MATERIALS AND METHODS

IL-6 Preparation-Recombinant human IL-6 was ex pressed in an Escherichia coli system and purified to homogeneity(8). The specific activity of the IL-6 was assessed in terms of B cell stimulatory activities utilizing B cell line SKW6-CL4. 125I-labeled IL-6 was prepared using Bolton Hunter reagent(14). The specific radioactivity was determined to be 6.16•~ 013cpm/g. Construction of Plasmids-PBSF2R. 236(1) was digest ed by SphI and the resulting 1,205 by fragment coding IL-6R cDNA was inserted in mp18(Amersham). sIL-6R cDNA was prepared using an in vitro Mutagenesis System (Amersham) with a synthetic nucleotide oligomer, 5• L-ATA TTCTCTAGAGAGATTCT. Thus, the termination codon was introduced at amino acid position 345. For the COS cell expression, the cDNA was inserted in the expression plasmid pSVL (Pharmacia) to give pSVL344. For the CHO cell expression, the dihydrofolate reductase(dhfr) cDNA (9) was inserted into the PvuII site of the plasmid pECE (10), to give the plasmid pECEdhfr. The HindIII-SaiI fragment coding sIL-6R was inserted in the pECEdhfr to give the CHO cell expressin plasmid pECEdhfr344. Transient Expression of sIL-6R Gene-COST cells were transfected with the plasmid by the calcium phosphate method(11). Culture supernatant was collected on day 3. A mock control was prepared using pSVL vector without the insert. sIL-6R Assay-EIA-RIA strip plate-8(Costar) was coated with 100ƒÊl of anti-IL-6 receptor antibody MT18 (12)(2ƒÊg/ml) in 0.1 M sodium-hydrogen carbonate buffer (pH 9.6) at 4• Ž overnight. The wells were blocked with 100 ƒÊ l of 1% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.05% Tween 20 for 2h at room temperature, then washed; 100ƒÊl of test sample was added. After a 2-h incubation at room temperature, the wells were washed, and incubated with 100ƒÊl of 125I labeled IL-6 (200cpm/ƒÊl) for 2h at room temperature. Following the last wash of the wells, radioactivities of each well were counted with a ƒÁ-counter(Aloka, Tokyo). Transfection of CHO Cells and Selection of the Trans formants-DXB-11(13) cells, a dhfr-CHO cell line, were transfected by calcium phosphate precipitation(11) with 10ƒÊg of pECEdhfr344. The cells had been cultured in a selective medium(nucleoside-deficient ƒ¿-MEM with 10% dialyzed fetal calf serum(FCS), 1mM glutamine, 100U/1To whom correspondence should be addressed.