Leptin regulates functional capacities of polymorphonuclear neutrophils

Recent studies report that leptin may be able to modulate some functions of cells involved in non-specific immune response. We recently found that a functional leptin receptor is present on polymorphonuclear neutrophils (PMNs) and may be able to influence their oxidative capacities. We demonstrate here for the first time that leptin is also able to stimulate chemotaxis of PMNs and exerts by itself a chemoattractive effect comparable to that of well-known formyl-methionyl-leucyl-phenylalanine, and a stimulating effect on intracellular hydrogen peroxide production, without modification of phagocytosis     

Dual vascular effects of leptin via endothelium: hypothesis and perspective

Secreted by adipocytes, leptin is a hormone which regulates appetite and metabolism. Leptin secretion is proportional to the fat mass, and thus leptin concentration is raised in most obese subjects. In recent years, more and more biological effects have been attributed to leptin; one of the most well-known effects is the effect of leptin on the vascular tone. Obesity is very often associated with hypertension, and it has been known that leptin affects the blood pressure by activating the sympathetic nervous system and causing endothelial cell (EC) dysfunction. However, there has been strong evidence that leptin is able to dilate blood vessels. Such vasodilation has been shown to be EC-dependent and EC-independent. Further, both nitric oxide-dependent and nitric oxide-independent mechanisms have been reported. In this mini-review, we summarize the heterogeneous mechanisms by which leptin causes relaxation of vascular smooth muscle. We also argue that while leptin may act as a direct dilator on the vasculature in healthy subjects, hyperleptinemia in obese subjects gradually dysregulates blood pressure control by deteriorating EC functions. How these dual effects of leptin on EC might be related to EC ionic channels is also discussed.     

Intracellular Salmonella typhimurium induce lysis of human polymorphonuclear leukocytes which is not associated with the Salmonella virulence plasmid

The interaction between Salmonella typhimurium and human polymorphonuclear leukocytes (PMNs) was analyzed in vitro. Three S. typhimurium strains, the wild-type strain OU5043, its isogenic virulence plasmid-cured strain OU5048, and LT2, which represented the types that exhibited three mouse virulence levels, respectively, were used in this study. There was no correlation between the recovery of intracellular S. typhimurium from PMNs and the presence or absence of the virulence plasmid, or the strains' mouse virulence level. When the oxygen-dependent response of PMNs upon phagocytosis of S. typhimurium was examined by checking the intracellular reduction of nitroblue tetrazolium (NBT), the fraction of PMNs that reduced NBT on phagocytosis of the three strains was around 80%, whereas it was 58% with Escherichia coli, 95% with phorbol 12-myristate 13-acetate and 15% with a negative control. Thus there were no significant differences among the three Salmonella strains in terms of their ability to induce the oxidative response in PMNs. Microscopic analysis of Salmonella-infected PMNs indicated that the intracellular Salmonella induced lysis of PMNs. Both OU5043 and OU5048 exhibited a significant intracellular cytotoxic effect on PMNs after 24 hr of infection and this effect was not associated with the presence or absence of the virulence plasmid. On the other hand, lysis of PMNs was related to the intracellular survival of Salmnonella, as ofloxacin, an antibiotic, appeared to be able to protect human PMNs from Salmonella-induced cytotoxicity when this agent was added into the medium to inactivate the intracellular organism. The ability to induce lysis of PMNs by either wild-type or plasmid-cured strains of S. typhimurium may play a crucial role in the pathogenesis of non-typhoid Salmonella. The contribution of pSTV to human salmonellosis is likely to be limited. Furthermore, early institution of antibiotics with a high intracellular activity against Salmonella, such as fluoroquinolones, may be useful to prevent the dissemination of Salmonella infection.     

Leptin and prolactin modulate the expression of SOCS-1 in association with interleukin-6 and tumor necrosis factor-alpha in mammary cells: a role in differentiated secretory epithelium

Leptin and its receptors have been shown to be expressed in several tissues, suggesting that this protein might be effective not only at the CNS level but also peripherally. We have previously reported that leptin and its long form receptor are expressed in the mouse mammary epithelial cell line HC11. In this study, we report a specific relationship among leptin, prolactin (PRL), interleukin-6 (IL-6), and tumor necrosis-alpha (TNF-alpha) in the modulation of the suppressor of cytokine signaling 1 (SOCS-1). Furthermore, we show that leptin and PRL are able to effectively enhance SOCS-1 gene expression in the HC11 cell line. Finally, high concentrations of leptin (100 nM) and/or PRL significantly (p<0.05) reduce the inhibitory effect of IL-6 (10 and 100 ng/ml) and TNF-alpha (10 and 100 ng/ml) on beta-casein gene expression in HC11 cells transfected with pbetacCAT, a chimeric rat-beta casein gene promoter-cloramphenicol acetyl transferase (CAT) gene construct. These results provide evidence that leptin may be an important mediator in regulating mammary gland growth and development and that this role may be related to the immune factors that are involved in inflammation.     

Transcytosis of gastric leptin through the rat duodenal mucosa

Leptin is secreted into the gastric juice by epithelial Chief cells and reaches the duodenum in a biologically intact active form. We assessed the possibility that this gastric leptin crosses the intestinal mucosa by transcytosis through enterocytes to reach blood circulation. Endogenous gastric leptin secretion was triggered by cholinergic stimulation. In another set of experiments, recombinant leptin was inserted in vivo into the duodenal lumen. Plasma levels of leptin were assessed by enzyme immunoassay and Western blot, and duodenal tissue was processed for immunocytochemistry. We first observed that leptin was found inside duodenal enterocytes from fed rats but not inside those from fasted ones. Stimulation of gastric secretion by a cholinergic agent led to rapid increases in plasma leptin levels (202 +/- 39%) except when the pylorus was clamped. Insertion of recombinant leptin into the duodenal lumen raised plasma leptin concentrations (558 +/- 34%) quite rapidly, whereas carrier solution without leptin had no effect. The use of FITC-tagged leptin reinforced these results. Light and electron microscopy revealed the cellular compartments involved in its transcytosis, namely, the enterocyte microvilli, the endocytotic vesicles, the Golgi complex, and the basolateral interdigitations. Leptin was also present in the lamina propria, in capillary endothelial cell plasmalemmal vesicles, and in capillary lumina. These results demonstrate that gastric exocrine leptin is internalized by duodenal enterocytes and delivered to the lamina propria and blood circulation. It may thus be able to play important paracrine and endocrine functions for the control of gastric emptying and nutrient absorption.     

Effect of endocellular cryoprotectant upon polymorphonuclear neutrophil function during storage at low temperature.

The effect of cryoprotective agents dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon the function of polymorphonuclear neutrophils (PMNs) during storage between 0 ° and 4 °C was investigated.Increasing concentration of each cryoprotectant caused an increasing inhibition of chemotaxis with complete inhibition at 16.7%. At this concentration most PMNs were still able to exclude trypan blue dye. Chemotaxis was not inhibited if PMNs were exposed to 4.2 or 8.3% concentrations of cryoprotectants for 1 hr, and washed subsequently. However, the recovery of chemotaxis was not observed at 16.7% after 1 hr exposure to cryoprotectants. Moreover, a considerable number of PMNs could not exclude the dye. This would indicate that cells become fragile with cryoprotectants at a high concentration and the PMNs are easily damaged by washing. With 20 hr exposure PMNs, the inhibitory effect on chemotaxis was removed by washing when a 4.2% agent was used, but using an 8.3% agent, chemotaxis was not restored but PMNs exposed to DMSO displayed almost the same chemotaxis as a control. On the other hand, the ability of PMNs to ingest bacteria was not so markedly inhibited as the chemotaxis. With 1 hr exposure to cryoprotectants, the ingestive ability was hardly affected within 8.3%. As for 20 hr exposure, the same ingestive ability as that of a control was observed in all cases using a 4.2% concentration. However, using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability.Judging from the above findings, DMSO would be suitable as a crypotrotective agent although the problem on toxicity remains to be resolved.     

Modulation of hypothalamic PTP1B in the TNF-α-induced insulin and leptin resistance

We have associated functional and molecular studies of insulin and leptin to investigate the effect of TNF-α on central insulin and leptin signaling in rats pre-treated with PTP1B-ASO. The icv infusion of TNF-α-induced an increase in PTP1B protein expression and activity, and attenuated insulin and leptin sensitivity and signaling in the hypothalamus. However, TNF-α was able to completely blunt the leptin and insulin effect in rats treated with PTP1B-ASO, suggesting that TNF-α does not require PTP1B to fully attenuate the leptin and insulin effects. In addition, our data also show that other mechanisms of insulin and leptin resistance are activated in the hypothalamus by TNF-α.     

Leptin enhances,via AP-1, expression of aromatase in the MCF-7 cell line

Leptin, a product of adipocytes, is involved in the regulation of body weight and results strongly correlated to body fat content. An excess of fat mass represents a breast cancer risk factor particularly in postmenopausal women, where estrogen production by adipose tissue through its own aromatase activity stimulates tumor progression. Leptin stimulates estrogen production through the increase of aromatase expression and activity in human luteinized granulosa cells and adipose stromal cells. In the present study, we have examined the possible link that exists between leptin and breast cancer, focusing our attention on the direct effect of leptin on aromatase activity, which may enhance estrogen production and induce tumor cell growth stimulation. We have shown that leptin enhances aromatase mRNA expression, aromatase content, and its enzymatic activity in MCF-7. Aromatase expression appears to be regulated by tissue-specific promoter. It has been demonstrated that promoters II and 1.3 are the major promoters that drive aromatase expression in MCF-7. Transient transfection experiments using vector containing human aromatase promoters II and 1.3 sequence fused with luciferase reporter gene demonstrated that leptin is able to activate this promoter. In the presence of either mitogen-activated protein kinase inhibitor PD 98059 or ERK2 dominant negative as well as in the presence of STAT3 dominant negative, the stimulatory effects of leptin on aromatase promoter, enzymatic activity, and aromatase protein content were inhibited. Functional studies of mutagenesis and electrophoretic mobility shift assay revealed that the AP-1 motif is important in determining the up-regulatory effects induced by leptin on aromatase expression in MCF-7.     

Human neutrophils as a source of nociceptin: a novel link between pain and inflammation

Nociceptin is a neuropeptide sharing sequence homology with classical opioid peptides but with a distinct pharmacological profile. Through activation of its receptor, NociR, nociceptin has been linked with several physiological functions in the central nervous system including memory, locomotion, and processing of pain signals. Recently, peripheral blood neutrophils (PMNs) were demonstrated to express a functional NociR, a result suggesting that additional functions of the neuropeptide remain to be elucidated. The present study investigated the possibility that PMNs may be a source of nociceptin and whether the neuropeptide elicits PMN early responses. We observed the presence of nociceptin in the synovial fluids from arthritic patients, an inflammatory milieu typically containing high numbers of PMNs. In addition, freshly isolated PMNs were found to express and secrete nociceptin following degranulation, identifying these inflammatory cells as a novel source of the neuropeptide. Incubation of PMNs with nociceptin elicited a specific pattern of cellular protein phosphorylation on tyrosine residues in a rapid and transient fashion. Moreover, nociceptin prevented intracellular accumulation of cAMP in fMLP-stimulated PMNs, an effect mimicked by the specific NociR synthetic agonist, Ro 64-6198. Taken together, these results show that nociceptin/NociR is present and functional in human neutrophils, and the results identify a novel dialogue pathway between neural and immune tissues.     

Short-term effects of leptin on skeletal muscle protein metabolism in the rat

We have examined the short-term effects of leptin on protein metabolism in the rat. Indeed, an intravenous leptin administration (100 microg/kg body weight), which resulted in no changes in circulating insulin in the time interval studied, induced a decrease in the incorporation of (14)C-leucine to (14)C-skeletal muscle protein. No changes were observed in relation to muscle protein degradation (either measured in vivo following isotope preloading or in vitro as tyrosine released into the incubation medium) and gene expression associated with the different proteolytic systems (cathepsin B, m-calpain and ubiquitin-proteasome system). The effects of leptin on amino acid incorporation into muscle protein do not seem to be direct because incubation of isolated EDL muscles in the presence of 10 microg/ml of leptin did not modify either the protein incorporation or the oxidation of (14)C-leucine. It may, therefore, be suggested that leptin is able to influence protein synthesis in skeletal muscle through the action of an unknown mediator.     

Surface protease of Treponema denticola hydrolyzes C3 and influences function of polymorphonuclear leukocytes

Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsin-like surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O(-)(2) by PMNs against T. denticola ATCC 35405 and dentilisin-deficient mutant K1. T. denticola ATCC 35405 induced production of O(-)(2), whereas dentilisin-deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O(-)(2), whereas K1 was relatively unaffected. We also used Immunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the alpha-chain of C3, producing iC3b. Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin activated PMNs via complement pathways and may play a role in establishing periodontal lesions.     

Reduction of obesity, as induced by leptin, reverses endothelial dysfunction in obese (Lep(ob)) mice.

Obesity is a major health care problem and is associated with significant cardiovascular morbidity. Leptin, a neuroendocrine hormone released by adipose tissue, is important in modulating obesity by signaling satiety and increasing metabolism. Moreover, leptin receptors are expressed on vascular endothelial cells (ECs) and mediate angiogenesis. We hypothesized that leptin may also play an important role in vasoregulation. We investigated vasoregulatory mechanisms in the leptin-deficient obese (ob/ob) mouse model and determined the influence of leptin replacement on endothelial-dependent vasorelaxant responses. The direct effect of leptin on EC nitric oxide (NO) production was also tested by using 4, 5-diaminofluorescein-2 diacetate staining and measurement of nitrate and nitrite concentrations. Vasoconstrictor responses to phenylephrine, norepinephrine, and U-46619 were markedly enhanced in aortic rings from ob/ob mice and were modulated by NO synthase inhibition. Vasorelaxant responses to ACh were markedly attenuated in mesenteric microvessels from ob/ob mice. Leptin replacement resulted in significant weight loss and reversal of the impaired endothelial-dependent vasorelaxant responses observed in ob/ob mice. Preincubation of ECs with leptin enhanced the release of NO production. Thus leptin-deficient ob/ob mice demonstrate marked abnormalities in vasoregulation, including impaired endothelial-dependent vasodilation, which is reversed by leptin replacement. These findings may be partially explained by the direct effect of leptin on endothelial NO production. These vascular abnormalities are similar to those observed in obese, diabetic, leptin-resistant humans. The ob/ob mouse may, therefore, be an excellent new model for the study of the cardiovascular effects of obesity.     

瘦素对GH3细胞分泌和凋亡的影响

本文旨在探讨瘦素(leptin)对垂体瘤GH3细胞的生长激素(growth hormone,GH)分泌的作用及可能机制。我们观察了leptin对GH3细胞生长激素的分泌、细胞的增殖和凋亡的影响,结果显示:leptin(1、10和100 nmol/L)对GH3细胞的基础GH分泌有抑制作用(P<0.05),并存在剂量依赖效应。用10 nmol/L的leptin作用30 min、1和3 h对GH分泌无明显影响,而作用1、2和3 d则可抑制GH分泌(P<0.05)。应用噻唑蓝(MTT)比色分析法和流式细胞仪研究leptin对GH3细胞增殖和凋亡的影响,我们发现leptin对GH3细胞的增殖有抑制作用,并存在剂量依赖效应;同时leptin可减低GH3细胞的S期细胞比例,而G1期的细胞比例明显增加,进入2相和4相的凋亡细胞比例增加。上述结果表明,leptin可抑制GH3 细胞的基础GH分泌,其作用可能是通过抑制GH3细胞的DNA合成,促进GH3细胞的凋亡,从而影响GH的分泌。     

A detailed study on the role of sex steroid milieu in determining plasma leptin concentrations in adult male and female rats.

We examined the effects of sex steroid milieu on plasma leptin levels in adult male and female rats. Since the body weight is known to influence leptin concentrations, the hormone was measured in rats with a very similar body weight (about 250 g) throughout this study. Plasma leptin levels were significantly higher in female than in male rats. Orchidectomy (ODX) caused a significant rise in leptin, and replacement of a physiological dose of testosterone (T) completely abolished the effect of ODX. Since the effect of tamoxifen (estrogen antagonist) coadministered with T on leptin levels in ODX rats was the same as that of T alone, it was suggested that the suppressive effect of T on leptin may be mediated by the androgenic potency of T, but not by its aromatized product, estradiol. In female rats, plasma leptin concentrations did not change significantly during the estrous cycle. Furthermore, leptin levels were not affected either by ovariectomy alone or by the administration after ovariectomy of physiological doses of estradiol, progesterone, or both. This is the first study to demonstrate in rats with a very similar body weight the existence of a clear sexual difference in plasma leptin levels, and also a suppressive action of T on the adipocyte hormone concentrations.     

Antifungal drugs affecting the chemotaxis of polymorphonuclear neutrophils

Investigations on the chemotaxis of polymorphonuclear neutrophils (PMNs) towards a cytoplasmic extract of Trichophyton rubrum in the presence and absence of antifungal drugs are described. It is shown that with griseofulvin, clotrimazole, econazole, ketoconazole, miconazole and natamycin at 1 mg l(-1), the number of PMNs migrating was significantly reduced. After 3 h of exposure to 10 mg l(-1), not one of the drugs tested had any discernable effect on the viability of the PMNs, or the complement. The anti-inflammatory activity of the drugs is discussed and whilst the chemosuppression of PMN chemotaxis may be an undesirable feature in a drug used to treat systemic mycoses, it is unlikely to have any adverse effect in the therapy of the dermatophytoses.     

Inhibition of Mg2+-dependent adhesion of polymorphonuclear leukocytes by serum hemopexin: differences in divalent-cation dependency of cell adhesion in the presence and absence of serum

Circulating and nonadherent polymorphonuclear leukocytes (PMNs) become activated to attain adhesive state in an integrin-dependent manner by various stimuli, and perform a variety of microbicidal functions such as phagocytosis and superoxide production. We found that, in the absence of serum, a physiological concentration of hemopexin has a strong inhibitory action on Mg(2+)-dependent adhesion of PMA-activated PMNs to fibrinogen- and serum-coated surfaces. Under these conditions, Ca(2+) had no effect on Mg(2+)-dependent adhesion or the adhesion-inhibitory activity of hemopexin. In contrast, PMNs suspended in serum containing sufficient amounts of hemopexin to inhibit adhesion showed marked adherence, which was inhibited by EGTA. Next, we prepared a small-molecule fraction of serum by ultrafiltration followed by boiling. PMA-activated PMNs was found to adhere in the presence of both hemopexin and the small-molecule fraction, and the adhesion was enhanced by exogenous Ca(2+). EGTA abolished the effect of the small molecule fraction. The data suggest that serum contains adhesion-promoting factor(s) which allows PMNs to adhere despite the presence of hemopexin and that Ca(2+) is required for adhesion-promoting activity. Further study of hemopexin may provide clues for new therapeutic strategies aimed at interfering with PMN adhesion to control inflammation and tissue injury.     

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity Together with Polymorphonuclear Leukocytes (PMNs) in Chronic Myeloid Leukemia Patients

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.