Abstract
Macrophages become polarized by cues in their environment and this polarization causes a functional change in their behavior. Two main subsets of polarized macrophages have been described. M1, or “classically activated” macrophages, are pro-inflammatory and M2, or “alternatively activated” macrophages, are anti-inflammatory. In this study, we investigated the motility and force generation of primary human macrophages polarized down the M1 and M2 pathways using chemokinesis assays and traction force microscopy on polyacrylamide gels. We found that M1 macrophages are significantly less motile and M2 macrophages are significantly more motile than unactivated M0 macrophages. We also showed that M1 macrophages generate significantly less force than M0 or M2 macrophages. We further found that M0 and M2, but not M1, macrophage force generation is dependent on ROCK signaling, as identified using the chemical inhibitor Y27632. Finally, using the chemical inhibitor blebbistatin, we found that myosin contraction is required for force generation by M0, M1, and M2 macrophages. This study represents the first investigation of the changes in the mechanical motility mechanisms used by macrophages after polarization.
Similar content being viewed by others
Abbreviations
- CCR7:
-
C-C chemokine receptor type 7
- CCL22:
-
C-C chemokine ligand type 22
- IFNγ:
-
Interferon-gamma
- IL-12, -23, -4, -10, -1β:
-
Interleukin-12, 23, 4, 10, 1β
- LPS:
-
Lipopolysaccharide
- M-CSF:
-
Macrophage colony stimulating factor
- MMP9:
-
Matrix metallopeptidase 9
- N-6:
-
N-6-((acryloyl)amino)hexanoic acid
- ROCK:
-
RhoA Kinase
- TNFα:
-
Tumor necrosis factor alpha
References
Ambarus, C. A., S. Krausz, M. van Eijk, J. Hamann, T. R. Radstake, K. A. Reedquist, P. P. Tak, and D. L. Baeten. Systematic validation of specific phenotypic markers for in vitro polarized human macrophages. J. Immunol. Methods 375:196–206, 2012.
Arnold, L., A. Henry, F. Poron, Y. Baba-Amer, N. van Rooijen, A. Plonquet, R. K. Gherardi, and B. Chazaud. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J. Exp. Med. 204:1057–1069, 2007.
Biswas, S. K., M. Chittezhath, I. N. Shalova, and J. Y. Lim. Macrophage polarization and plasticity in health and disease. Immunol. Res. 53:11–24, 2012.
Biswas, S. K., and A. Mantovani. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat. Immunol. 11:889–896, 2010.
Chioda, M., E. Peranzoni, G. Desantis, F. Papalini, E. Falisi, S. Solito, S. Mandruzzato, and V. Bronte. Myeloid cell diversification and complexity: an old concept with new turns in oncology. Cancer Metastasis Rev. 30:27–43, 2011.
Condeelis, J., and J. W. Pollard. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124:263–266, 2006.
Cougoule, C., E. Van Goethem, V. Le Cabec, F. Lafouresse, L. Dupre, V. Mehraj, J. L. Mege, C. Lastrucci, and I. Maridonneau-Parini. Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments. Eur. J. Cell Biol. 91:938–949, 2012.
Dembo, M. The LIBTRC User’s Guide for Version 2.4. Boston, 2010.
Dembo, M., and Y. L. Wang. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys. J. 76:2307–2316, 1999.
Dunn, G. A. Characterising a kinesis response: time averaged measures of cell speed and directional persistence. Agents Actions Suppl. 12:14–33, 1983.
Hao, N. B., M. H. Lu, Y. H. Fan, Y. L. Cao, Z. R. Zhang, and S. M. Yang. Macrophages in tumor microenvironments and the progression of tumors. Clin. Dev. Immunol. 2012:948098, 2012.
Hind, L. E., M. Dembo, and D. A. Hammer. Macrophage motility is driven by frontal-towing with a force magnitude dependent on substrate stiffness. Integr. Biol. (Camb) 7:447–453, 2015.
Hind, L. E., J. L. Mackay, D. Cox, and D. A. Hammer. Two-dimensional motility of a macrophage cell line on microcontact-printed fibronectin. Cytoskeleton (Hoboken) 71:542–554, 2014.
Jannat, R. A., M. Dembo, and D. A. Hammer. Traction forces of neutrophils migrating on compliant substrates. Biophys. J. 101:575–584, 2011.
Mantovani, A., and A. Sica. Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Curr. Opin. Immunol. 22:231–237, 2010.
Mantovani, A., S. Sozzani, M. Locati, P. Allavena, and A. Sica. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23:549–555, 2002.
McWhorter, F. Y., T. Wang, P. Nguyen, T. Chung, and W. F. Liu. Modulation of macrophage phenotype by cell shape. Proc. Natl. Acad. Sci. USA 110:17253–17258, 2013.
Nassiri, S., I. Zakeri, M. S. Weingarten, and K. L. Spiller. Relative expression of proinflammatory and antiinflammatory genes reveals differences between healing and nonhealing human chronic diabetic foot ulcers. J Invest Dermatol 135:1700–1703, 2015.
Oh, D. Y., H. Morinaga, S. Talukdar, E. J. Bae, and J. M. Olefsky. Increased macrophage migration into adipose tissue in obese mice. Diabetes 61:346–354, 2012.
Pelham, Jr, R. J., and Y. Wang. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94:13661–13665, 1997.
Pless, D. D., Y. C. Lee, S. Roseman, and R. L. Schnaar. Specific cell adhesion to immobilized glycoproteins demonstrated using new reagents for protein and glycoprotein immobilization. J. Biol. Chem. 258:2340–2349, 1983.
Reinhart-King, C. A., M. Dembo, and D. A. Hammer. The dynamics and mechanics of endothelial cell spreading. Biophys. J. 89:676–689, 2005.
Sharma, V. P., B. T. Beaty, A. Patsialou, H. Liu, M. Clarke, D. Cox, J. S. Condeelis, and R. J. Eddy. Reconstitution of in vivo macrophage-tumor cell pairing and streaming motility on one-dimensional micro-patterned substrates. Intravital 1:77–85, 2012.
Solinas, G., S. Schiarea, M. Liguori, M. Fabbri, S. Pesce, L. Zammataro, F. Pasqualini, M. Nebuloni, C. Chiabrando, A. Mantovani, and P. Allavena. Tumor-conditioned macrophages secrete migration-stimulating factor: a new marker for M2-polarization, influencing tumor cell motility. J. Immunol. 185:642–652, 2010.
Spiller, K. L., R. R. Anfang, K. J. Spiller, J. Ng, K. R. Nakazawa, J. W. Daulton, and G. Vunjak-Novakovic. The role of macrophage phenotype in vascularization of tissue engineering scaffolds. Biomaterials 35:4477–4488, 2014.
Vogel, D. Y., P. D. Heijnen, M. Breur, H. E. de Vries, A. T. Tool, S. Amor, and C. D. Dijkstra. Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation. J. Neuroinflamm. 11:23, 2014.
Worthylake, R. A., S. Lemoine, J. M. Watson, and K. Burridge. RhoA is required for monocyte tail retraction during transendothelial migration. J. Cell Biol. 154:147–160, 2001.
Acknowledgment
This work was supported by NIH GM1094287 and HL18208.
Conflict of Interest
Laurel E. Hind, Emily B. Lurier, Micah Dembo, Kara L. Spiller, and Daniel A. Hammer declare that they have no conflicts of interest.
Ethical Standards
All human subjects research was carried out in accordance with institutional guidelines and was approved by a University of Pennsylvania Institutional Review Board under HL18208. The authors performed no animal studies in this work.
Author information
Authors and Affiliations
Corresponding author
Additional information
Associate Editor Michael R. King oversaw the review of this article.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Supplementary Video 1
M0-M1-M2 macrophages migrating on 10,400 Pa polyacrylamide gels coated with 5 µg/mL fibronectin. Left: M0, Center: M1, Right: M2. Supplementary material 1 (AVI 10991 kb)
Rights and permissions
About this article
Cite this article
Hind, L.E., Lurier, E.B., Dembo, M. et al. Effect of M1–M2 Polarization on the Motility and Traction Stresses of Primary Human Macrophages. Cel. Mol. Bioeng. 9, 455–465 (2016). https://doi.org/10.1007/s12195-016-0435-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12195-016-0435-x