cell culture with FGF-2 to stimulate the differentiation and maturation of human-induced pluripotent stem cells or peripheral blood monocytes to myeloid cells [13-15]. Myeloid cells include monocytes such as macrophages and dendritic cells, and granulocytes such as neutrophils, eosinophils, and basophils. The differentiation into macrophages is commonly performed using media supplemented with GM-CSF or M-CSF. GM-CSF and M-CSF can increase the glycolytic activity of macrophages, as well as influence their polarization and shape [16-19]. GM-CSF, along with Lipopolysaccharide and IFN-γ, favor an M1-polarized macrophage of a “fried-egg” shape [19-22]. In the case of dendritic cells, GM-CSF and IL-4 can generate immature dendric cells [23,24]. Further maturation is achieved using a mixture of IL-1β, IL-6, TNF-α, PGE2, and IL-10 [24]. Finally, GM-CSF is used as a growth factor to differentiate stem cells into erythrocytes and megakaryocytes with IL-3 as well as to stimulate and promote the self-renewal of keratinocytes [4,25,26].

GM-CSF is involved in the pathogenesis of several inflammatory and immune disorders such as asthma, rheumatoid arthritis, and multiple sclerosis [1,27-29]. As such, several preclinical models and clinical models have evaluated that GM-CSF may be a viable therapeutic target [2,30]. GM-CSF-based vaccines have been shown to enhance the immune response to vaccines by promoting the maturation and activation of dendritic cells in preclinical models [31]. GM-CSF has also been approved for patients with cancer undergoing chemotherapy as well as for bone marrow transplantation to stimulate the production of myeloid cells. However, further insights are needed on GM-CSF pro-tumorigenic effects to harness its therapeutic potential [32,33]. Finally, due to its role in embryonic development, GM-CSF has been proposed as a culture media supplement for in vitro fertilization where its potential and effectiveness will be confirmed with more randomized controlled trials [34].

Background references

1. Lotfi, N. et al. Roles of GM-CSF in the pathogenesis of autoimmune diseases: An update. Front Immunol 10, 1265 (2019). doi: 10.3389/fimmu.2019.01265
2. Lee, K. M. C., Achuthan, A. A. & Hamilton, J. A. GM-CSF: A Promising Target in Inflammation and Autoimmunity. Immunotargets Ther 9, 225 (2020). doi: 10.2147/ITT.S262566
3. Burgess, A. W. & Metcalf, D. The Nature and Action of Granulocyte-Macrophage Colony Stimulating Factors. Blood 56, 947–958 (1980). PMID: 7002232
4. Robinson, B. E., McGrath, H. E. & Quesenberry, P. J. Recombinant murine granulocyte macrophage colony-stimulating factor has megakaryocyte colony-stimulating activity and augments megakaryocyte colony stimulation by interleukin 3. Journal of Clinical Investigation 79, 1648–1652 (1987). doi: 10.1172/JCI113002
5. Hamilton, J. A. Cytokines Focus GM-CSF in inflammation. Journal of Experimental Medicine 217, (2019). doi: 10.1084/jem.20190945
6. Hamilton, J. A., Stanley, E. R., Burgess, A. W. & Shadduck, R. K. Stimulation of macrophage plasminogen activator activity by colony-stimulating factors. J Cell Physiol 103, 435–445 (1980). doi: 10.1002/jcp.1041030309
7. Hansen, G. et al. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation. Cell 134, 496–507 (2008). doi: 10.1016/j.cell.2008.05.053
8. Breen, F. N., Hume, D. A. & Weidemann, M. J. Interactions among granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and IFN-gamma lead to enhanced proliferation of murine macrophage progenitor cells. The Journal of Immunology 147, 1542–1547 (1991). PMID: 1908878
9. Caracciolo, D. et al. Recombinant human macrophage colony-stimulating factor (M-CSF) requires subliminal concentrations of granulocyte/macrophage (GM)-CSF for optimal stimulation of human macrophage colony formation in vitro. Journal of Experimental Medicine 166, 1851–1860 (1987). doi: 10.1084/jem.166.6.1851
10. Kurzrock, R. Granulocyte-macrophage colony-stimulating factor. (2003). https://www.ncbi.nlm.nih.gov/books/NBK13400/
11. Ponomarev, E. D. et al. GM-CSF Production by Autoreactive T Cells Is Required for the Activation of Microglial Cells and the Onset of Experimental Autoimmune Encephalomyelitis. The Journal of Immunology 178, 39–48 (2007). doi: 10.4049/jimmunol.178.1.39
12. van Nieuwenhuijze, A. et al. GM-CSF as a therapeutic target in inflammatory diseases. Mol Immunol 56, 675–682 (2013). doi: 10.1016/j.molimm.2013.05.002
13. Rios, F. J., Touyz, R. M. & Montezano, A. C. Isolation and differentiation of human macrophages. Methods in Molecular Biology 1527, 311–320 (2017). doi: 10.1007/978-1-4939-6625-7_24
14. Greter, M. et al. GM-CSF Controls Nonlymphoid Tissue Dendritic Cell Homeostasis but Is Dispensable for the Differentiation of Inflammatory Dendritic Cells. Immunity 36, 1031–1046 (2012). doi: 10.1016/j.immuni.2012.03.027
15. Van De Laar, L., Coffer, P. J. & Woltman, A. M. Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune homeostasis and therapy. Blood 119, 3383–3393 (2012). doi: 10.1182/blood-2011-11-370130
16. Singh, P. et al. GM-CSF Enhances Macrophage Glycolytic Activity In Vitro and Improves Detection of Inflammation In Vivo. Journal of Nuclear Medicine 57, 1428 (2016). doi: 10.2967/jnumed.115.167387
17. Ying, W., Cheruku, P. S., Bazer, F. W., Safe, S. H. & Zhou, B. Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages. J Vis Exp 50323 (2013) doi:10.3791/50323. doi: 10.3791/50323
18. Jin, X. & Kruth, H. S. Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages. J Vis Exp 2016, 54244 (2016). doi: 10.3791/54244
19. Mia, S., Warnecke, A., Zhang, X. M., Malmström, V. & Harris, R. A. An optimized Protocol for Human M2 Macrophages using M-CSF and IL-4/IL-10/TGF-β Yields a Dominant Immunosuppressive Phenotype. Scand J Immunol 79, 305 (2014). doi: 10.1111/sji.12162
20. Orekhov, A. N. et al. Monocyte differentiation and macrophage polarization. Vessel Plus 3, 10 (2019). doi: 10.20517/2574-1209.2019.04
21. Yamane, K. & Leung, K. P. Rabbit M1 and M2 macrophages can be induced by human recombinant GM‐CSF and M‐CSF. FEBS Open Bio 6, 945 (2016). doi: 10.1002/2211-5463.12101
22. Ohradanova-Repic, A., Machacek, C., Fischer, M. B. & Stockinger, H. Differentiation of human monocytes and derived subsets of macrophages and dendritic cells by the HLDA10 monoclonal antibody panel. Clin Transl Immunology 5, e55 (2016). doi: 10.1038/cti.2015.39
23. Obermaier, B. et al. Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes. Biol Proced Online 5, 197 (2003). doi: 10.1251/bpo62
24. Hubo, M. et al. Costimulatory molecules on immunogenic versus tolerogenic human dendritic cells. Front Immunol 4, 43008 (2013). doi: 10.3389/fimmu.2013.00082
25. Tanaka, M., Dykes, P. J. & Marks, R. Keratinocyte Growth Stimulation by Granulocyte acrophage Colony-stimulating Factor (GM-CSF). Keio J Med 46, 184–187 (1997). doi: 10.2302/kjm.46.184
26. Li, H. et al. IL-1α, IL-6, and GM-CSF Are Downstream Mediators of IL-17A that Promote Asymmetric Stem Cell Self-Renewal in Human Keratinocytes. Journal of Investigative Dermatology 141, 458-462.e3 (2021). doi:10.1016/j.jid.2020.05.112
27. Cook, A. D. et al. Granulocyte macrophage colony-stimulating factor receptor α expression and its targeting in antigen-induced arthritis and inflammation. Arthritis Res Ther 18, (2016). doi: 10.1186/s13075-016-1185-9
28. Louis, C. et al. NK cell–derived GM-CSF potentiates inflammatory arthritis and is negatively regulated by CIS. J Exp Med 217, (2020). doi: 10.1084/jem.20191421
29. Saha, S. et al. Granulocyte–macrophage colony-stimulating factor expression in induced sputum and bronchial mucosa in asthma and COPD. Thorax 64, 671 (2009). doi: 10.1136/thx.2008.108290
30. Molfino, N. A. et al. Phase 2, randomised placebo-controlled trial to evaluate the efficacy and safety of an anti-GM-CSF antibody (KB003) in patients with inadequately controlled asthma. BMJ Open 6, (2016). doi: 10.1136/bmjopen-2015-007709
31. Zhao, W., Zhao, G. & Wang, B. Revisiting GM-CSF as an adjuvant for therapeutic vaccines. Cellular & Molecular Immunology 2018 15:2 15, 187–189 (2017). doi: 10.1038/cmi.2017.105
32. Hong, I. S. Stimulatory versus suppressive effects of GM-CSF on tumor progression in multiple cancer types. Exp Mol Med 48, e242 (2016). doi: 10.1038/emm.2016.64
33. Kumar, A., Taghi Khani, A., Sanchez Ortiz, A. & Swaminathan, S. GM-CSF: A Double-Edged Sword in Cancer Immunotherapy. Front Immunol 13, (2022). doi: 10.3389/fimmu.2022.901277
34. Armstrong, S., MacKenzie, J., Woodward, B., Pacey, A. & Farquhar, C. GM‐CSF (granulocyte macrophage colony‐stimulating factor) supplementation in culture media for women undergoing assisted reproduction. Cochrane Database Syst Rev 2020, (2020). doi: 10.1002/14651858.CD013497.pub2