Recombinant human resistin

NB4^PMN cells were used to study the effect of RETN on neutrophil function. The NB4^PMN cell line offers the advantage of standardization and reproducibility when compared with primary neutrophils [9 (link), 15 (link)]. Neutrophilic differentiation was achieved as previously described [9 (link)]. Briefly, NB4 cells were treated with 1 μM all-trans retinoic acid (ATRA) for 6 days in Roswell Park Memorial Institute medium (RPMI 1640) (VWR, Radnor, PA) with l-glutamine containing 10% fetal bovine serum (FBS) (Atlanta Biologicals; Flowery Branch, GA). Successful neutrophil differentiation was assessed by flow cytometry to monitor expression of CD11b, CD35, and CD71 surface markers (BD Biosciences, San Jose, CA). In some experiments, NB4^PMN cells were incubated for 1 h in human control serum (Atlanta Biologicals) containing 100 ng/mL of recombinant human resistin (R&D Systems, Minneapolis, MN) to replicate concentrations measured in patients with sepsis-induced AKI [15 (link)].

We used neutrophilic-differentiated NB4 cells (NB4^PMN) to study the effect of hyperresistinemia on neutrophil function in a standard and reproducible way. Neutrophilic differentiation was achieved by previously described methods [10 (link)]. Briefly, NB4 cells were treated with 1 μM all-trans retinoic acid (ATRA) for 6 days in culture media. Successful neutrophilic differentiation was assessed using CD11b, CD35, and CD71 surface markers (BD Biosciences) with flow cytometry. For some experiments, NB4^PMN were incubated for 1 h in human control serum (Atlanta Biologicals Flowery Branch, GA, USA) spiked with different concentrations of recombinant human resistin (R&D Systems, Minneapolis, MN, USA) at 20, 50, 100 ng/mL to replicate normal circulating vs. septic resistin concentrations.

THP-1 cells (monocytes) and NB4^PMN were incubated for 1 h in human serum in the presence and absence of 100 ng/mL recombinant human resistin (R&D Systems). Following the incubation period, cells were washed, counted, and mixed at different neutrophil-to-monocyte ratios for experimentation. The physiologic proportion of neutrophils to monocytes in human blood (which ranges from 3:1 to 7:1—we used 3:1 for convenience) was simulated, and this was then compared to a mixture of 1:1 neutrophils to monocytes. The total number of immune cells (monocytes and neutrophils) in each experimental sample was kept equal. S. aureus or P. aeruginosa were then added to the serum in the transwell plates and incubated at 37 °C for 1 h. The contents of each treatment were collected and subjected to the bacterial clearance assay.