In the context of periodontal disease, cysteine proteinases or gingipains from have been implicated in the hydrolysis of cytokines, including gamma interferon (IFN-). coincubation with sodium or potassium chloride answer. The N-terminal residues of IFN- were implicated in mediating the effect of K1K2, while antibody binding to loop 1 of K2 blocked the action of K1K2. The findings indicate the potential significance of structurally defined Kgp adhesin modules in the inactivation of IFN- but also the Apigenin inhibitor potential of K1K2 in locating the target for the catalytic domain name of Kgp. INTRODUCTION Periodontal diseases are characterized by the presence of inflammatory lesions and are customarily separated into infections affecting only the gingiva and those affecting the underlying structures of the periodontium (34). The transition from gingivitis to periodontitis is usually marked by the change in T-cell to B-cell predominance (9, 35). In this concept, it is clear that the balance of cytokines, bacterial factors such as protease production, and other host factors determine whether tissue destruction occurs or homeostasis is usually maintained. Among periodontal pathogens, most evidence points to a pathogenic role for (18, 37). The mechanisms underlying the differences in virulence between strains are not yet fully comprehended, but a group of cysteine proteinases, known as gingipains, play critical functions in hemagglutination, hemolysis, and disruption of cytokine networks (15, 21, 40, 41). Gingipains with either arginine (Arg-gingipain; RgpA and RgpB) or lysine (Lys-gingipain; Kgp) specificity are produced in large quantities by and (28, 29, 31). The predominant outer-membrane-associated high-molecular-weight forms of RgpA and Kgp form characteristic enzyme clusters comprising the catalytic domains linked to a number of hemagglutinin/adhesin modules (30). It is considered that the manner of secretion, processing, Apigenin inhibitor and attachment to the outer membrane utilizes the same pathway as for RgpB (26). Following extraction from may lie in its ability to upregulate class I and II major histocompatibility complex (MHC) antigens (6). Several studies have reported a significant increase in the gingival crevicular fluid (GCF) concentration of IFN- in those subjects with severe periodontitis (8, 27, 38). In contrast, measurement of IFN- in cultures of peripheral blood mononuclear cells (PBMCs) from periodontitis and control subjects has shown that IFN- levels in leukocyte cultures from periodontitis patients are consistently low (24). In this context, we previously exhibited that gingipains can degrade IFN-, and this may bias the immune response to a Th2 cytokine response (42). Currently, the precise events relating to recognition of cytokine networks by remain undetermined. The present study was conducted to determine the effects of hemagglutinin/adhesin domains of Kgp on IFN–regulated responses in K562 cells. The data demonstrate selectivity in the action of adhesin domains of Kgp in relation to the inhibition of Apigenin inhibitor IFN- action. Among these adhesin polypeptides, prolonged exposure to K1K2 can decrease IFN–regulated expression of HLA-1 and HLA-DR protein. Further, binding of K1K2 to IFN- resulted in decreased surface expression of IFN-R1 and IFN-R2 protein in K562 cells. MATERIALS AND METHODS Reagents. Bovine submaxillary mucin, fetal calf serum (FCS), l-cysteine, sodium dodecyl sulfate (SDS), strain W83 were prepared as previously described (20, 21). Cytokines and Abs. Nonglycosylated recombinant human gamma interferon (rIFN-) and glycosylated natural gamma interferon were purchased from Sigma-Aldrich (Castle Hill, New South Wales, Australia). Monoclonal antibody (MAb) against IFN- (clone 25718) was obtained from R&D Systems (Minneapolis, MN). Fluorescein isothiocyanate (FITC)Canti human HLA-1 MAb (clone W6/32) was purchased from Sigma-Aldrich (Castle Hill, New South Wales, Australia). Monoclonal anti-human HLA class II DR Ab (clone TAL.1B5) Rabbit Polyclonal to VN1R5 was purchased from Dako (Sydney). Polyclonal antibodies specific for amino acids 466 to 485 mapping at the COOH-terminal domain name of the human IFN- R chain (IFN-R1) or amino acids 318 to 337 mapping at the COOH-terminal domain name for IFN- R chain (IFN-R2) were obtained from Santa Cruz Biotechnology. ELISA. Binding of adhesin domains to rIFN- or IFN- peptides was tested using an enzyme-linked immunosorbent assay (ELISA) method. IFN- peptide (peptide 1-14 or MKYTSYILAFQLCI [pI 8.8] at the N terminus) was synthesized by Mimotopes with purity of 80%. Briefly, 1 g of adhesin domains was used to coat the wells of a.