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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

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

F Mehraban, S Kasturi

Arthritis Rheum. 1998 Mar;41(3):515-24.

PMID: 9506580

Abstract:

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.
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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4247016	Anti-EP4 Prostanoid Receptor antibody produced in rabbit			Price

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