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We have recently demonstrated the presence and involvement of IL-18 in rheumatoid arthritis (RA) synovitis. Moreover, blockade of IL-18 in vivo is protective in arthritis models. We sought to demonstrate for the first time the production and intracellular processing of IL-18 by human neutrophils. Thereafter we investigated novel processing pathways and potential regulatory mechanisms for IL-18 bioactivity that could operate in synovium.
Matched peripheral blood (PB) and synovial fluid (SF) neutrophils were isolated by ficoll density gradients. IL-18 was detected in neutrophil total protein lysates by western blotting. Serum free neutrophil culture supernatants were incubated with recombinant IL-18 prior to HPLC fractionation and assessed for biological activity using IL-18 sensitive KG-1 cells.
Western blotting of neutrophil lysates isolated from PB and SF of rheumatoid and psoriatic arthritis patients demonstrated the presence of a number of IL-18 specific bands ranging in molecular weight from 22 to 6 kD in size, representing pro, mature and possible IL-18 cleavage products. HPLC purified culture supernatants from PB and SF neutrophils contain heat sensitive enzymatic activity capable of in vitro cleavage of both recombinant pro and mature IL-18. This caspase independent cleavage of IL-18 resulted in the generation of biologically active fragments capable of modulating IL-18 induced IFN-g production by KG-1 cells.
These data demonstrate for the first time that modified fractions of IL-18 may be biologically active, suggesting the existence of a novel regulatory mechanism in the IL-1 cytokine family. In light of their rapid accumulation in large numbers within RA joints, our data further suggest that both neutrophils and IL-18 play important roles in disease pathogenesis.