We believe this to be the first report to demonstrate that ghrelin, via functional cell surface GHS-R, exerts both specific and selective inhibitory effects on the expression and production of the inflammatory cytokines IL-1β, IL-6, and TNF-α by human PBMCs and T cells. GHS-Rs on primary and cultured human T cells, similar to other classical GPCRs, elicit a potent intracellular calcium release upon ligation with their natural ligand, ghrelin, and are preferentially associated with GM1+
lipid rafts upon cellular activation. We also observed that, consistent with expression of functional GHS-R ghrelin on T cells, ghrelin actively induces actin polymerization within human T cells. Similar to treatment with chemokines (SDF-1α), ghrelin treatment led to the cellular polarization of leukocytes and actin distribution changes from a linear cortical pattern in resting lymphocytes to more concentrated patterns at the leading edge and contact zones in polarized and activated T cells (19
). These GPCR-like redistribution patterns also support a potential role for GHS-R in immune cell signaling and trafficking.
There is increasing evidence that the immune system — in particular the production of inflammatory cytokines by leukocytes — may play an important role in the development of anorexia-cachexia syndrome (11
). The cytokines considered to be the most relevant to inflammatory anorexia include IL-1β, IL-6, and TNF-α. Peripherally administered ghrelin has been shown to block IL-1β–induced anorexia (29
) and produces positive energy balance by promoting food intake and decreasing energy expenditure. Our current data demonstrates an inhibitory effect of ghrelin on proinflammatory cytokine expression, supporting a possible regulatory role for ghrelin and GHS-R in controlling cytokine-induced anorexia. Moreover, the combination of IL-1β and leptin has also been shown to inhibit ghrelin expression in stomach (29
), and stomach ghrelin expression is increased in leptin-deficient mice. Leptin and ghrelin are considered to exert mutually antagonistic effects on the food intake at the hypothalamic level (4
). Leptin, a member of gp130 family of cytokines, induces a strong Th1 response (25
) and is regarded as a proinflammatory inducer (22
). Leptin’s actions on food intake are controlled, in part, by an increase in the level of IL-1β in the hypothalamus (32
). Similarly, anorectic effects of IL-1 are mediated via increasing leptin levels (33
). However, the relationship between leptin and ghrelin at the level of immune cells is completely unknown.
We demonstrate here that leptin can directly induce the mRNA expression and secretion of IL-1β, IL-6, and TNF-α by human T cells and PBMCs. Leptin and several other gp130 ligands including LIF, CNTF, and IL-6 all appear to exert similar effects on host metabolism (34
). Moreover, IL-6–deficient mice, in a fashion similar to leptin-deficient mice, develop obesity (36
). While leptin has been shown to be associated with cachexia, leptin levels are not elevated in many cancer-associated wasting conditions (37
), most likely due to a systemic decline in adipose tissue. However, cachexia seen in chronic heart failure patients is associated with hyperleptinemia (38
). In contrast, ghrelin attenuates cachexia associated with chronic heart failure in rats (39
), and the GHS-R analogue, GHRP-2, counteracts protein hypercatabolism, skeletal muscle proteolysis, and osteoporosis in critically ill patients with wasting condition (40
). It has recently been reported that an increase in the level of circulating leptin within a murine MS model regulates inflammatory anorexia and disease susceptibility (41
). Moreover, fasting-induced suppression of leptin levels dramatically attenuates the onset of experimental autoimmune encephalomyelitis (EAE) in this model (41
). Given that fasting is associated with a decrease in serum leptin and a strong increase in circulating ghrelin levels (5
), it seems feasible that the observed anti-inflammatory effects of fasting in this murine MS model may also be mediated, in part, by ghrelin. Furthermore, it has recently been demonstrated that NPY attenuates Th1-mediated induction of EAE (42
). Given that fasting and ghrelin induce NPY, it seems likely that the orexigenic axis might play a critical role in regulating endogenous inflammatory responses. Ghrelin has now been recognized to have pleiotropic functions in a variety of organ systems; however, studies by Smith and colleagues (43
) have demonstrated no physiological abnormalities in a ghrelin knockout mouse, suggesting involvement of some possible compensatory mechanisms regulated by other orexigens.
Because regulation of hunger is most critical for the survival of species, a complex circuitry of compensatory and overlapping mechanisms has evolved to protect the host against deficiency in one or more of these regulators. Similar genetic approaches to study the other potent orexigens such as NPY and agouti-related peptide have also failed to yield a definite phenotype (44
). Therefore, additional controlled studies in orexigens or their receptor knockout mouse models in response to stress, inflammation, or pathogenic challenge might shed more light and reveal additional unique and overlapping functions of these orexigens.
To date, ghrelin has only been reported to be produced by endocrine-like cells in the stomach and subsequently released into the peripheral circulation. Through a number of analytical techniques, we demonstrate here that ghrelin is endogenously produced and secreted by both T cells and PBMCs in a fashion similar to many immune-derived cytokines. The majority of T cells examined from human donors were found to constitutively express low levels of endogenous ghrelin, which is significantly increased upon cellular activation. This high percentage of ghrelin-positive cells may also be due to the fact that our anti-ghrelin antibody recognized both the mature as well as the 117-amino-acid preproghrelin forms. However, the preproghrelin antibody does not bind the mature peptide, and subcellular localization revealed tight colocalization of preproghrelin in the Golgi apparatus. Activated T cells express and secrete the ghrelin protein, which strongly suggests that prepro peptide must be actively cleaved in T cells to yield the active ghrelin peptide. Similar to several cytokines (e.g., TGF-β) and hormones (e.g., thyroid stimulating hormone), these precursor proteins are synthesized and subsequently stored for immediate cleavage and use when needed. Furthermore, we also demonstrate the expression and secretion of the mature form of ghrelin from T cells after activation via TCR ligation. Gastrectomy results in only a 35–50% decline in circulating ghrelin, and ghrelin levels increase to two thirds of pre-gastrectomy levels in human subjects, which suggests that other tissues compensate for maintaining the peripheral ghrelin levels (27
). Secretion of ghrelin from T cells suggests that immune cell–derived ghrelin might make up part of residual concentration of circulating ghrelin. In addition, ghrelin is also regarded as the only known hormone where the hydroxyl group of the third serine residue is acylated by n
-octanoic acid, and this acylation is critical for some of the biological activities of this polypeptide (1
). N-terminal acylated peptides are known to preferentially aggregate in cholesterol rich microdomains (45
), and, interestingly, we observed that ghrelin immunoreactivity in activated T cells is highly colocalized within cholesterol-rich GM1+
domains. These results suggest that ghrelin may be selectively targeted to the plasma membrane to facilitate interaction with its own transmembrane receptor to optimally mediate receptor-ligand interactions. Such a pathway would have strong implications regarding the role of ghrelin in the control of immune responses. In addition, it seems likely that localized production of ghrelin may play a critical role in the immediate control of ongoing and leptin-mediated responses within the local microenvironment.
LPS-induced endotoxemia in mice is a well-recognized model for inducing septic shock and is also associated with anorexia due to excessive production of proinflammatory mediators. In spite of a large body of data, the causes of systemic inflammatory response syndrome (SIRS) remain unknown, and various therapeutic approaches have yielded minimal beneficial results (30
). LPS directly acts on mononuclear cells, but the resultant endotoxemia also affects a wide variety of cells and systems and is associated with a refractory catabolic state. Because ghrelin receptors are ubiquitously expressed in monocytes (Figure ), B cells, and dendritic cells (data not shown) and that ghrelin inhibits inflammatory cytokines in human mononuclear cells as well as monocytes, we utilized an established murine model of LPS-induced endotoxemia (46
). We demonstrate that ghrelin infusions in LPS-challenged mice lead to a significant inhibition of proinflammatory cytokines IL-1α and IL-β, IL-6, and TNF-α in circulation as well as in liver, spleen, lungs, and mesenteric lymph nodes. In addition, LPS-induced endotoxemia results in inhibition of ghrelin secretion (47
), and ghrelin infusion increases body weight in septic animals (48
). Considering the data presented herein, it seems plausible that inhibition of ghrelin secretion after LPS challenge might exacerbate the ongoing inflammatory insult and promote development of a catabolic state. Furthermore, we demonstrated that LPS-induced inflammatory anorexia is also significantly reduced in ghrelin-treated mice. These data strongly support possible inclusion of ghrelin and synthetic GHS as potential candidates in treatment of SIRS. Ghrelin might also have a regulatory role in chronic conditions such as Helicobacter pylori
infection, where persisting gastric inflammation is associated with lower ghrelin levels (49
) and correction of infection leads to up regulation of ghrelin secretion.
Our current studies suggest that ghrelin functions as a vital counterregulatory signal in the immune system, controlling not only activation-induced cytokine expression but also leptin-induced expression of these same inflammatory mediators. The reciprocal regulatory effects of these hormones on expression of IL-1β, IL-6, and TNF-α by immune cells may have widespread implications in the development of wasting diseases, aging, and frailty. Proposed interventions to lower ghrelin levels or to block GHS-R for treatment of obesity may result in a potentiation of ongoing inflammatory insults or lead to immune dysregulation. On the contrary, the novel anti-inflammatory actions of ghrelin within the immune system may have potential benefits in management of anorexia-cachexia syndrome associated with a wide range of inflammatory conditions and cancer.