Identification of serum proteins that bind with S100A8, S100A9 and S100A8/A9: clinical significance of using proteins for monitoring the postoperative condition of liver recipients.

BACKGROUND: Serum proteins that non-specifically bind with human S100A8/A9 (h-S100A8/A9) have been proposed. Our aim was to isolate and identify these proteins, and verify their clinical significance for monitoring the postoperative condition of liver recipients, and further to discuss the transportation of human fibronectin (h-FN) with h-S100A8/A9 and its functional role in vivo. METHODS: To isolate the serum proteins, recombinant human S100A8, S100A9 and S100A8/A9 affinity columns were used. Proteins were identified by mass spectrometry. Two enzyme-linked immunosorbent assays (ELISA) were used to measure h-S100A8/A9 and h-FN in the sera of liver recipients. Flow cytometry was employed to detect h-S100A8/A9 and h-FN on immunological cells. Western blotting was used to confirm serum constituents using antibodies specific to each constituent. RESULTS: One of the proteins was identified with h-FN, and its fluctuation pattern in the serum of the recipient was in contrast to that of CRP. Flow cytometry showed a positive reaction for h-S100A8/A9 and h-FN on neutrophils and monocytes, indicating that both proteins exist on these cells. CONCLUSIONS: The h-FN could be transported with S100A8/A9 in blood and/or on immunological cells, and effectively prevent further attack by various internal oxidants or repair damaged liver tissue in vivo.

[Possible mechanism for regulation of inflammatory responses with the S100A8/A9 protein].

We have described a possible mechanism for the regulation of excessive inflammatory responses with S100A8/A9 protein in damaged rat livers. Recombinant human S100A8(r-S100A8) and S100A9 (r-S100A9) were expressed in E. coli cells, and their heterodimer (r-S100A8/A9) with 90% approximate purity was also prepared successfully. The effect of the r-S100A8/A9 on suppression of acute inflammatory changes in rat livers with LPS-induced damage was microscopically observed. Indeed, the liver damage diminished as the dose of the r-S100A8/A9 increased, and the minimum requirement of the protein was estimated to be 1,000 microg/rat in this study. Observation of superoxide anions was positively observed in control rats treated with LPS alone, but almost not in the livers of rats treated with the r-S 100A8/A9 1h after injection of LPS. This fact strongly suggests that the r-S100A8/A9 could indirectly suppress production of such internal oxidants according to unknown pathway (s) in acute inflammation. Expression of mRNAs of several kinds of inflammatory cytokines, such as TNF-alpha, IL-6 and IL-1beta, was also significantly suppressed, which was of much note. Therefore, the possibility that the r-S100A8/A9 partly inhibits the process of signal transduction of inflammatory responses in the immunological cells leading to down regulation of inflammatory changes in vivo was suggested in this study. Conclusively, S100A8/A9 is not necessarily an inflammatory-induced factor, and preferably effective on suppression of excessive inflammatory reaction in vivo dose-dependently, although the mechanism is still unclear.

[Possibility of formation of the S100A8/A9-proinflammatory cytokine complexes in vivo in acute inflammation and their functional roles].

We previously hypothesized that S100A8/A9 binds with several kinds of proinflammatory cytokines, such as TNF-alpha, IL-6 and IL-1beta, to form the S100A8/A9-proinflammatory cytokine complexes in vivo in acute inflammation, leading to subsidence of inflammatory responses. Our goal was to verify the presence of these complexes in liver tissues of rats with lipopolysaccharide (LPS) induced damage. We firstly prepared two kinds of the full-length cDNA encoding amino acid sequences of human S100A8 and S100A9 proteins, and constructed their pCold-I expression vectors. The recombinant S100A8 and S100A9 were successfully expressed in E. coli, and then purified by Ni-agarose columns, respectively. The S100A8/A9 was noncovalently synthesized in 2.0 mol/1 Tris-NaOH solution (pH 12) using the purified S100A8 and S100A9. After purification, this heterodimer (1 mg) was intraperitoneally injected into a rat 1h after injection of LPS. Two kinds of ELISA systems were used to detect the S100A8/A9-inflammatory cytokine complexes in the rat liver tissue. As determined by the ELISA-A and B, the reaction was apparently positive and quantitative. Immunohistochemistry provided such complexes-positive cells in the liver with damage. The S100A8/A9-positive cells almost corresponded to the cytokines-positive ones morphologically, strongly suggested the presence of the S100A8/A9-proinflammatory cytokine complexes. In conclusion, the possibility that these complexes were formed in vivo and accumulated to the immunological cells, such as macrophages and/or activated neutrophils, was indicated. Our effort is currently addressed to isolate the S100A8/A9-proinflammatory cytokine complexes using biochemical techniques, and to comprehensively resolute their clinical significance in the differential diagnosis of inflammatory diseases.