GnRH analogue remarkably down-regulates inflammatory proteins in peritoneal fluid proteome of women with endometriosis.

OBJECTIVE: To determine the changes in the peritoneal fluid proteome of women with endometriosis determined by the administration of gonadotropin-releasing hormone analogues (GnRH-a). STUDY DESIGN: Peritoneal fluid samples were collected during laparoscopy from patients under GnRH-a and from women who did not receive any type of hormonal treatment in the 6 months before surgery. Samples were subjected to 2-D gel electrophoresis and compared by computerized analysis. Protein spots differentially expressed between the study groups were identified by liquid chromatography tandem mass spectrometry. RESULTS: More than 470 protein spots were analyzed. Several proteins with significant alterations were found. The down-regulated molecules were isoforms of alpha 2-HS glycoprotein, alpha 1-antitrypsin, S100-A8, haptoglobin alpha chain and vitamin D-binding protein. No protein spot had significantly higher expression in peritoneal fluid of women under GnRH-a than in untreated patients. CONCLUSION: Several inflammatory molecules present in peritoneal fluid are down-regulated during treatment with GnRH-a; administration of this drug reduces the inflammation in the peritoneal cavity.

Selective inhibition of the C5a chemotactic cofactor function of the vitamin D binding protein by 1,25(OH)2 vitamin D3.

The Vitamin D binding protein (DBP) is a multifunctional plasma protein that can significantly enhance the chemotactic response to complement fragment C5a. The chemotactic cofactor function of DBP requires cell surface binding in order to mediate this process. The goal of this study was to investigate the effect of ligating DBP with its two primary physiological ligands, Vitamin D and G-actin, on both binding to neutrophils and the ability to enhance chemotaxis to C5a. There was no difference in neutrophil binding between of the holo (bound) forms versus the apo (unbound) form of radioiodinated DBP, indicating that the cell binding region of DBP is likely distinct from the Vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D3, and G-actin plus 25(OH)D3 bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH)2D3) completely eliminated the chemotactic cofactor function of DBP. Dose-response curves demonstrated that as little as 1pM 1,25(OH)2D3 significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH)2D3 needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B4 was not altered by 1,25(OH)2D3, indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase (AP) with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH)2D3. These results indicate that the cell binding and co-chemotactic functions of DBP are not altered when the protein binds G-actin and/or Vitamin D. Furthermore, the co-chemotactic signal from DBP can be eliminated or counteracted by 1,25(OH)2D3.

Elastase controls the binding of the vitamin D-binding protein (Gc-globulin) to neutrophils: a potential role in the regulation of C5a co-chemotactic activity.

The vitamin D-binding protein (DBP) binds to the plasma membranes of numerous cell types and mediates a diverse array of cellular functions. DBP bound to the surface of leukocytes serves as a co-chemotactic factor for C5a, significantly enhancing the chemotactic activity of pM concentrations of C5a. This study investigated the regulation of DBP binding to neutrophils as a possible key step in the process of chemotaxis enhancement to C5a. Using radioiodinated DBP as a probe, neutrophils released 70% of previously bound DBP into the extracellular media during a 60-min incubation at 37 degrees C. This was suppressed by serine protease inhibitors (PMSF, Pefabloc SC), but not by metallo- or thiol-protease inhibitors. DBP shed from neutrophils had no detectable alteration in its m.w., suggesting that a serine protease probably cleaves the DBP binding site, releasing DBP in an unaltered form. Cells treated with PMSF accumulate DBP vs time with over 90% of the protein localized to the plasma membrane. Purified neutrophil plasma membranes were used to screen a panel of protease inhibitors for their ability to suppress shedding of the DBP binding site. Only inhibitors to neutrophil elastase prevented the loss of membrane DBP-binding capacity. Moreover, treatment of intact neutrophils with elastase inhibitors prevented the generation of C5a co-chemotactic activity from DBP. These results indicate that steady state binding of DBP is essential for co-chemotactic activity, and further suggest that neutrophil elastase may play a critical role in the C5a co-chemotactic mechanism.