Ceruloplasmin has two nearly identical sites that bind myeloperoxidase.

Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120degrees, which, in turn, exerts tension on MPO and results in conformational change.

Protective role of HO-1 and carbon monoxide in ethanol-induced hepatocyte cell death and liver injury in mice.

BACKGROUND & AIMS: Alcoholic liver disease is associated with inflammation and cell death. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with anti-apoptotic and anti-inflammatory properties. Here we tested the hypothesis that induction of HO-1 or treatment with a carbon monoxide releasing molecule (CORM) during chronic ethanol exposure protects and/or reverses ethanol-induced liver injury. METHODS: Female C57BL/6J mice were allowed free access to a complete liquid diet containing ethanol or to pair-fed control diets for 25days. Mice were treated with cobalt protoporphyrin (CoPP) to induce HO-1 expression during ethanol feeding or once liver injury had been established. Mice were also treated with CORM-A1, a CO-releasing molecule (CORM), after ethanol-induced liver injury was established. The impact of HO-1 induction on ethanol-induced cell death was investigated in primary cultures of hepatocytes. RESULTS: Induction of HO-1 during or after ethanol feeding, as well as treatment with CORM-A1, ameliorated ethanol-induced increases in AST and expression of mRNAs for inflammatory cytokines. Treatment with CoPP or CORM-A1 also reduced hepatocyte cell death, indicated by decreased accumulation of CK18 cleavage products and reduced RIP3 expression in hepatocytes. Exposure of primary hepatocyte cultures to ethanol increased their sensitivity to TNFα-induced cell death; this response was attenuated by necrostatin-1, an inhibitor of necroptosis, but not by caspase inhibitors. Induction of HO-1 with CoPP or CORM-3 treatment normalized the sensitivity of hepatocytes to TNFα-induced cell death after ethanol exposure. CONCLUSIONS: Therapeutic strategies to increase HO-1 and/or modulate CO availability ameliorated chronic ethanol-induced liver injury in mice, at least in part by decreasing hepatocellular death.

A novel murine model of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) induced by immunization with a spermine binding protein (p25) peptide.

The pathophysiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is poorly understood. Inflammatory and autoimmune mechanisms may play a role. We developed a murine model of experimental autoimmune prostatitis (EAP) that mimics the human phenotype of CP/CPPS. Eight-week-old mice were immunized subcutaneously with prostate-specific peptides in an emulsion of complete Freund's adjuvant. Mice were euthanized 10 days after immunization, and lymph node cells were isolated and assessed for recall proliferation to each peptide. P25 99-118 was the most immunogenic peptide. T-cell and B-cell immunity and serum levels of C-reactive protein and nitrate/nitrite levels were evaluated over a 9-wk period. Morphometric studies of prostate, 24-h micturition frequencies, and urine volume per void were evaluated. Tactile referred hyperalgesia was measured using von Frey filaments to the pelvic region. The unpaired Student's t-test was used to analyze differences between EAP and control groups. Prostates from p25 99-118-immunized mice demonstrated elevated gene expression levels of TNF-α, IL-17A, IFN-γ, and IL-1ß, not observed in control mice. Compared with controls, p25 99-118-immunized mice had significantly higher micturition frequency and decreased urine output per void, and they demonstrated elevated pelvic pain response. p25 99-118 immunization of male SWXJ mice induced prostate-specific autoimmunity characterized by prostate-confined inflammation, increased micturition frequency, and pelvic pain. This autoimmune prostatitis model provides a useful tool for exploring the pathophysiology and new treatments.

Protective role of macrophage-derived ceruloplasmin in inflammatory bowel disease.

OBJECTIVE: Intestinal microflora and inflammatory cell infiltrates play critical roles in the pathogenesis of acute colitis. Ceruloplasmin is an acute-phase plasma protein produced by hepatocytes and activated macrophages, and has ferroxidase with bactericidal activities. The goal is to understand the role of ceruloplasmin in colitis progression in a genetically modified murine model. DESIGN: Experimental colitis was induced in ceruloplasmin null (Cp(-/-)) and wild-type (WT) mice by dextran sulphate sodium administration. The role of ceruloplasmin was further evaluated by transplantation of WT macrophages into Cp(-/-) mice. RESULTS: Cp(-/-) mice rapidly lost weight and were moribund by day 14, while WT mice survived at least 30 days. Colon culture supernatants from Cp(-/-) mice exhibited elevated levels of TNFα, KC and MCP-1, indicative of increased inflammation and neutrophil and macrophage infiltration. Elevated leucocytes and severe histopathology were observed in Cp(-/-) mice. Elevated protein carbonyl content was detected in colons from Cp(-/-) mice suggesting ceruloplasmin antioxidant activity might contribute to its protective function. Unexpectedly, intraperitoneal administration of human ceruloplasmin into Cp(-/-) mice did not afford protection. Bone marrow transplantation from WT mice or injection of isolated peripheral blood monocytes markedly reduced severity of colitis and morbidity in Cp(-/-) mice. CONCLUSION: Macrophage-derived ceruloplasmin contributes importantly to protection against inflammation and tissue injury in acute and chronic experimental colitis. The findings suggest that defects in ceruloplasmin expression or processing may influence the onset or progression of inflammatory bowel disease in patients.