Leptin Modulates Inflammatory Cytokine and Neuroendocrine Responses to Endotoxin in the Primate

Endocrinology, Oct 2003

Xiao, Ennian, Xia-Zhang, Linna, Vulliémoz, Nicolas R., Ferin, Michel, Wardlaw, Sharon L.

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Leptin Modulates Inflammatory Cytokine and Neuroendocrine Responses to Endotoxin in the Primate

Endocrinology Leptin Modulates Inflammatory Cytokine and Neuroendocrine Responses to Endotoxin in the Primate ENNIAN XIAO 0 1 LINNA XIA-ZHANG 0 1 NICOLAS R. VULLI E´MOZ 0 1 MICHEL FERIN 0 1 SHARON L. WARDLAW 0 1 0 Abbreviations: HPA , Hypothalamic-pituitary-adrenal; IL-1ra, IL-1 receptor antagonist; SOCS, suppressor-of-cytokine signaling 1 Departments of Medicine and Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons , New York, New York 10032 , USA Leptin, which plays a crucial role in regulating energy balance, can also modulate the inflammatory response. Although leptin-deficient rodents are more sensitive to the toxic effects of bacterial endotoxin, it is unknown if leptin can modulate inflammatory cytokine or neuroendocrine responses to inflammation in a primate model. We have therefore studied the effects of leptin on plasma cytokine and hypothalamic-pituitary-adrenal responses to endotoxin (5 g iv) in nine ovariectomized rhesus monkeys. Human leptin (50 g/h) or saline was infused iv for 16 h before and 4 h after endotoxin injection; mean plasma leptin increased from 3.6 1.0 ng/ml to 18 1.7 ng/ml (P < 0.001). Leptin infusion had no effect on baseline plasma cytokine and hormone levels before endotoxin injection. As expected, endotoxin stimulated TNF- , IL-6, IL-1 receptor antagonist (IL-1ra), ACTH, and cortisol in the salineinfused animals (P < 0.001). There was a significant attenua- - T HERE IS INCREASING evidence that the adipocytederived hormone leptin, which plays a crucial role in regulating energy balance, can also modulate the inflammatory response. Leptin is a member of the cytokine superfamily and its receptor, which is related to the class I cytokine receptors, is present in bone marrow and spleen and on peripheral monocytes and lymphocytes ( 1 ). Leptin deficiency is associated with impaired T cell immunity and increased sensitivity to monocyte-/macrophage-activating stimuli ( 1, 2 ). Rodents with genetic leptin deficiency are more sensitive to the lethal effects of bacterial endotoxin and TNF- ; similar increased sensitivity to endotoxin occurs during fasting when leptin levels are suppressed ( 3–5 ). In both cases, the increased susceptibility to endotoxic shock can be reversed by leptin administration. It is unknown whether physiological levels of leptin can modulate inflammatory cytokine responses to inflammation in a primate model. Leptin levels are, however, known to be increased in critically ill, septic patients and high levels of leptin are positively correlated with survival ( 6 – 8 ). We have therefore studied the effects of leptin infusion, compared with saline infusion, on plasma cytokine, TNF- , IL-6, IL-1 receptor antagonist (IL1ra), responses to endotoxin in female rhesus monkeys. Because proinflammatory cytokines are potent activators of the hypothalamic-pituitary-adrenal (HPA) (9), we have also examined the effects of leptin on this neuroendocrine response to endotoxin. tion of the IL-6 (P < 0.005) and cortisol (P < 0.001) responses (repeated measures ANOVA) to endotoxin in the leptin-infused animals. There was a significant reduction (by paired analysis) in the responses of the leptin compared with salinetreated animals: 47% for TNF- , 48% for IL-6, 30% for IL1ra, 42% for ACTH, and 22% for cortisol (P < 0.05). We conclude that an increase in circulating leptin, within the physiological range of our monkey colony, can blunt the inflammatory cytokine and hypothalamic-pituitary-adrenal responses to an inflammatory challenge. These results, coupled with our recent finding that endotoxin stimulates leptin release in the monkey, demonstrate that leptin can be both released in response to inflammatory cytokines and act to attenuate the responses to these cytokines. (Endocrinology 144: 4350 – 4353, 2003) Materials and Methods Animals Nine adult female rhesus monkeys (Macaca mulatta), weighing 5–9 kg, were used in these experiments. Monkeys were housed in individual cages in a temperature-controlled room (19 –22 C) with a 12-h light, 12-h dark photocycle and were fed 20 Purina Monkey Chow biscuits ( 6.8 g each) twice daily at 1000 h and 1500 h. This was supplemented with fresh fruit or vegetables daily. All animals were ovariectomized at least 2 months before the studies to eliminate fluctuations in estradiol levels, because estradiol has been shown to affect cytokine and neuroendocrine responses to endotoxin. All protocols were approved by the Columbia University Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health guide for the care and use of laboratory animals. Experimental protocol The night before each experiment, monkeys were briefly sedated with 5–7 mg/kg ketamine and catheters were placed in both femoral veins for leptin infusion and for blood collection, respectively. The animals were then seated in a plastic primate chair to which they had previously been adapted, which restrained them for the period of the experiment and endotoxin (lipopolysaccharide Escherichia coli 055:B5, Sigma, St. Louis, MO), 5 g, was injected iv the next morning. Animals were considered adapted to the chair if they sat quietly in the chair and if cortisol levels were less than 50 g/dl. Human leptin (50 g/h) (National Hormone & Peptide Program) or normal saline was infused iv for 16 h before and for 4 h after endotoxin injection. Each monkey was studied twice in random order with at least 3 wk between experiments. Monkeys were seated in the chair after eating their afternoon meal. The morning meal was withheld, but the animals received small amounts of fresh fruit in the chair during the experiment. Hormone and cytokine assays Leptin was assayed with a double antibody primate RIA kit that uses an antiserum to human leptin that cross-reacts fully with monkey leptin (Linco Research Inc., St. Charles, MO). ACTH was measured in unextracted plasma by immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). Serum cortisol was assayed in unextracted plasma by solid-phase RIA (ICN Biochemicals Inc., Costa Mesa, CA). IL-6 and IL-1ra were assayed by specific monoclonal sandwich immunoassays with human ELISA kits (R&D Systems, Minneapolis, MN), which we have validated for use in the rhesus monkey ( 10 ). TNF- was assayed with a rhesus monkey ELISA kit (Biosource International, Inc., Camarillo, CA). Data analysis The effects of endotoxin injection on hormone and cytokine responses in monkeys were analyzed by ANOVA with repeated measures and with Bonferroni-Dunn post hoc analysis. Areas under the hormone and cytokine response curves were calculated by trapezoid analysis, and the responses to endotoxin in the leptin and saline-treated animals were compared by paired t test. Analyses were performed with statistical software (Statview, Abacus Concepts Inc., Berkeley, CA). Results The mean plasma leptin level measured just before endotoxin injection was 3.6 1.0 ng/ml during the saline infusion and increased to 18 1.7 ng/ml during the leptin infusion (P 0.0001). Basal levels of IL-6 and TNF- were below the limits of assay detection during both saline and leptin infusions. Mean basal IL-1ra levels were 267 46 pg/ml after saline vs. 416 122 pg/ml after leptin (P 0.22). Baseline plasma ACTH and cortisol levels before endotoxin injection were also not significantly different after leptin compared with saline infusion. As expected, endotoxin stimulated TNF- , IL-6, IL-1ra, ACTH, and cortisol release in the salineinfused animals (P 0.001). There was a significant attenuation (repeated measures ANOVA) of the IL-6 (P 0.005) and cortisol (P 0.001) responses to endotoxin in the leptininfused animals (Figs. 1 and 2). When the mean areas under the respective cytokine and hormone response curves were calculated, there was a significant reduction (by paired analysis) in the responses of the leptin compared with salinetreated animals: 47% for TNF- (P 0.01), 48% for IL-6 (P 0.02), 30% for IL1ra (P 0.05), 42% for ACTH (P 0.05), and 22% for cortisol (P 0.05) (Figs. 1 and 2). Mean peak cytokine and hormone responses in the saline compared with the leptin-treated animals were: 446 97 vs. 248 111 pg/ml for TNF- (P 0.005), 2750 575 vs. 1370 344 pg/ml for IL-6 (P 0.02), 52.8 9.97 vs. 35.7 8.34 ng/ml for IL-1ra (P 0.15), 519 129 vs. 210 60 pg/ml for ACTH (P 0.05), 80 6.3 vs. 63 3.2 g/dl for cortisol (P 0.05). Discussion The results of this study show that an increase in circulating leptin, within the physiological range of our monkey colony, can blunt the inflammatory cytokine and HPA responses to an inflammatory challenge. These results, coupled with our recent finding that endotoxin stimulates leptin release in the primate ( 11 ), demonstrate that leptin, which is a member of the cytokine superfamily, can be both released in response to inflammatory cytokines and act to attenuate the responses to these cytokines. These studies are in agreement with reports in the rodent showing that leptin deficiency is associated with increased sensitivity to the toxic effects of endotoxin. Mice fasted for 48 h were more sensitive to the toxic effects of endotoxin and had a 5-fold greater increase in serum TNF- levels than the fed controls; these effects were blunted by leptin replacement during the 48-h period of fasting ( 5 ). Leptin-deficient ob/ob mice were also more sensitive to the toxic effects of endotoxin, an effect that was reversed by leptin administration starting 40 h before and FIG. 1. Mean ( SEM) plasma IL-6, TNF- , and IL-1ra responses to endotoxin [lipopolysaccharide (LPS)] injection in nine monkeys studied twice following either a saline infusion (solid circles) or a leptin infusion (open triangles). Leptin was infused for 16 h before LPS injection and for 4 h afterward. There was a significant attenuation of the cytokine responses in the leptintreated animals. Mean plasma leptin levels, measured before LPS injection at time 0, increased significantly by 5-fold during leptin infusion (lower right). *, P 0.0001. continuing for 32 h after endotoxin injection ( 3 ). In that study, no significant differences in endotoxin-induced levels of IL-1 or TNF- were detected in plasma at 2 or 6 h after endotoxin injection; however, a blunted induction of IL-10 and IL-1ra was observed in ob/ob mice. Leptin administration has also been reported to protect leptin-deficient mice from the toxic effects of TNF- but had no protective effect in normal mice ( 4 ). In our primate study, leptin infusion for 16 h before and 4 h after endotoxin injection appeared to cause a decrease in the release of the proinflammatory cytokines, TNF- and IL-6, because plasma levels were significantly attenuated. A significant attenuation of IL-1ra levels was also noted but the magnitude of this change was less than for TNF- and IL-6. Baseline IL-1ra levels tended to be higher in the leptin-treated monkeys, but this was not significant. Leptin has been reported to increase IL-1ra secretion from human monocytes in vitro ( 12 ). Such an effect could serve to blunt subsequent inflammatory responses. Our data do not support this as a potential mechanism for the leptin-induced effects in the current study. It is unknown if a longer period of leptin treatment in vivo would have caused a significant stimulation of plasma IL-1ra levels. Accumulating evidence suggests that leptin can modulate immune and inflammatory responses by multiple mechanisms. Leptin and its receptor are structurally and functionally related to the IL-6 cytokine family. Leptin activates signal transduction, like other members of this family, by stimulating the JAK-STAT pathway. Leptin also induces the expression of SOCS-3, a member of the suppressor-of-cytokine signaling (SOCS) proteins, which inhibits cytokine signal transduction ( 13 ). The long form of the leptin receptor is expressed by tissues and cells of the immune system, including bone marrow, spleen, monocytes/macrophages, CD4 and CD8 T lymphocytes, and CD34 cells ( 1, 14 ). Direct effects of leptin on lymphocyte and monocyte proliferation have been demonstrated in vitro ( 2, 14 ). Leptin administration has been shown to reverse the impaired T cell responses that are found during starvation and to modulate T lymphocyte activation toward the Th1 phenotype by stimulating the synthesis of IL-2 and interferon- ( 2 ). Direct effects of leptin on the activation of human monocytes have also been demonstrated in vitro with some evidence for a proinflammatory effect under certain conditions ( 15 ). The majority of the in vivo evidence, however, is consistent with an attenuation of the inflammatory cytokine response to endotoxin in both the rodent and primate. Physiological levels of leptin can thus modulate the inflammatory response to an endotoxin challenge by altering the production of proinflammatory and antiinflammatory cytokines and may also affect cytokine signaling by a variety of mechanisms, including activation of SOCS-3. Leptin is also known to stimulate the expression of POMC in the hypothalamus, which is processed to -MSH, a neuropeptide that has well-documented antiinflammatory effects ( 16 –18 ). -MSH can modulate the inflammatory response by acting on melanocortin receptors both within the brain and on immune cells in the periphery ( 19 ). At least one mechanism by which -MSH antagonizes the effects of the inflammatory cytokines is by blocking the activation of the nuclear transcription factor nuclear factor- B ( 18, 20 ). It is currently unknown if -MSH plays a role in mediating any of the antiinflammatory effects that have been reported with leptin. The attenuation of the ACTH and cortisol responses to endotoxin that were observed in the leptin-treated monkeys may be secondary to the attenuated release of proinflammatory cytokines that are known to mediate the stimulatory effects of endotoxin on the HPA axis ( 9 ). Leptin itself, however, can also affect the HPA axis. Normally, an inverse relationship exists between plasma leptin and corticosteroid levels in both the human and the rodent ( 21, 22 ). The HPA axis is activated during starvation when leptin levels are low and in animals with genetic leptin deficiency. In both cases, the activation of the HPA axis can be attenuated by leptin injection (21). Based on the present study, no conclusions can be drawn about leptin’s ability to directly modulate the HPA response to an inflammatory challenge given the attenuated proinflammatory cytokine response to endotoxin in the leptin treated monkeys. Leptin has also been shown to attenuate the ACTH and corticosterone responses to immobilization stress in the mouse ( 23 ). The design of the present study in the monkey actually incorporates a mild form of immobilization stress. Baseline cortisol levels in the chaired monkeys before endotoxin injection were elevated compared with levels obtained from monkeys unrestrained in their cages ( 11 ). Leptin treatment, however, did not affect baseline ACTH or cortisol levels before endotoxin injection. Plasma leptin levels are known to increase in the human, monkey, and rodent after endotoxin administration, but the time course is quite different in the rodent compared with the primate ( 11, 24 –26 ). In the rodent, endotoxin stimulates leptin gene expression in adipose tissue and leptin release into peripheral blood within 2– 6 h after injection ( 24, 25 ). In contrast, in the human and in the monkey, no effect is seen during the initial 7-h period after endotoxin injection ( 11, 27–29 ), but a stimulatory effect becomes evident by 16 –24 h after injection (11). The reasons for these species differences are currently unknown. It thus appears that endotoxin stimulates leptin release, which may in turn play a role in downregulating the inflammatory response. There is evidence that these mechanisms may play an important role during an episode of sepsis in the human. Leptin levels are known to be increased in critically ill septic patients and high levels of leptin are positively correlated with survival ( 6 – 8 ). In one study, plasma leptin levels were 3-fold higher in patients who survived the septic episode than in nonsurvivors. In another study, both leptin and IL-6 levels were independent predictors of death ( 7 ). A strong negative correlation between mean 24 h plasma IL-6 and plasma leptin was also noted ( 8 ). 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Xiao, Ennian, Xia-Zhang, Linna, Vulliémoz, Nicolas R., Ferin, Michel, Wardlaw, Sharon L.. Leptin Modulates Inflammatory Cytokine and Neuroendocrine Responses to Endotoxin in the Primate, Endocrinology, 2003, 4350-4353, DOI: 10.1210/en.2003-0532