Analysis of osteoclast-activating factor produced by synovial tissues from rheumatoid arthritis mutilans

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The synovial tissue was obtained from the knee joint of patient with rheumatoid arthritis mutilans during total knee replacement. Cells were isolated by collagenase treatment and cultured. After one week of culture, the culture supernatant was collected and concentrated, and subjected to gel filtration using a Sephadex G-15 column. The sample was fractionated by molecular weight gradient and added to osteoclasts cultured on an ivory fragment, and the formation of resorption holes was compared between osteoclasts in the presence and absence of the sample. Osteoclasts treated with the sample formed three-fold more resorption holes than osteoclasts without treatment.The samples that promoted the formation of resorption holes were applied on electrophoresis to examine the molecular weights of contained substances, and about six peaks ranging from 0.5 kD to 12 kD were detected. The effect of each peak fraction on the formation of resorption holes by osteoclasts was examinedas described above, and the fraction containing an approximately 1 kD molecular weight protein significantly promoted the formation of resorption holes by osteoclasts compared with the control.This protein fraction was further fractioneted by electrical polarity using a DE52 column, and the fractions were added to osteoclasts to confirm the activity of promoting resorptionhole formation, and a fraction containing the active protein was obtained was obtained.The protein was isolated from the fraction by reverse-phase high performance liquid chromatography, and each protein obtained from three peaks was added to osteoclasts. Protein from one of three peaks activated the formation of resorption holes by osteoclasts.We are analysing the amino acid sequence of the protein obtained from this peak using a gas phase protein sequencer, but we have not yet determined the protein structure.