Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8

Nature Methods volume 3, pages 287–293 (2006)Cite this article

Abstract

Differentiation mechanisms and inflammatory functions of neutrophils and macrophages are usually studied by genetic and biochemical approaches that require costly breeding and time-consuming purification to obtain phagocytes for functional analysis. Because Hox oncoproteins enforce self-renewal of factor-dependent myeloid progenitors, we queried whether estrogen-regulated Hoxb8 (ER-Hoxb8) could immortalize macrophage or neutrophil progenitors that would execute normal differentiation and normal innate immune function upon ER-Hoxb8 inactivation. Here we describe methods to derive unlimited quantities of mouse macrophages or neutrophils by immortalizing their respective progenitors with ER-Hoxb8 using different cytokines to target expansion of different committed progenitors. ER-Hoxb8 neutrophils and macrophages are functionally superior to those produced by many other ex vivo differentiation models, have strong inflammatory responses and can be derived easily from embryonic day 13 (e13) fetal liver of mice exhibiting embryonic-lethal phenotypes. Using knockout or small interfering RNA (siRNA) technologies, this ER-Hoxb8 phagocyte maturation system represents a rapid analytical tool for studying macrophage and neutrophil biology.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: ER-Hoxb8 and ER-Hoxa9 function conditionally at the biochemical and cellular levels.
Figure 2: SCF ER-Hoxb8 progenitors execute neutrophil differentiation and GM-CSF ER-Hoxb8 progenitors execute macrophage differentiation.
Figure 3: Lineage-specific gene expression in SCF ER-Hoxb8 neutrophil progenitors and GM-CSF ER-Hoxb8 macrophage progenitors.
Figure 4: SCF ER-Hoxb8 progenitors behave as GMP, retaining an ability to differentiate into eosinophils and macrophages.
Figure 5: Re-expression of TRAF3 restores the signaling defect in CpG-induced transactivation of Ifn and IL-10 genes in Traf3−/− macrophages produced by ER-Hoxb8.

Similar content being viewed by others

References

  1. Karin, M. & Greten, F.R. NF-κB: linking inflammation and immunity to cancer development and progression. Nat. Rev. Immunol. 5, 749–759 (2005).

    Article  CAS  Google Scholar 

  2. Weisberg, S.P. et al. Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest. 112, 1796–1808 (2003).

    Article  CAS  Google Scholar 

  3. Pineault, N., Helgason, C.D., Lawrence, H.J. & Humphries, R.K. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp. Hematol. 30, 49–57 (2002).

    Article  CAS  Google Scholar 

  4. Lawrence, H.J. et al. Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood 89, 1922–1930 (1997).

    CAS  PubMed  Google Scholar 

  5. Thorsteinsdottir, U. et al. Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood 99, 121–129 (2002).

    Article  CAS  Google Scholar 

  6. Calvo, K.R., Sykes, D.B., Pasillas, M. & Kamps, M.P. Hoxa9 immortalizes a granulocyte-macrophage colony-stimulating factor–dependent promyelocyte capable of biphenotypic differentiation to neutrophils or macrophages, independent of enforced meis expression. Mol. Cell. Biol. 20, 3274–3285 (2000).

    Article  CAS  Google Scholar 

  7. Knoepfler, P.S., Sykes, D.B., Pasillas, M. & Kamps, M.P. HoxB8 requires its Pbx-interaction motif to block differentiation of primary myeloid progenitors and of most cell line models of myeloid differentiation. Oncogene 20, 5440–5448 (2001).

    Article  CAS  Google Scholar 

  8. Fischbach, N.A. et al. HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo. Blood 105, 1456–1466 (2005).

    Article  CAS  Google Scholar 

  9. Owens, B.M. & Hawley, R.G. HOX and non-HOX homeobox genes in leukemic hematopoiesis. Stem Cells 20, 364–379 (2002).

    Article  CAS  Google Scholar 

  10. Krishnaraju, K., Hoffman, B. & Liebermann, D.A. The zinc finger transcription factor Egr-1 activates macrophage differentiation in M1 myeloblastic leukemia cells. Blood 92, 1957–1966 (1998).

    CAS  PubMed  Google Scholar 

  11. Liebermann, D.A. & Hoffman, B. Differentiation primary response genes and proto-oncogenes as positive and negative regulators of terminal hematopoietic cell differentiation. Stem Cells 12, 352–369 (1994).

    Article  CAS  Google Scholar 

  12. Kharbanda, S. et al. Expression of the early growth response 1 and 2 zinc finger genes during induction of monocytic differentiation. J. Clin. Invest. 88, 571–577 (1991).

    Article  CAS  Google Scholar 

  13. Hacker, H. et al. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature 439, 204–207 (2006).

    Article  Google Scholar 

  14. Gaines, P., Chi, J. & Berliner, N. Heterogeneity of functional responses in differentiated myeloid cell lines reveals EPRO cells as a valid model of murine neutrophil functional activation. J. Leukoc. Biol. 77, 669–679 (2005).

    Article  CAS  Google Scholar 

  15. Engelke, U., Wang, D.M. & Lipsick, J.S. Cells transformed by a v-Myb-estrogen receptor fusion differentiate into multinucleated giant cells. J. Virol. 71, 3760–3766 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Zeisig, B.B. et al. Hoxa9 and Meis1 are key targets for MLL-ENL–mediated cellular immortalization. Mol. Cell. Biol. 24, 617–628 (2004).

    Article  CAS  Google Scholar 

  17. Sykes, D.B. & Kamps, M.P. Estrogen-dependent E2a/Pbx1 myeloid cell lines exhibit conditional differentiation that can be arrested by other leukemic oncoproteins. Blood 98, 2308–2318 (2001).

    Article  CAS  Google Scholar 

  18. Lipsick, J.S. & Wang, D.M. Transformation by v-Myb. Oncogene 18, 3047–3055 (1999).

    Article  CAS  Google Scholar 

  19. Chen, J., Kremer, C.S. & Bender, T.P. A Myb dependent pathway maintains Friend murine erythroleukemia cells in an immature and proliferating state. Oncogene 21, 1859–1869 (2002).

    Article  CAS  Google Scholar 

  20. Lyon, J.J. & Watson, R.J. Conditional inhibition of erythroid differentiation by c-Myb/oestrogen receptor fusion proteins. Differentiation 59, 171–178 (1995).

    Article  CAS  Google Scholar 

  21. White, J.R. & Weston, K. Myb is required for self-renewal in a model system of early hematopoiesis. Oncogene 19, 1196–1205 (2000).

    Article  CAS  Google Scholar 

  22. Kumar, A., Lee, C.M. & Reddy, E.P. c-Myc is essential but not sufficient for c-Myb–mediated block of granulocytic differentiation. J. Biol. Chem. 278, 11480–11488 (2003).

    Article  CAS  Google Scholar 

  23. Wood, M.A., McMahon, S.B. & Cole, M.D. An ATPase/helicase complex is an essential cofactor for oncogenic transformation by c-Myc. Mol. Cell 5, 321–330 (2000).

    Article  CAS  Google Scholar 

  24. Hock, H. et al. Intrinsic requirement for zinc finger transcription factor Gfi-1 in neutrophil differentiation. Immunity 18, 109–120 (2003).

    Article  CAS  Google Scholar 

  25. Nemeth, M.J., Cline, A.P., Anderson, S.M., Garrett-Beal, L.J. & Bodine, D.M. Hmgb3 deficiency deregulates proliferation and differentiation of common lymphoid and myeloid progenitors. Blood 105, 627–634 (2005).

    Article  CAS  Google Scholar 

  26. Li, M., Zhou, J.Y., Ge, Y., Matherly, L.H. & Wu, G.S. The phosphatase MKP1 is a transcriptional target of p53 involved in cell cycle regulation. J. Biol. Chem. 278, 41059–41068 (2003).

    Article  CAS  Google Scholar 

  27. Yang, A.H. et al. Chromosome segregation defects contribute to aneuploidy in normal neural progenitor cells. J. Neurosci. 23, 10454–10462 (2003).

    Article  CAS  Google Scholar 

  28. Sykes, D.B. & Kamps, M.P. Estrogen-regulated conditional oncoproteins: tools to address open questions in normal myeloid cell function, normal myeloid differentiation, and the genetic basis of differentiation arrest in myeloid leukemia. Leuk. Lymphoma 44, 1131–1139 (2003).

    Article  CAS  Google Scholar 

  29. Wang, G.G., Pasillas, M.P. & Kamps, M.P. Meis1 programs transcription of FLT3 and cancer stem cell character, using a mechanism that requires interaction with PBX and a novel function of the Meis1 C terminus. Blood 106, 254–264 (2005).

    Article  CAS  Google Scholar 

  30. Park, J.M. et al. Signaling pathways and genes that inhibit pathogen-induced macrophage apoptosis–CREB and NF-κB as key regulators. Immunity 23, 319–329 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported by Public Health Service grant (National Institutes of Health CA56876) awarded to M.P.K. We thank D. Young for assistance with FACS analysis, and G. Chun and W. Westra at Scripps Research Foundation for collaborating in the spectral karyotype analysis.

Author information

Authors and Affiliations

  1. Department of Pathology & Molecular Pathology Graduate Program, School of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, 92093, CA, USA

    Gang G Wang, Katherine R Calvo, Martina P Pasillas, David B Sykes & Mark P Kamps

  2. Biomedical Sciences Graduate Program, School of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, 92093, CA, USA

    Gang G Wang

  3. Laboratory of Pathology, National Health Institutes, National Cancer Institute, 10 Center Drive, Bldg. 10, 2N212, Bethesda, 20892, Maryland, USA

    Katherine R Calvo

  4. Department of Medicine, Harvard Massachusetts General Hospital, 55 Fruit Street, Boston, 02114, Massachusetts, USA

    David B Sykes

  5. Department of Infectious Diseases, St. Jude Children's Research Hospital, Room E8062, 332 North Lauderdale Street, Memphis, 38105, Tennessee, USA

    Hans Häcker

Authors
  1. Gang G Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katherine R Calvo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martina P Pasillas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David B Sykes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hans Häcker
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mark P Kamps
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark P Kamps.

Ethics declarations

Competing interests

A patent is pending on the system described in this article.

Supplementary information

Supplementary Fig. 1

SKY karyotype analysis of Hoxb8-ER macrophage progenitors cultured 2.5 years. (TIFF 912 kb)

Supplementary Fig. 2

Verification of changes observed on Affymetrix arrays. (TIFF 1162 kb)

Supplementary Fig. 3

A concerted program of phagocytic differentiation follows inactivation of Hoxb8-ER in both neutrophil and macrophage progenitors. (TIFF 2397 kb)

Supplementary Fig. 4

Inflammatory signaling pathways are preserved in macrophages derived from Hoxb8-ER GM-CSF progenitors. (TIFF 940 kb)

Supplementary Table 1

Expression of genes encoding transcription factors, cell cycle regulators, and proteins involved in myeloid innate immunity, assessed during neutrophil differentiation of SCF Hoxb8-ER progenitors and during macrophage differentiation of GM-CSF Hoxb8-ER progenitors. (PDF 37 kb)

Supplementary Table 2

Gene down-regulation in SCF Hoxb8-ER neutrophil progenitors and in GM-CSF Hoxb8-ER macrophage progenitors during terminal differentiation. (PDF 19 kb)

Supplementary Table 3

Gene up-regulation in SCF Hoxb8-ER neutrophil progenitors and in GM-CSF Hoxb8-ER macrophage progenitors during terminal differentiation. (PDF 35 kb)

Supplementary Table 4

Inflammatory response of GM-CSF Hoxb8-ER macrophages to LPS or BLP. (PDF 30 kb)

Supplementary Methods (DOC 73 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, G., Calvo, K., Pasillas, M. et al. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat Methods 3, 287–293 (2006). https://doi.org/10.1038/nmeth865

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth865

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing