Abstract:Transfection of either the C5a receptor or the formylpeptide receptor into undifferentiated U937

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°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.

ObjectiveTo prospectively assess the efficacy, general safety, and joint safety of fasinumab, an anti–nerve growth factor monoclonal antibody, in osteoarthritis (OA) hip and/or knee pain.MethodsPatients with moderate‐to‐severe OA pain (knee or hip) and history of inadequate response or intolerance to analgesics were randomized to receive fasinumab (at 1 mg, 3 mg, 6 mg, or 9 mg) or placebo every 4 weeks over 16 weeks and were followed up to week 36. Efficacy end points were the change from baseline to week 16 in the pain and physical function subscale scores of the Western Ontario and McMaster Universities OA Index (WOMAC), and patient global assessment (PGA) of OA. Joints were monitored at scheduled assessments (by plain film radiography and magnetic resonance imaging) during treatment and follow‐up, and if prompted, at the time of active joint symptoms.ResultsOf the 421 patients randomized, 342 completed the 36‐week study. All doses of fasinumab yielded statistically significant and clinically important reductions in pain compared to placebo (least squares mean difference in WOMAC pain subscale scores at week 16 ranging −0.78 to −1.40), without any clear dose dependence. Physical function and PGA scores improved in parallel. Treatment‐emergent adverse event rates were 17% with fasinumab and 10% with placebo, and 4% and 1% of patients, respectively, discontinued treatment. Arthropathies (25 in total, 7% of fasinumab‐treated patients and 1% of placebo‐treated patients) occurred in a dose‐dependent manner, with 2 occurring in patients receiving the lowest dose of fasinumab and 10 in patients receiving the highest dose. Most of the arthropathies (16 of 25) were discovered with scheduled radiographs and not based on symptoms. Destructive arthropathy (in 1 of 337 treated patients) occurred in 1 patient who was receiving 6 mg fasimumab.ConclusionFasinumab provided improvements in OA pain and function, even in those benefitting little from previous analgesics. The observed benefit‐to‐risk relationship favors further clinical development to explore the lowest doses of fasinumab in patients with knee or hip OA.

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 Gactin, 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 likely is distinct from the vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D 3 , and G-actin plus 25(OH) D 3 bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of vitamin D (1,25(OH) 2 D 3 ) completely eliminated the chemotactic cofactor function of DBP. Dose response curves demonstrated that as little as 1 pM 1,25(OH) 2 D 3 significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH) 2 D 3 needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B 4 was not altered by 1,25(OH) 2 D 3 indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH) 2 D 3 . 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) 2 D 3 .