Ghrelin expression and function in T cells. (A) The purified human T cells were stained with incubated with control IgG and did not show nonspecific binding, whereas acylated ghrelin was found to be expressed in T cells. Nuclei are labeled with DAPI and acyl ghrelin was visualized by anti–guinea pig acyl ghrelin antibody conjugated to Alexa Fluor-594. Greater than 50% of the cells are labeled for acyl ghrelin (red). Given that these images are acquired using an epifluorescent microscope, certain dim acyl ghrelin^{lo cells} appear negative in the merge with DAPI. Thus, in the merge image distribution may appear less that 50%. The FACS analysis of peripheral blood mononuclear cells (PBMCs) for acylated ghrelin (B) allows the laser to pick MFIs of varying ghrelin expression (including weakly stained cells) on T cells. Images were acquired by Spot Advanced software on a Zeiss Axiovert S100 Microscope under 100× objective (Carl Zeiss, Thornwood, NY). (B) The human PBMCs were double labeled with anti-CD3 antibody conjugated to phycoerythrin and stained with acyl ghrelin conjugated to Alexa Fluor-488 and analyzed on FACS Calibur (Becton Dickinson, San Jose, CA). A representative plot from duplicate runs of 3 healthy donors is shown. Control antibody staining was not significant in any donor tested. Here, 46% of the PBMCs labeled positive for CD3 and 34% of the PBMCs expressed CD3⁺ and acylated ghrelin indicating that greater than 70% of the T cells express acylated ghrelin. Approximately 18% of the CD3⁻ ghrelin-positive cells most likely represents B and natural killer (NK) cells and monocytes. (C) Primary human T cells were stimulated by plate-bound anti-CD3 and -CD28, and lipid raft fractions were analyzed for acyl ghrelin expression by dot blot analysis. (D) GM1⁺ lipid rafts in activated T cells were visualized by cholera toxin conjugated to Alexa Fluor-594; the activated human T cells display polarized expression of acyl ghrelin and colocalized with lipid raft. (E) The lipid raft and cytoplasmic fractions were pooled and equalized for protein, and ghrelin was analyzed using ELISA. (F) Ghrelin siRNA causes significant reduction in ghrelin production from primary human T cells. (G) Ghrelin knockdown in activated T cells increases proinflammatory cytokine secretion. We failed to observe any influence of the siRNA on T-cell proliferation or cell viability and thus we believe these variables have no influence on the cytokine inhibition observed (data not shown). (H) Total protein lysates from control and ghrelin siRNA–transfected cells were analyzed for phosphorylation of IkB. Control siRNA–transfected cells were treated with 100 ng/mL ghrelin for 5 minutes.