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Cytotechnology

, Volume 68, Issue 4, pp 921–933 | Cite as

Establishment and characterization of DB-1: a leptin receptor-deficient murine macrophage cell line

  • Lea H. Dib
  • M. Teresa Ortega
  • Tonatiuh Melgarejo
  • Stephen K. Chapes
Original Research

Abstract

Metabolic and immune mediators activate many of the same signal transduction pathways. Therefore, molecules that regulate metabolism often affect immune responses. Leptin is an adipokine that exemplifies this interplay. Leptin is the body’s major nutritional status sensor, but it also plays a key role in immune system regulation. To provide an in vitro tool to investigate the link between leptin and innate immunity, we immortalized and characterized a leptin receptor-deficient macrophage cell line, DB-1. The cell line was created using bone marrow cells from leptin receptor-deficient mice. Bone marrow cells were differentiated into macrophages by culturing them with recombinant mouse macrophage colony stimulating factor, and passaged when confluent for 6 months. The cells spontaneously immortalized at approximately passage 20. Cells were cloned twice by limiting dilution cloning prior to characterization. The macrophage cell line is diploid and grows at a linear rate for 4–5 days before reaching the growth plateau. The cells are MAC-2 and F4/80 positive and have phagocytic activity similar to primary macrophages from wild-type and leptin receptor-deficient mice. DB-1 cells were responsive to stimulation with interferon-γ as measured by increase in Nos2 transcript levels. In addition, DB-1 macrophages are not responsive to the chemotactic signaling of adipocyte conditioned media nor leptin when compared to primary WT macrophages. We believe that DB-1 cells provide a dependable tool to study the role of leptin or the leptin receptor in obesity-associated inflammation and immune system dysregulation.

Keywords

Macrophages Leptin receptor Immortal cell line 

Notes

Acknowledgments

This project has been supported by the American Heart Association Grant 0950036G, NIH Grants AI088070, RR17686, RR17708, NASA grant NNX08BA91G and The Kansas State Agricultural Experiment Station. This is Kansas Agricultural Experiment Station Publication #11-255-J.

References

  1. Ahima RS, Flier JS (2000) Leptin. Annu Rev Physiol 62:413–437CrossRefGoogle Scholar
  2. Armstrong JW, Nelson KA, Simske SJ, Luttges MW, Iandolo JJ, Chapes SK (1993) Skeletal unloading causes organ-specific changes in immune cell responses. J Appl Physiol 75:2734–2739Google Scholar
  3. Batra A, Okur B, Glauben R, Erben U, Ihbe J, Stroh T, Fedke I, Chang HD, Zeitz M, Siegmund B (2009) Leptin: a critical regulator of CD4+ T-cell polarization in vitro and in vivo. Endocrinology 151:56–62CrossRefGoogle Scholar
  4. Baumann H, Morella KK, White DW, Dembski M, Bailon PS, Kim H, Lai CF, Tartaglia LA (1996) The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc Natl Acad Sci USA 93:8374–8378CrossRefGoogle Scholar
  5. Beharka AA, Armstrong JW, Chapes SK (1998) Macrophage cell lines derived from major histocompatibility complex II-negative mice. In Vitro Cell Dev Biol 34:499–507CrossRefGoogle Scholar
  6. Caldefie-Chezet F, Poulin A, Vasson MP (2003) Leptin regulates functional capacities of polymorphonuclear neutrophils. Free Radic Res 37:809–814CrossRefGoogle Scholar
  7. Chan J, Leenen PJ, Bertoncello I, Nishikawa SI, Hamilton JA (1998) Macrophage lineage cells in inflammation: characterization by colony-stimulating factor-1 (CSF-1) receptor (c-Fms), ER-MP58, and ER-MP20 (Ly-6C) expression. Blood 92:1423–1431Google Scholar
  8. Chan JL, Heist K, DePaoli AM, Veldhuis JD, Mantzoros CS (2003) The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men. J Clin Invest 111:1409–1421CrossRefGoogle Scholar
  9. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP (1996) Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84:491–495CrossRefGoogle Scholar
  10. Cheung CC, Thornton JE, Kuijper JL, Weigle DS, Clifton DK, Steiner RA (1997) Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology 138:855–858CrossRefGoogle Scholar
  11. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, Caro JF (1996) Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 334:292–295Google Scholar
  12. Curat CA, Miranville A, Sengenes C, Diehl M, Tonus C, Busse R, Bouloumie A (2004) From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes. Diabetes 53:1285–1292CrossRefGoogle Scholar
  13. Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, Saper CB (1998) Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 395:535–547CrossRefGoogle Scholar
  14. Falagas ME, Kompoti M (2006) Obesity and infection. Lancet Infect Dis 6:438–446CrossRefGoogle Scholar
  15. Gainsford T, Willson TA, Metcalf D, Handman E, McFarlane C, Ng A, Nicola NA, Alexander WS, Hilton DJ (1996) Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells. Proc Natl Acad Sci USA 93:14564–14568CrossRefGoogle Scholar
  16. Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K (2010) Development of monocytes, macrophages, and dendritic cells. Science 327:656–661CrossRefGoogle Scholar
  17. Gruen ML, Hao M, Piston DW, Hasty AH (2007) Leptin requires canonical migratory signaling pathways for induction of monocyte and macrophage chemotaxis. Am J Physiol Cell Physiol 293:C1481–C1488CrossRefGoogle Scholar
  18. Hsu A, Aronoff DM, Phipps J, Goel D, Mancuso P (2007) Leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia. Clin Exp Immunol 150:332–339CrossRefGoogle Scholar
  19. Ikejima S, Sasaki S, Sashinami H, Mori F, Ogawa Y, Nakamura T, Abe Y, Wakabayashi K, Suda T, Nakane A (2005) Impairment of host resistance to Listeria monocytogenes infection in liver of db/db and ob/ob mice. Diabetes 54:182–189CrossRefGoogle Scholar
  20. Inouye KE, Shi H, Howard JK, Daly CH, Lord GM, Rollins BJ, Flier JS (2007) Absence of CC chemokine ligand 2 does not limit obesity-associated infiltration of macrophages into adipose tissue. Diabetes 56:2242–2250CrossRefGoogle Scholar
  21. Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, Kitazawa R, Kitazawa S, Miyachi H, Maeda S, Egashira K, Kasuga M (2006) MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest 116:1494–1505CrossRefGoogle Scholar
  22. Krishnan EC, Trost L, Aarons S, Jewell WR (1982) Study of function and maturation of monocytes in morbidly obese individuals. J Surg Res 33:89–97CrossRefGoogle Scholar
  23. La Cava A, Matarese G (2004) The weight of leptin in immunity. Nat Rev Immunol 4:371–379CrossRefGoogle Scholar
  24. Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI, Friedman JM (1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632–635CrossRefGoogle Scholar
  25. Leenen PJ, Jansen AM, van Ewijk W (1986) Murine macrophage cell lines can be ordered in a linear differentiation sequence. Differentiation 32:157–164CrossRefGoogle Scholar
  26. Leenen PJ, Melis M, Slieker WA, Van Ewijk W (1990) Murine macrophage precursor characterization. II. Monoclonal antibodies against macrophage precursor antigens. Eur J Immunol 20:27–34CrossRefGoogle Scholar
  27. Leenen PJ, de Bruijn MF, Voerman JS, Campbell PA, van Ewijk W (1994a) Markers of mouse macrophage development detected by monoclonal antibodies. J Immunol Methods 174:5–19CrossRefGoogle Scholar
  28. Leenen PJ, de Bruijn MF, Voerman JS, Campbell PA, Van Ewijk W (1994b) Markers of mouse macrophage development detected by monoclonal antibodies. J Immunol Methods 174:5–19CrossRefGoogle Scholar
  29. Loffreda S, Yang SQ, Lin HZ, Karp CL, Brengman ML, Wang DJ, Klein AS, Bulkley GB, Bao C, Noble PW, Lane MD, Diehl AM (1998) Leptin regulates proinflammatory immune responses. FASEB J 12:57–65Google Scholar
  30. MacMicking J, Xie QW, Nathan C (1997) Nitric oxide and macrophage function. Annu Rev Immunol 15:323–350CrossRefGoogle Scholar
  31. Mancuso P, Gottschalk A, Phare SM, Peters-Golden M, Lukacs NW, Huffnagle GB (2002) Leptin-deficient mice exhibit impaired host defense in Gram-negative pneumonia. J Immunol 168:4018–4024CrossRefGoogle Scholar
  32. Matarese G, Moschos S, Mantzoros CS (2005) Leptin in immunology. J Immunol 174:3137–3142CrossRefGoogle Scholar
  33. Mattioli B, Straface E, Quaranta MG, Giordani L, Viora M (2005) Leptin promotes differentiation and survival of human dendritic cells and licenses them for Th1 priming. J Immunol 174:6820–6828CrossRefGoogle Scholar
  34. Metcalf D, Merchav S, Wagemaker G (1982) Commitment of GM-CSF or M-CSF of bipotential GM progenitor cells to granulocyte or macrophage formation. In: Baum SJ, Ledley GD, Thierfelder S (eds) Experimental hematology today. Karger, BaselGoogle Scholar
  35. Mordica WJ, Woods KM, Clem RJ, Passarelli AL, Chapes SK (2009) Macrophage cell lines use CD81 in cell growth regulation. In Vitro Cell Dev Biol Anim 45:213–225CrossRefGoogle Scholar
  36. Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM (2006) Prevalence of overweight and obesity in the United States, 1999–2004. JAMA 295:1549–1555CrossRefGoogle Scholar
  37. Ordway D, Henao-Tamayo M, Smith E, Shanley C, Harton M, Troudt J, Bai X, Basaraba RJ, Orme IM, Chan ED (2008) Animal model of Mycobacterium abscessus lung infection. J Leukoc Biol 83:1502–1511CrossRefGoogle Scholar
  38. Ortega MT, Xie L, Mora S, Chapes SK (2011) Evaluation of macrophage plasticity in brown and white adipose tissue. Cell Immunol 271:124–133CrossRefGoogle Scholar
  39. Otero M, Lago R, Lago F, Casanueva FF, Dieguez C, Gomez-Reino JJ, Gualillo O (2005) Leptin, from fat to inflammation: old questions and new insights. FEBS Lett 579:295–301CrossRefGoogle Scholar
  40. Papathanassoglou E, El-Haschimi K, Li XC, Matarese G, Strom T, Mantzoros C (2006) Leptin receptor expression and signaling in lymphocytes: kinetics during lymphocyte activation, role in lymphocyte survival, and response to high fat diet in mice. J Immunol 176:7745–7752CrossRefGoogle Scholar
  41. Park S, Rich J, Hanses F, Lee JC (2009) Defects in innate immunity predispose C57BL/6J-Leprdb/Leprdb mice to infection by Staphylococcus aureus. Infect Immun 77:1008–1014CrossRefGoogle Scholar
  42. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefGoogle Scholar
  43. Raso GM, Pacilio M, Esposito E, Coppola A, Di Carlo R, Meli R (2002) Leptin potentiates IFN-gamma-induced expression of nitric oxide synthase and cyclo-oxygenase-2 in murine macrophage J774A.1. Br J Pharmacol 137:799–804CrossRefGoogle Scholar
  44. Schuler MF, Jefferson LS, Gorodecki J, Glaser R, Dietz J, Lipton A (1977) Selective growth of transformed cell lines by rat liver perfusate. Cancer Res 37:1662–1665Google Scholar
  45. Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG (1997) Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46:2119–2123CrossRefGoogle Scholar
  46. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moriarty A, Moore KJ, Smutko JS, Mays GG, Wool EA, Monroe CA, Tepper RI (1995) Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271CrossRefGoogle Scholar
  47. Thornton JE, Cheung CC, Clifton DK, Steiner RA (1997) Regulation of hypothalamic proopiomelanocortin mRNA by leptin in ob/ob mice. Endocrinology 138:5063–5066CrossRefGoogle Scholar
  48. Tian Z, Sun R, Wei H, Gao B (2002) Impaired natural killer (NK) cell activity in leptin receptor deficient mice: leptin as a critical regulator in NK cell development and activation. Biochem Biophys Res Commun 298:297–302CrossRefGoogle Scholar
  49. Vaughan T, Li L (2010) Molecular mechanism underlying the inflammatory complication of leptin in macrophages. Mol Immunol 47:2515–2518CrossRefGoogle Scholar
  50. Vindelov LL, Christensen IJ, Nissen NI (1983) A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3:323–327CrossRefGoogle Scholar
  51. Wauters M, Considine RV, Van Gaal LF (2000) Human leptin: from an adipocyte hormone to an endocrine mediator. Eur J Endocrinol 143:293–311CrossRefGoogle Scholar
  52. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112:1796–1808CrossRefGoogle Scholar
  53. Weisberg SP, Hunter D, Huber R, Lemieux J, Slaymaker S, Vaddi K, Charo I, Leibel RL, Ferrante AW Jr (2006) CCR2 modulates inflammatory and metabolic effects of high-fat feeding. J Clin Invest 116:115–124CrossRefGoogle Scholar
  54. Xie L, Ortega MT, Mora S, Chapes SK (2010) Interactive changes between macrophages and adipocytes. Clin Vaccine Immunol 17:651–659CrossRefGoogle Scholar
  55. Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, Ross JS, Tartaglia LA, Chen H (2003) Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 112:1821–1830CrossRefGoogle Scholar
  56. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Lea H. Dib
    • 1
    • 2
  • M. Teresa Ortega
    • 2
  • Tonatiuh Melgarejo
    • 1
  • Stephen K. Chapes
    • 2
  1. 1.Department of Human NutritionKansas State UniversityManhattanUSA
  2. 2.Division of BiologyKansas State UniversityManhattanUSA

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