Class A scavenger receptor expression and function in eight novel tadpole cell lines from the green frog (Lithobates clamitans) and the wood frog (Lithobates sylvatica)

  • Nguyen T. K. Vo
  • Joshua Everson
  • Levi Moore
  • Stephanie J. DeWitte-OrrEmail author
Original Article


A total of eight tadpole cell lines were established from green frogs (Lithobates clamitans) and wood frogs (Lithobates sylvatica). The five green frog cell lines were named GreenTad-HF1, GreenTad-HF2, GreenTad-HF3, GreenTad-HE4, and GreenTad-gill. The three wood frog cell lines were named WoodTad-HE1, WoodTad-Bone, and WoodTad-rpe. DNA barcoding confirmed the cell lines to be from the correct species and the growth characteristics (optimal temperature and FBS requirement) were elucidated. In order to begin studying the innate immune capacity for each cell line, class A scavenger receptor expression and function were next explored. All cell lines expressed genes for at least 3 of the 5 class A scavenger receptor (SR-A) family members, but the gene expression patterns varied between cell lines. MARCO was only expressed in GreenTad-HE4 and WoodTad-Bone, while only GreenTad-HF3 did not express SCARA5 and only WoodTad-rpe did not express SR-AI. Acetylated low density lipoprotein (AcLDL) is a well-defined ligand for SR-As and WoodTad-rpe was the only cell line to which it was unable to bind. In the other seven tadpole cell lines, the SR-A competitive ligands (dextran sulfate, fucoidan, polyinosinic acid) blocked AcLDL binding whereas the SR-A non-competitive ligand counterparts (chondroitin sulfate, fetuin, polycytidylic acid, respectively) did not. Overall, these new eight cell lines can become important tools in the study of innate immunity in general and SR-A functions in particular in green frogs and wood frogs.


Frog Class A scavenger receptor acLDL Cell line Tadpole Innate immunity 



The research was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada in the form of a Discovery grant and the Ontario Ministry of Research, Innovation and Science (MRIS)’s Early Researcher Award (ERA) grant to SDO.


  1. Acton S, Resnick D, Freeman M, Ekkel Y, Ashkenas J, Krieger M (1993) The collagenous domains of macrophage scavenger receptors and complement component C1q mediate their similar, but not identical, binding specificities for polyanionic ligands. J Biol Chem 268:3530Google Scholar
  2. Baid K, Nellimarla S, Huynh A, Boulton S, Guarné A, Melacini G, Collins SE, Mossman KL (2018) Direct binding and internalization of diverse extracellular nucleic acid species through the collagenous domain of class A scavenger receptors. Immunol Cell Biol 96:922–934CrossRefGoogle Scholar
  3. Bols NC, Pham PH, Dayeh VR, Lee LEJ (2017) Invitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout. Vitro Cell Dev Biol Anim 53:383–405CrossRefGoogle Scholar
  4. Bowdish DM, Gordon S (2009) Conserved domains of the class A scavenger receptors: evolution and function. Immunol Rev 227:19–31CrossRefGoogle Scholar
  5. Catenazzi A (2015) State of the world’s amphibians. Annu Rev Environ Resour 40:91–119CrossRefGoogle Scholar
  6. Chinchar VG (2002) Ranaviruses (family Iridoviridae): emerging cold-blooded killers. Arch Virol 147:447–470CrossRefGoogle Scholar
  7. Cooper JK, Sykes G, King S, Cottrill K, Ivanova NV, Hanner R, Ikonomi P (2007) Species identification in cell culture: a two-pronged molecular approach. Vitro Cell Dev Biol Anim 43:344–351CrossRefGoogle Scholar
  8. Dansako H, Yamane D, Welsch C, McGivern DR, Hu F, Kato N, Lemon SM (2013) Class A scavenger receptor 1 (MSR1) restricts hepatitis C virus replication by mediating toll-like receptor 3 recognition of viral RNAs produced in neighboring cells. PLoS Pathog 9:e1003345CrossRefGoogle Scholar
  9. Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE, Speare R (1999) Emerging infectious diseases and amphibian population declines. Emerg Infect Dis 5:735–748CrossRefGoogle Scholar
  10. Densmore CL, Green DE (2007) Diseases of amphibians. ILAR J 48:235–254CrossRefGoogle Scholar
  11. DeWitte-Orr SJ, Leong JAC, Bols NC (2007) Induction of antiviral genes, Mx and vig-1, by dsRNA and Chum salmon reovirus in rainbow trout monocyte/macrophage and fibroblast cell lines. Fish Shellfish Immunol 23(3):670–682CrossRefGoogle Scholar
  12. DeWitte-Orr SJ, Collins SE, Bauer CM, Bowdish DM, Mossman KL (2010) An accessory to the ‘Trinity’: SR-As are essential pathogen sensors of extracellular dsRNA, mediating entry and leading to subsequent type I IFN responses. PLoS Pathog 6:e1000829CrossRefGoogle Scholar
  13. Dieudonne A, Torres D, Blanchard S, Taront S, Jeannin P, Delneste Y, Pichavant M, Trottein F, Gosset P (2012) Scavenger receptors in human airway epithelial cells: role in response to double-stranded RNA. PLoS ONE 7:e41952CrossRefGoogle Scholar
  14. Doi T, Higashino K, Kurihara Y, Wada Y, Miyazaki T, Nakamura H, Uesugi S, Imanishi T, Kawabe Y, Itakura H (1993) Charged collagen structure mediates the recognition of negatively charged macromolecules by macrophage scavenger receptors. J Biol Chem 268:2126–2133Google Scholar
  15. Fukuda M, Ohtani K, Jang SJ, Yoshizaki T, Mori K, Motomura W, Yoshida I, Suzuki Y, Kohgo Y, Wakamiya N (2011) Molecular cloning and functional analysis of scavenger receptor zebrafish CL-P1. Biochim Biophys Acta 1810:1150–1159CrossRefGoogle Scholar
  16. Goldstein J, Ho Y, Basu S, Brown M (1979) Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci USA 76:333CrossRefGoogle Scholar
  17. Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16(3):183–190Google Scholar
  18. Grogan LF, Robert J, Berger L, Skerratt LF, Scheele BC, Castley JG, Newell DA, McCallum HI (2018) Review of the amphibian immune response to chytridiomycosis, and future directions. Front Immunol 9:2536CrossRefGoogle Scholar
  19. Han HJ, Tokino T, Nakamura Y (1998) CSR, a scavenger receptor-like protein with a protective role against cellular damage causedby UV irradiation and oxidative stress. Hum Mol Genet 7:1039–1046CrossRefGoogle Scholar
  20. Jiang Y, Oliver P, Davies KE, Platt N (2006) Identification and characterization of murine SCARA5, a novel class A scavenger receptor that is expressed by populations of epithelial cells. J Biol Chem 281:11834–11845CrossRefGoogle Scholar
  21. Kraal G, van der Laan L, Elomaa O, Tryggvason K (2000) The macrophage receptor MARCO. Microbes Infect 2:313–316. CrossRefGoogle Scholar
  22. Krieger M (1997) The other side of scavenger receptors: pattern recognition for host defense. Curr Opin Lipidol 8:275–280CrossRefGoogle Scholar
  23. MacLeod DT, Nakatsuji T, Yamasaki K, Kobzik L, Gallo RL (2013) HSV-1 exploits the innate immune scavenger receptor MARCO to enhance epithelial adsorption and infection. Nat Commun 4:1963CrossRefGoogle Scholar
  24. Murphy JE, Tedbury PR, Homer-Vanniasinkam S, Walker JH, Ponnambalam S (2005) Biochemistry and cell biology of mammalian scavenger receptors. Atherosclerosis 182:1–15CrossRefGoogle Scholar
  25. Nakamura K, Funakoshi H, Miyamoto K, Tokunaga F, Nakamura T (2001) Molecular cloning and functional characterization of a human scavenger receptor with C-type lectin (SRCL), a novel member of a scavenger receptor family. Biochem Biophys Res Commun 280:1028–1035CrossRefGoogle Scholar
  26. Ohtani K, Suzuki Y, Eda S, Kawai T, Kase T, Keshi H, Sakai Y, Fukuoh A, Sakamoto T, Itabe H, Suzutani T, Ogasawara M, Yoshida I, Wakamiya N (2001) The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells. J Biol Chem 276:44222–44228CrossRefGoogle Scholar
  27. Poynter SJ, Weleff J, Soares AB, DeWitte-Orr SJ (2015) Class-A scavenger receptor function and expression in the rainbow trout (Oncorhynchus mykiss) epithelial cell lines RTgutGC and RTgill-W1. Fish Shellfish Immunol 44:138–146CrossRefGoogle Scholar
  28. Poynter SJ, Monjo AL, DeWitte-Orr SJ (2018) Identification of three class A scavenger receptors from rainbow trout (Oncorhynchus mykiss): SCARA3, SCARA4, and SCARA5. Fish Shellfish Immunol 76:121–125CrossRefGoogle Scholar
  29. Rafferty KA Jr (1969) Mass culture of amphibian cells: methods and observations concerning stability of cell type. In: Mizell M (ed) Biology of amphibian tumors. Springer, Berlin, pp 52–81CrossRefGoogle Scholar
  30. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage K, Ueda O, Sakaguchi H, Higashi T, Suzuki T, Takashima Y, Kawabe Y, Cynshi O, Wada Y, Honda M, Kurihara H, Aburatani H, Doi T, Matsumoto A, Azuma S, Noda T, Toyoda Y, Itakura H, Yazaki Y, Kodama T (1997) A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature 386:292–296CrossRefGoogle Scholar
  31. Vo NTK, Bender AW, Ammendolia DA, Lumsden JS, Dixon B, Bols NC (2015a) Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septicemia virus (VHSV IVb) and to protection by synthetic dsRNA. Fish Shellfish Immunol 45:83–93CrossRefGoogle Scholar
  32. Vo NTK, Bender AW, Lee LEJ, Lumsden JS, Lorenzen N, Dixon B, Bols NC (2015b) Development of a walleye cell line and use to study the effects of temperature on infection by viral hemorrhagic septicaemia virus (VHSV) group IVb. J Fish Dis 38:121–136CrossRefGoogle Scholar
  33. Vo NTK, Guerreiro M, Yaparla A, Grayfer L, DeWitte-Orr SJ (2019a) Class A scavenger receptors are used by frog virus 3 during its cellular entry. Viruses 11(2):E93. CrossRefGoogle Scholar
  34. Vo NTK, Moore LC, Leis E, DeWitte-Orr SJ (2019b) Class A scavenger receptors mediate extracellular dsRNA sensing, leading to downstream antiviral gene expression in a novel American toad cell line, BufoTad. Dev Comp Immunol 92:140–149CrossRefGoogle Scholar
  35. Whelan FJ, Meehan CJ, Golding GB, McConkey BJ, Bowdish DM (2012) The evolution of the class A scavenger receptors. BMC Evol Biol 12:227CrossRefGoogle Scholar
  36. Wright K (2003) Cholesterol, corneal lipidosis, and xanthomatosis in amphibians. Vet Clin N Am Exot Anim Pract 6:155–167CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Health SciencesWilfrid Laurier UniversityWaterlooCanada
  2. 2.Department of BiologyWilfrid Laurier UniversityWaterlooCanada

Personalised recommendations