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 proper ties on the IL-1 pathway. Methods. High-affinity IL-1R sites were iden tified in a human chondrosarcoma cell line by means of I-125-IL-1 beta binding, A 1-kilobase complementary DNA (cDNA) encoding the ligand-bi nding 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 s ynovial cell line (HIG-82) and a stably transfected cell population wa s selected. The production of sIL-1R was confirmed in the medium of tr ansfected cells using I-125-IL-1 beta binding, S-35 labeling of transf ected cultures, followed by immunoprecipitation and gel electrophoresi s, was used to characterize the size of the recombinant sIL-1R, Strome lysin and IL-1 alpha steady-state messenger RNA (mRNA) levels were ass essed by Northern blotting, Prostaglandin E-2 (PGE(2)) release was mea sured by enzyme-linked immunosorbent assay. Results. IL-1R on the surf ace of HIG-82 cells bound I-125-IL-1 beta with an equilibrium dissocia tion constant (K-d) of 67.3 +/- 7.8 pM (mean +/- SD), Transfection of the sIL-1R expression vector into a synovial cell line in vitro result ed in the appearance of an sIL-1R protein that bound I-125-IL-1 beta w ith high affinity in the medium(K-d = 108 +/- 5 pM), Two protein bands (M(r)42 kd and 47 kd) were immunoprecipitated with an antibody agains t type 1 T cell-derived sIL-1R, Expression of sIL-1R was accompanied b y a marked decrease in both stromelysin and IL-1 alpha steady-state mR NA levels, In conjunction, there was a significant inhibition of basal and IL-1-stimulated PGE(2) released by sIL-1R-producing cells, Conclu sion. The data suggest that gene transfer of type 1 sIL-1R into the sy novium may be an effective means of inhibiting IL-l-induced metallopro teinase expression and inflammatory responses.