Measurement of both native and inactivated forms ofa1 proteinase inhibitor in human inflammatory extracellular fluids

Abstract

Background: Inactivation of the elastase inhibitor, a1 proteinase inhibitor (a1PI), may be of pathogenic significance in inflammatory diseases like periodontal disease. Two key mechanisms of inactivation appear to be (a) the formation of an a1PI-elastase complex and (b) proteolytic cleavage by elastase or other enzymes such as metalloproteinases of host origin or enzymes of bacterial origin. Based on the different heat stabilities of the intact, complexed and proteolytically cleaved forms of a1PI, an enzyme-linked immunosorbent assay (ELISA) that allowed the simultaneous measurement of native and inactive forms of a1PI was developed. Methods: The ELISA method described employs a commercially available antibody and represents a rapid, reproducible and sensitive method for studying a1PI inactivation in human inflammatory diseases. The assay was applied to normal human plasma and to human extracellular fluids obtained from patients with inflammatory diseases such as adult periodontitis and rheumatoid arthritis. Samples from patients with osteoarthritis, a "non-inflammatory" joint disease, were also studied. Results: The findings expressed as the mean percentage (ᓄ) of the total a1PI that was inactivated were as follows: gingival crevicular fluid from adult periodontitis patients: 73.5ᱶ.6% (n=12); normal human plasma: 8.4ᴮ9% (n=13); knee-joint synovial fluid (SF) from rheumatoid arthritis patients: 12.5ᴮ5% (n=15); plasma from rheumatoid arthritis patients: 8.0ᱮ8% (n=15); knee-joint SF from osteoarthritis patients: 8.6Ḯ2% (n=14); plasma from osteoarthritis patients: 5.7ᴮ8% (n=14). The results obtained by ELISA were in good agreement with those obtained by the semi-quantitative method of SDS-PAGE and Western blotting. Conclusions: We have shown that the differential heat stability of a1PI may be utilised as the basis for a rapid, sensitive and reproducible ELISA assay of a1PI inactivation. In gingival crevicular fluid from periodontal disease patients, a1PI is mainly inactivated and the extent of this inactivation is much higher than in inflammatory fluids from other chronic diseases such as rheumatoid arthritis. This assay could be useful in monitoring the progression of periodontal disease.