An alternative form of IL-18 in human blood plasma: Complex formation withIgM defined by monoclonal antibodies

Monoclonal Abs 21 and 132 were raised against human functionally inactive r IL-18, and plasma IL-18 levels were determined by the sandwich ELISA establ ished with these mAbs. Plasma IL-18, designated type 2, was detected by thi s ELISA, and the levels found were not consistent with those obtained with the commercially available kit for determination of functionally active IL- 18 (type 1). Type 1 was detected in all volunteers, whereas type 2 was dete cted in similar to 30% of healthy subjects, and the levels of type 2 in the ir blood plasma were high (25-100 ng/ml) compared with those of type 1 (0.0 5-0.3 ng/ml). We purified IL-18 type 2 from blood plasma of volunteers with high IL-18 type 2 concentrations, and its M-r was determined to be 800 kDa by SDS-PAGE and molecular sieve HPLC. The purified 800-kDa protein, either caspase-1-treated or untreated, expressed no or marginal IL-18 function in terms of potentiation of NK-mediated cytolysis and IFN-gamma induction, an d it barely bound IL-18R-positive cells. N-terminal amino acid analysis ind icated that the purified protein was IgM containing a minimal amount of IL- 18 proform and its fragment. Again, the purified IgM from IL-18 type2-posit ive volunteers exhibited cross-reaction with mAb 21 against IL-18. This ban d was not detected with 125-2H, an mAb against functionally active IL-18. H ence, human IgM carries functionally inactive IL-18 forming a disulfide-bri dged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type 1 IL-18 in blood circulation in similar to 30% normal su bjects.