Molecular and Cellular Biochemistry

, Volume 344, Issue 1–2, pp 55–63 | Cite as

Effect of toll-like receptor activation on thymosin beta-4 production by chicken macrophages

  • Lakshmi Kannan
  • Narayan C. Rath
  • Rohana Liyanage
  • Jackson O. LayJr.
Article

Abstract

Thymosin beta-4 (Tβ4) is an actin-binding intracellular peptide that promotes wound healing, tissue remodeling, and angiogenesis. The mechanism of Tβ4 secretion to the extracellular environment is not understood. The macrophage is a rich source of Tβ4 which also participates in wound healing process. The objective of this study was to find how Tβ4 may be externalized. Using activation of macrophage through their toll-like receptors (TLR), the changes in cellular Tβ4 was studied. A naturally transformed chicken macrophage cell line HTC was treated with different TLR agonists and the cellular Tβ4 changes was determined at 6 and 24 h after stimulations using stable isotope labelling of amino acids in cell culture (SILAC) and mass spectrometry. Real time PCR was used to determine changes in gene expression. The results showed that TLR agonists such as peptidoglycan (PGN) or lipopolysacharide (LPS) caused depletions in cellular Tβ4 peptide along with its detection in the cell culture supernatant at 24 h. These TLR agonists also induced the expression of interleukins-1β, -6, and nitric oxide synthase genes at 6 h but failed to modulate Tβ4 gene at that time point indicating that the Tβ4 externalization was not associated with its production. To find whether Tβ4 externalization was associated with cell death, we measured the lactate dehydrogenase (LDH) activity of the conditioned media as an indicator of cell damage. The results showed that the TLR agonists which induced depletion of intracellular Tβ4 at 24 h also increased the LDH content of the conditioned media, suggesting that the Tβ4 in the extracellular media most likely originated from dying macrophages.

Keywords

Macrophage Thymosin beta-4 SILAC Mass spectrometry Toll-like receptor 

Abbreviations

AU

Arbitrary units

CpG-ODN

CpG oligodeoxynucleotide

DHB

2,5-dihydroxybenzoic acid

FGN

Flagellin

H

Heavy isotope 13C-lysine label

IL

Interleukin

iNOS

Inducible nitric oxide synthase

L

Light lysine label

LOX

Loxoribine

LPS

Lipopolysaccharide

MALDI-TOF

Matrix-assisted laser desorption ionization-time of flight

MS

Mass spectrometry

m/z

Mass/charge

PGN

Peptidoglycan

poly I:C

Poly (inosinic:cytidilic acid)

qPCR

Quantitative polymerase chain reaction

RT-PCR

Reverse transcription-polymerase chain reaction

SILAC

Stable isotope labelling of amino acids in cell culture

TLR

Toll-like receptor

References

  1. 1.
    Goldstein AL (2007) History of the discovery of the thymosins. Ann N Y Acad Sci 1112:1–13CrossRefPubMedGoogle Scholar
  2. 2.
    Huff T, Muller CS, Otto AM et al (2001) beta-Thymosins, small acidic peptides with multiple functions. Int J Biochem Cell Biol 33:205–220CrossRefPubMedGoogle Scholar
  3. 3.
    Safer D, Elzinga M, Nachmias VT (1991) Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable. J Biol Chem 266:4029–4032PubMedGoogle Scholar
  4. 4.
    Sun HQ, Kwiatkowska K, Yin HL (1995) Actin monomer binding proteins. Curr Opin Cell Biol 7:102–110CrossRefPubMedGoogle Scholar
  5. 5.
    Smart N, Rossdeutsch A, Riley PR (2007) Thymosin beta4 and angiogenesis: modes of action and therapeutic potential. Angiogenesis 10:229–241CrossRefPubMedGoogle Scholar
  6. 6.
    Malinda KM, Sidhu GS, Mani H et al (1999) Thymosin beta4 accelerates wound healing. J Invest Dermatol 113:364–368CrossRefPubMedGoogle Scholar
  7. 7.
    Goldstein AL, Hannappel E, Kleinman HK (2005) Thymosin beta4: actin-sequestering protein moonlights to repair injured tissues. Trends Mol Med 11:421–429CrossRefPubMedGoogle Scholar
  8. 8.
    Philp D, Goldstein AL, Kleinman HK (2004) Thymosin beta4 promotes angiogenesis, wound healing, and hair follicle development. Mech Ageing Dev 125:113–115CrossRefPubMedGoogle Scholar
  9. 9.
    Tang YQ, Yeaman MR, Selsted ME (2002) Antimicrobial peptides from human platelets. Infect Immun 70:6524–6533CrossRefPubMedGoogle Scholar
  10. 10.
    Smith J, Speed D, Law AS et al (2004) In-silico identification of chicken immune-related genes. Immunogenetics 56:122–133CrossRefPubMedGoogle Scholar
  11. 11.
    Mannherz HG, Hannappel E (2009) The beta-thymosins: Intracellular and extracellular activities of a versatile actin binding protein family. Cell Motil Cytoskeleton 66:839–851CrossRefPubMedGoogle Scholar
  12. 12.
    Kannan L, Rath NC, Liyanage R et al (2007) Identification and characterization of thymosin beta-4 in chicken macrophages using whole cell MALDI-TOF. Ann N Y Acad Sci 1112:425–434CrossRefPubMedGoogle Scholar
  13. 13.
    Ross JA, Auger M (2002) The biology of the macrophage. In: Burke B, Lewis CE (ed) The macrophage. Oxford University Press, Oxford, UK, pp 1–72Google Scholar
  14. 14.
    Rath NC, Kannan L, Liyanage R et al (2007) Thymosin beta in macrophage. J Endocrinol Reprod 11:55–61Google Scholar
  15. 15.
    Crowther M, Brown NJ, Bishop ET et al (2001) Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors. J Leukoc Biol 70:478–490PubMedGoogle Scholar
  16. 16.
    Kaisho T, Akira S (2006) Toll-like receptor function and signaling. J Allergy Clin Immunol 117:979–987CrossRefPubMedGoogle Scholar
  17. 17.
    Hume DA (2006) The mononuclear phagocyte system. Curr Opin Immunol 18:49–53CrossRefPubMedGoogle Scholar
  18. 18.
    Xie H, Raybourne RB, Babu US et al (2003) CpG-induced immunomodulation and intracellular bacterial killing in a chicken macrophage cell line. Dev Comp Immunol 27:823–834CrossRefPubMedGoogle Scholar
  19. 19.
    Rath NC, Parcells MS, Xie H et al (2003) Characterization of a spontaneously transformed chicken mononuclear cell line. Vet Immunol Immunopathol 96:93–104CrossRefPubMedGoogle Scholar
  20. 20.
    Ong S-E, Mann M (2007) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protocols 1:2650–2660CrossRefGoogle Scholar
  21. 21.
    Brownlie R, Zhu J, Allan B et al (2009) Chicken TLR21 acts as a functional homologue to mammalian TLR9 in the recognition of CpG oligodeoxynucleotides. Mol Immunol 46:3163–3170CrossRefPubMedGoogle Scholar
  22. 22.
    Kannan L, Liyanage R, Lay JO Jr et al (2009) Evaluation of beta defensin 2 production by chicken heterophils using direct MALDI mass spectrometry. Mol Immunol 46:3151–3156CrossRefPubMedGoogle Scholar
  23. 23.
    Kogut MH, Iqbal M, He H et al (2005) Expression and function of Toll-like receptors in chicken heterophils. Dev Comp Immunol 29:791–807CrossRefPubMedGoogle Scholar
  24. 24.
    Kannan L, Rath NC, Liyanage R et al (2009) Direct screening identifies mature beta-defensin 2 in avian heterophils. Poult Sci 88:372–379CrossRefPubMedGoogle Scholar
  25. 25.
    Vanderlinde RE (1985) Measurement of total lactate dehydrogenase activity. Ann Clin Lab Sci 15:13–31PubMedGoogle Scholar
  26. 26.
    Grant DS, Rose W, Yaen C et al (1999) Thymosin beta4 enhances endothelial cell differentiation and angiogenesis. Angiogenesis 3:125–135CrossRefPubMedGoogle Scholar
  27. 27.
    Sosne G, Szliter EA, Barrett R et al (2002) Thymosin beta 4 promotes corneal wound healing and decreases inflammation in vivo following alkali injury. Exp Eye Res 74:293–299CrossRefPubMedGoogle Scholar
  28. 28.
    Frohm M, Gunne H, Bergman AC et al (1996) Biochemical and antibacterial analysis of human wound and blister fluid. Eur J Biochem 237:86–92CrossRefPubMedGoogle Scholar
  29. 29.
    Huang WQ, Wang QR (2001) Bone marrow endothelial cells secrete thymosin beta4 and AcSDKP. Exp Hematol 29:12–18CrossRefPubMedGoogle Scholar
  30. 30.
    Bock-Marquette I, Saxena A, White MD et al (2004) Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432:466–472CrossRefPubMedGoogle Scholar
  31. 31.
    Gondo H, Kudo J, White JW et al (1987) Differential expression of the human thymosin-beta 4 gene in lymphocytes, macrophages, and granulocytes. J Immunol 139:3840–3848PubMedGoogle Scholar
  32. 32.
    Hannappel E, Xu GJ, Morgan J et al (1982) Thymosin beta 4: a ubiquitous peptide in rat and mouse tissues. Proc Natl Acad Sci USA 79:2172–2175CrossRefPubMedGoogle Scholar
  33. 33.
    Serhan CN, Brain SD, Buckley CD et al (2007) Resolution of inflammation: state of the art, definitions and terms. FASEB J 21:325–332CrossRefPubMedGoogle Scholar
  34. 34.
    Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386CrossRefPubMedGoogle Scholar
  35. 35.
    Ong SE, Mann M (2007) Stable isotope labeling by amino acids in cell culture for quantitative proteomics. Methods Mol Biol 359:37–52CrossRefPubMedGoogle Scholar
  36. 36.
    Chettibi S, Lawrence AJ, Young JD et al (1994) Dispersive locomotion of human neutrophils in response to a steroid-induced factor from monocytes. J Cell Sci 107(Pt 11):3173–3181PubMedGoogle Scholar
  37. 37.
    Jain AK, Moore SM, Yamaguchi K et al (2004) Selective nonsteroidal anti-inflammatory drugs induce thymosin beta-4 and alter actin cytoskeletal organization in human colorectal cancer cells. J Pharmacol Exp Ther 311:885–891CrossRefPubMedGoogle Scholar
  38. 38.
    Young JD, Lawrence AJ, MacLean AG et al (1999) Thymosin beta 4 sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids. Nat Med 5:1424–1427CrossRefPubMedGoogle Scholar
  39. 39.
    Soler C, Valdes R, Garcia-Manteiga J et al (2001) Lipopolysaccharide-induced apoptosis of macrophages determines the up-regulation of concentrative nucleoside transporters Cnt1 and Cnt2 through tumor necrosis factor-alpha-dependent and -independent mechanisms. J Biol Chem 276:30043–30049CrossRefPubMedGoogle Scholar
  40. 40.
    Marriott HM, Ali F, Read RC et al (2004) Nitric oxide levels regulate macrophage commitment to apoptosis or necrosis during pneumococcal infection. FASEB J 18:1126–1128PubMedGoogle Scholar
  41. 41.
    Xaus J, Comalada M, Valledor AF et al (2000) LPS induces apoptosis in macrophages mostly through the autocrine production of TNF-alpha. Blood 95:3823–3831PubMedGoogle Scholar

Copyright information

© US Government 2010

Authors and Affiliations

  • Lakshmi Kannan
    • 1
    • 2
    • 3
  • Narayan C. Rath
    • 1
  • Rohana Liyanage
    • 4
  • Jackson O. LayJr.
    • 4
  1. 1.PPPSRU/Agricultural Research Service/USDA, Poultry Science CenterUniversity of ArkansasFayettevilleUSA
  2. 2.Cell and Molecular Biology ProgramUniversity of ArkansasFayettevilleUSA
  3. 3.Department of MedicineBeth Israel Deaconess Medical Center, Harvard Medical SchoolBostonUSA
  4. 4.Department of Chemistry and BiochemistryUniversity of ArkansasFayettevilleUSA

Personalised recommendations