Open AccessGene 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
Author(s) -
Mehraban Fuad,
Kasturi Sriram
Publication year - 1998
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/1529-0131(199803)41:3<515::aid-art18>3.0.co;2-o
Subject(s) - microbiology and biotechnology , transfection , biology , cell culture , complementary dna , interleukin 1 receptor , receptor , expression vector , expression cloning , recombinant dna , cytokine , interleukin , biochemistry , gene , immunology , genetics
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 125 I‐IL‐1β 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 125 I‐IL‐1β binding. 35 S labeling of transfected cultures, followed by immunoprecipitation and gel electrophoresis, was used to characterize the size of the recombinant sIL‐1R. Stromelysin and IL‐1α steady‐state messenger RNA (mRNA) levels were assessed by Northern blotting. Prostaglandin E 2 (PGE 2 ) release was measured by enzyme‐linked immunosorbent assay. Results IL‐1R on the surface of HIG‐82 cells bound 125 I‐IL‐1β with an equilibrium dissociation constant ( K d ) of 67.3 ± 7.8 p M (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 125 I‐IL‐1β with high affinity in the medium ( K d = 108 ± 5 p M ). Two protein bands ( M r 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‐1α steady‐state mRNA levels. In conjunction, there was a significant inhibition of basal and IL‐1‐stimulated PGE 2 released by sIL‐1R‐producing cells. Conclusion 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.