The functions of pregnancy-associated plasma protein A in rheumatoid arthritis

Pregnancy associated plasma protein A (PAPP-A) is produced in increasing amounts with advancing pregnancy, a major source being the placenta. It was originally described as a homotetramer with ~ 200,000 M[r] subunits. An alternative structure has since been proposed of a disulphide bridged complex consisting of equimolar amounts of the PAPP-A subunit and the proform of eosinophil major basic protein (pro MBP). The increasing levels of PAPP-A produced during pregnancy and reports of possible proteinase inhibitory activity and immunomodulatory activity suggested that it may be an antiarthritic agent, involved in the amelioration of rheumatoid arthritis (RA) observed during pregnancy.PAPP-A has been purified from late pregnancy plasma using ammonium sulphate precipitation, gel filtration chromatography, ion exchange chromatography and affinity chromatography. The purity has been confirmed by SDS PAGE and western blot analysis. The purified PAPP-A has been used to investigate functions of PAPP-A which may support or refute a role in the amelioration of the symptoms of RA during pregnancy.Potential immunomodulatory actions of PAPP-A may be mediated by the inhibition of cytokine production or action. The effects of PAPP-A on the production of proinflammatory cytokines and also on the production of prostaglandin E[2] (PGE[2]), an inflammatory mediator, were studied using the THP 1 human monocytic cell line and the MG-63 human osteoblast- like cell line. PGE[2] was measured by radioimmunoassay and the cytokines IL-1, IL-6 and TNFalpha using commercial ELISA kits from Biosource International. IL-1alpha stimulated the production of PGE[2] and IL-6 in the MG-63 cells, whereas PAPP-A had no significant effect on basal or IL-1alpha stimulated IL-6 and PGE[2] production. In THP 1 cells, LPS, as expected, stimulated the production of PGE[2], IL-l? , IL-6 and TNFalpha. PAPP-A alone also significantly stimulated their production at higher concentrations, having a much greater effect at higher concentrations than LPS alone. PAPP-A, when incubated with LPS, produced a greater than additive effect on the production of PGE[2], but in combination with LPS had no significant effect on the levels of IL-6 obtained with LPS alone.Binding of PAPP-A to IL-1alpha and TGFbeta was studied using [125]I-cytokines. Formation of PAPP-A cytokine complexes was determined using either native SDS PAGE or gel filtration chromatography. No evidence of cytokine binding was obtained. The binding of PAPP-A to human cartilage was also investigated, since this may facilitate any protective effects of PAPP-A, such as proteinase inhibition, on cartilage. Cartilage binding was studied using immunohistochemical methods. The results indicated that PAPP-A does bind to cartilage, suggesting a possible protective role.The effect of PAPP-A on retinoic acid stimulated resorption of bovine nasal cartilage was studied and cartilage proteoglycan release measured as glycosaminoglycans using the dimethylemethylene blue assay. PAPP-A appeared to stimulate the resorption of cartilage.A key issue was whether PAPP-A functions as a proteinase inhibitor thus preventing connective tissue breakdown. Inhibition of elastase, cathepsin B, plasmin and trypsin was studied using colorimetric and fluorescent peptide substrates. PAPP-A was shown to competitively inhibit elastase, possibly due to the electrostatic interactions between the negatively charged PAPP-A and positively charged elastase. It did not inhibit the other enzymes studied,A monoclonal antibody to PAPP-A was produced by immunization of mice with the purified PAPP-A. This is currently been evaluated as to its potential use in a screening ELISA assay for monitoring PAPP-A levels.Overall the effects of PAPP-A on monocytic cell cytokine and PGE2 production and its stimulation of cartilage proteoglycan breakdown suggest it does not have a major role in the remission of RA during pregnancy. However this work has demonstrated functions of PAPP-A not previously reported and confirmed earlier observations of inhibitory activity towards elastase. Further work is required to determine the key functions of PAPP-A during pregnancy.