Reduction of T cell-derived ghrelin enhances proinflammatory cytokine expression: implications for age-associated increases in inflammation - PubMed (original) (raw)

Reduction of T cell-derived ghrelin enhances proinflammatory cytokine expression: implications for age-associated increases in inflammation

Vishwa D Dixit et al. Blood. 2009.

Abstract

Ghrelin (Grln) is a peptide hormone that is predominantly produced in the stomach and stimulates appetite and induces growth hormone (GH) release. We have previously reported that ghrelin is also expressed in T cells and exerts prothymic and anti-inflammatory effects. However, the biologic relevance of T cell-derived ghrelin remains to be determined. Here, we report that acylated-bioactive ghrelin is expressed in human T cells and preferentially segregates within the lipid raft domains upon TCR ligation. The RNA interference (RNAi)-mediated down-regulation of ghrelin in primary human T cells activates IkB, and increases Th1 cytokines and IL-17 secretion. Ghrelin expression declines with increasing age in spleen and T cells and exogenous ghrelin administration in old mice reduces proinflammatory cytokines. These findings demonstrate that ghrelin functions in an autocrine and paracrine capacity to regulate proinflammatory cytokine expression in human and murine T cells and may contribute in regulating "inflamm-aging."

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Figures

Figure 1

Figure 1

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 ghrelinlo 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.

Figure 2

Figure 2

Age-related reduction in ghrelin expression and inflammation. Nonfractionated splenic T cells from (A) 2-month-old and (B) 24-month-old BALB/C mice were separated using negative selection and labeled with ghrelin; nuclei were counterstained with DAPI. (C) The ghrelin mRNA expression in spleen was studied by real-time PCR analysis. The threshold amplification values (Ct) of 4 to 6 mice per group were collapsed and normalized to GAPDH and are presented as fold change. (D) Ghrelin was infused at a concentration of 1.25 μg/hour (levels corresponding to fasting ghrelin peripheral concentration of young mice) for 2 weeks (n = 8), whereas control BALB/c mice were implanted with osmotic pumps containing the PBS vehicle alone. The circulating cytokines were measured using a bead based multiplex assay kit in serum of 20-month-old mice. In all cases, the differences between sham control– and ghrelin-treated mice were significantly different (P < .05). In contrast to the studies by Xia et al, we failed to observe any inhibition of T-cell proliferation or alterations in T-cell viability in response to ghrelin treatment that may account for the inhibitory effects of ghrelin on cell activation (data not shown).

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