Construction and characterization of a replication-deficient adenovirus expressing rat-soluble interleukin-6 receptor.
BACKGROUND
The pleiotropic cytokine interleukin-6 mediates its multiple effects at the cell level through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130). A soluble form of gp80 (sIL-6R or gp55) is found released from the surface of cells and appears to possess interleukin-6 (IL-6) agonist activity. Increases in circulating levels of sIL-6R have been reported in different pathological conditions but the precise role of this protein in vivo remains unknown.


METHODS
The cDNA encoding the extracellular domain of the rat IL-6R (sIL-6R) with an appropriate leader sequence has been cloned into the E1 region of an adenovirus vector under the control of the hCMV promoter (Ad5.sIL-6R).


RESULTS
Infection of different human or rodent cell lines with Ad5.sIL-6R leads to extended production of recombinant sIL-6R protein into the culture media. The kinetics of transgene expression depends both on the cell type and the species. sIL-6R produced in this manner is biologically active as it confers responsiveness of human hepatoma cells (HepG2) to rat IL-6 stimulation. Adenovirus vectors have been shown to be highly effective for transient delivery of cytokines in vivo. Antibodies against recombinant rat soluble IL-6R were generated and an ELISA developed that allowed us to quantify sIL-6R concentrations. The sIL-6R expressing adenovirus vector has been instilled intratracheally into rats and induced an increase in lung sIL-6R concentration from Day 1 up to Day 10. We demonstrate the potency of our system to deliver in vivo or in vitro soluble cytokine receptors in a prolonged but transient manner.
Thibault
V
V
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Terlain
B
B
Graham
F L
FL
Gauldie
J
J
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004261:DNA Replication; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D019947:Receptors, Interleukin-6 / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility; D014132:Trachea
D018076:DNA, Complementary; D004274:DNA, Recombinant; D012333:RNA, Messenger; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins