Gene transfer of type 1 interleukin-1 receptor extracellular-domain complementary DNA into rabbit synovial cell line HIG-82 results in cellular blockade of interleukin-1 signal transduction.
OBJECTIVE
To produce, by means of expression cloning, a soluble type 1 interleukin-1 receptor (sIL-1R), and to assess its inhibitory properties on the IL-1 pathway.


METHODS
High-affinity IL-1R sites were identified in a human chondrosarcoma cell line by means of 125I-IL-1beta binding. A 1-kilobase complementary DNA (cDNA) encoding the ligand-binding domain of the type 1 IL-1R was cloned by using polymerase chain reaction, and the cDNA was inserted into a mammalian expression vector pRc/CMV. The sIL-1R expression vector was transfected into a rabbit synovial cell line (HIG-82) and a stably transfected cell population was selected. The production of sIL-1R was confirmed in the medium of transfected cells using 125I-IL-1beta binding. 35S labeling of transfected cultures, followed by immunoprecipitation and gel electrophoresis, was used to characterize the size of the recombinant sIL-1R. Stromelysin and IL-1alpha steady-state messenger RNA (mRNA) levels were assessed by Northern blotting. Prostaglandin E2 (PGE2) release was measured by enzyme-linked immunosorbent assay.


RESULTS
IL-1R on the surface of HIG-82 cells bound 125I-IL-1beta with an equilibrium dissociation constant (Kd) of 67.3 +/- 7.8 pM (mean +/- SD). Transfection of the sIL-1R expression vector into a synovial cell line in vitro resulted in the appearance of an sIL-1R protein that bound 125I-IL-1beta with high affinity in the medium (Kd = 108 +/- 5 pM). Two protein bands (Mr 42 kd and 47 kd) were immunoprecipitated with an antibody against type 1 T cell-derived sIL-1R. Expression of sIL-1R was accompanied by a marked decrease in both stromelysin and IL-1alpha steady-state mRNA levels. In conjunction, there was a significant inhibition of basal and IL-1-stimulated PGE2 released by sIL-1R-producing cells.


CONCLUSIONS
The data suggest that gene transfer of type 1 sIL-1R into the synovium may be an effective means of inhibiting IL-1-induced metalloproteinase expression and inflammatory responses.
Mehraban
F
F
Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4946, USA.
Kasturi
S
S
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015232:Dinoprostone / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019278:Matrix Metalloproteinase 3 / Q000235:genetics; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000235:genetics*; D015398:Signal Transduction / Q000502:physiology*; D013583:Synovial Membrane / Q000166:cytology* / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D007375:Interleukin-1; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D017472:Receptors, Interleukin-1; D019278:Matrix Metalloproteinase 3; D015232:Dinoprostone