An alternative form of IL-18 in human blood plasma: complex formation with IgM defined by monoclonal antibodies.
Monoclonal Abs 21 and 132 were raised against human functionally inactive rIL-18, and plasma IL-18 levels were determined by the sandwich ELISA established with these mABS: Plasma IL-18, designated type 2, was detected by this 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 detected in approximately 30% of healthy subjects, and the levels of type 2 in their blood plasma were high (25-100 ng/ml) compared with those of type 1 (0.05-0.3 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, and it barely bound IL-18R-positive cells. N-terminal amino acid analysis indicated 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-positive volunteers exhibited cross-reaction with mAb 21 against IL-18. This band was not detected with 125-2H, an mAb against functionally active IL-18. Hence, human IgM carries functionally inactive IL-18 forming a disulfide-bridged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type 1 IL-18 in blood circulation in approximately 30% normal subjects.
Shida
K
K
Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan.
Shiratori
I
I
Matsumoto
M
M
Fukumori
Y
Y
Matsuhisa
A
A
Kikkawa
S
S
Tsuji
S
S
Okamura
H
H
Toyoshima
K
K
Seya
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D001666:Binding Sites, Antibody; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood*; D007369:Interferon Inducers / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D007371:Interferon-gamma / Q000096:biosynthesis; D018664:Interleukin-12 / Q000502:physiology; D020382:Interleukin-18 / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D020033:Protein Isoforms / Q000097:blood / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D007369:Interferon Inducers; D020382:Interleukin-18; D046911:Macromolecular Substances; D020033:Protein Isoforms; D011994:Recombinant Proteins; D018664:Interleukin-12; D007371:Interferon-gamma