Bikunin (BK) is a Kunitz-type proteinase inhibitor responsible for most of the antitryptic activity of urine and so is known as the urinary trypsin inhibitor. As its excretion increases in inflammatory conditions, it is often considered to be a positive acute phase protein (APP). However, the gene for BK is downregulated in inflammation. In human plasma the major part of BK is covalently linked through a glycosaminoglycan chain to one or two homologous peptide heavy chains, thus forming high molecular weight proteinase inhibitors called pre-alpha-inhibitor (PalphaI) and inter-alpha-inhibitor (IalphaI), respectively. The C-terminal parts of these heavy chains are very sensitive to proteolysis. Neutrophil proteinases in particular are able to release from IalphaI and PalphaI BK (M, about 25,000) which retains its antitryptic activity and is quickly excreted in urine. It was therefore an early supposition that the higher urinary excretion of BK occurring during inflammatory diseases should be, at least in some respect, related to a partial proteolysis of IalphaI and PalphaI. In this study we observed that BK, determined as antitryptic activity, was clearly increased in urine from 35 patients with inflammatory diseases varying in origin and severity (76.5 +/- 75.5 IU/g vs. reference value <10 IU/g creatinine). This increase seems mainly to be associated with polymorphonuclear leukocyte activation, monitored by human leukocyte elastase (HLE) determination rather than with the acute phase response assessed by C-reactive protein (CRP) measurement. For all the patients we found that the urinary levels of BK and serum concentration of intact IalphaI correlated inversely (r=-0.36; p=0.03), in agreement with the presumed precursor-product relationship linking IalphaI and BK. We also proved that urinary BK was significantly higher, and serum IalphaI was significantly lower, in samples with plasma HLE values above the reference: 90 microg/l. Taken together, our results demonstrate that BK, the urinary excretion of which is increased in inflammatory conditions, originates, at least partly, from IalphaI and PalphaI by proteolytic cleavage. Consequently, urinary BK determination provides information on the severity of systemic proteolysis occurring in inflammation. We also demonstrated that during inflammatory diseases IalphaI and PalphaI concentrations in serum are dependent on their increased utilization as well as on the regulation of their biosynthesis.

Inter-alpha -inhibitor (I alphaI) and pre-alpha -inhibitor (P alphaI) are the main members of a set of multichain serine proteinase inhibitors. Present in human plasma, they may be involved in control of the inflammatory process. They are composed of homologous heavy chains (H1 and H2 for I alphaI; H3 for P alphaI) covalently linked by a protein-glycosaminoglycan-protein cross-link to bikunin, which is a chondroitin 4-sulfate proteoglycan. During the acute-phase response, biosynthesis of I alphaI and P alphaI is downregulated and upregulated, respectively.In this work, we provide evidence that, in inflammatory diseases, the chondroitin sulfate chain of bikunin increases in size proportionally to the severity of the inflammatory response. As a consequence, all I alphaI-related components that contain bikunin are structurally modified. Therefore, the changes in glycosylation of the acute-phase proteins are not restricted to N-linked glycans but also affect glycosaminoglycans.The implications of these findings are discussed with regard to biosynthesis and biological role, especially the anti-inflammatory effects of I alphaI-related proteinase inhibitors.

BACKGROUND Pre-alpha-inhibitor (PalphaI) is a human plasma serine-proteinase inhibitor that is structurally related to inter-alpha-inhibitor (IalphaI). It is composed of a heavy chain named H3 covalently linked to bikunin by means of a glycosaminoglycan chain. We developed an ELISA procedure making it possible to measure PalphaI for the first time and we investigated its levels in sera from patients with inflammatory diseases. MATERIALS AND METHODS We generated rabbit anti-H3 immunoglobulins, which were used on solid phase and biotinylated antibikunin immunoglobulins to detect trapped PalphaI. RESULTS We demonstrate that PalphaI is more susceptible than IalphaI to in vitro proteolysis by stimulated neutrophils. However, the degradation products thus released as well as the other members of the IalphaI family present in serum do not affect the ELISA test. In a panel of control sera we observed PalphaI concentrations of 25.6 +/- 7.8 mg L-1 (mean +/- SD; n = 30). These values increased to 64.2 +/- 16.06 mg L-1 (mean +/- SD; n = 15) in patients with inflammatory diseases, concording with the positive acute-phase protein nature of PalphaI. However, for all these patients, the serum concentrations of PalphaI and C-reactive protein poorly correlated (r = 0.476; P = 0.076). Indeed, four patients had a relatively weaker increase in their PalphaI level than that of C-reactive protein. More often than not their plasma elastase content was then elevated. CONCLUSION During inflammatory diseases plasma PalphaI levels may be dependent on increased synthesis in combination with enhanced catabolism, perhaps implicating neutrophil or other proteinases.