Leptin, the satiety hormone, appears to act as a link between nutritional status and immune function. It has been shown to elicit a number of immunoregulatory effects, including the promotion of T cell proliferative responses, and the induction of proinflammatory cytokines. Leptin deficiency is associated with an increased susceptibility to infection. As polymorphonuclear neutrophils (PMN) play a major role in innate immunity and host defense against infection, this study evaluated the influence of leptin on PMN activation. The presence of leptin receptor in human PMN was determined both at mRNA and protein levels, and the effect of leptin on PMN activation, as assessed by CD11b expression, was evaluated using flow cytometry. In contrast to monocytes, which express both the short and long forms of the leptin receptor (Ob-Ra and Ob-Rb, respectively), PMN expressed only Ob-Ra. Leptin up-regulated the expression of CD11b, an early marker of PMN activation, on PMN in whole blood, yet it had no effect on purified PMN, even those treated by submaximal doses of TNF-α or PMA. The kinetics of leptin-induced activation in whole blood were consistent with an indirect effect mediated by monocytes, and 71% of the leptin-stimulatory effect on PMN was blocked by a TNF-α inhibitor. Leptin-mediated induction of CD11b expression was observed when purified PMN were coincubated with purified monocytes. In conclusion, although leptin activates PMN, it does so indirectly via TNF-α release from monocytes. These findings provide an additional link among the obesity-derived hormone leptin, innate immune function, and infectious disease.
Leptin is a multifunctional peptide hormone primarily derived from adipocytes that plays an important role in the regulation of food intake, energy expenditure, and the control of body weight (1). Leptin regulates appetite and energy expenditure at the level of the hypothalamus (2), and a genetic deficiency in leptin or its receptor results in extreme obesity (3, 4). The administration of recombinant human leptin has major and sustained beneficial effects on the obesity and neuroendocrine/metabolic dysfunction associated with congenital human leptin deficiency (5).
Leptin and its receptor share structural similarities to the cytokines IL-2, IL-6, and IL-15 and the class I cytokine receptor family on hemopoietic cells (6). The observations that leptin deficiency may in part be responsible for the immune impairment that accompanies malnutrition (7, 8, 9), and is associated with an increased frequency of infection (10, 11) have stimulated interest into the effects of leptin on immunity (6, 12).
PBMC and CD4+ T cells express mRNA for the leptin receptor (7). We and others have previously shown that leptin stimulates IL-6, TNF-α, and IL-1 receptor antagonist release from human monocytes (13, 14, 15), and restores the normal function of murine monocytes from leptin-deficient mice (16). The possibility that leptin contamination by endotoxin is responsible for this effect has previously been excluded (14). Leptin also increases Th1 (IFN-γ and IL-2) and suppresses Th2 (IL-4) cytokine production from activated human lymphocytes (7). To date, the majority of studies have focused on the effects of leptin on adaptive immunity, particularly T lymphocyte function. Polymorphonuclear neutrophils (PMN)3 play a major role in innate immunity and host defense against infection, and an essential component of their activation is expression of CD11b on their cell surface. CD11b (Mac-1, complement receptor 3) is an α subunit of the CD11/CD18 heterodimeric complex, which is one of a subfamily of four cell surface integrin receptors sharing a common β-chain (β2 or CD18). CD11b is involved in neutrophil localization at inflammatory sites and phagocytosis of iC3b- or IgG-coated particles, and its expression is rapidly up-regulated on neutrophil activation (17). CD11b is therefore a critical complex for normal host defense (18). Leptin restores the expression of CD11b on neutrophils from leptin-deficient mice (19). However, the direct effect of leptin on PMN has not been examined. Given the association of leptin deficiency with an increased susceptibility to infection (20, 21, 22), and that PMN play a major role in innate immunity and host defense against infection, this study evaluated the expression of leptin receptors on peripheral blood PMN and the influence of leptin on PMN expression of CD11b.
Materials and Methods
All materials were purchased from Sigma-Aldrich (Poole, U.K.), unless otherwise stated.
Peripheral blood (20 ml) was collected into sterile tubes containing EDTA or heparin from healthy adult male volunteers aged 23–47 years. Volunteers gave informed written consent, and the studies were approved by the local ethics committee. All experiments were performed in triplicate.
PMN were rapidly isolated by density-gradient centrifugation. Whole blood was layered over a double density gradient of equal volumes of Histopaque-1077 and Histopaque-1119 and centrifuged (700 × g, 30 min, 20°C). The PMN phase (1.077 < density < 1.119 g/ml) was harvested and cells were washed once with PBS. Contaminating erythrocytes were lysed by 30-s hypotonic lysis with 0.2% w/v NaCl. Samples were returned to physiological osmolarity by the addition of 1.6% w/v NaCl. Samples were centrifuged at 250 × g for 5 min, and isolated PMN were washed with PBS, counted, and resuspended in PBS containing: 1 mM of calcium, 0.5 mM of magnesium, 20 mM of glucose, and 0.5% w/v BSA to a final count of 106 cells/ml. The purity of PMN was greater than 95%, as determined by cytospin analysis.
For the preparation of monocytes, the mononuclear cell layer (1.024 < density < 1.077 g/ml) was aspirated, and the cells were washed twice with PBS, after which they were resuspended in RPMI 1640 and incubated in either a 10-cm culture dish or 6-well plate at 37°C for 1 h. Nonadherent cells were removed by extensive washing with PBS, and adherent monocytes were either treated with TRIzol for RNA extraction or were mixed with isolated neutrophils at the same ratio found in whole blood. The purity of isolated monocytes was greater than 96%, as determined by flow cytometric analysis.
Flow cytometric analysis of leptin receptor on isolated PMN
Isolated PMN were resuspended in PBS containing 0.1% BSA (wash buffer) at a density of 1 × 107 cells/ml. A total of 106 cells (100 μl) was transferred to 12 × 75-mm polycarbonate tubes (BD Biosciences, Oxford, U.K.) and incubated with 2 μg of an anti-leptin receptor Ab (9F8) (14
RT-PCR analysis of leptin receptor gene expression
Leptin receptor gene expression on isolated PMN and peripheral blood monocytes was assessed using RT-PCR, essentially as described previously (14). Total RNA was extracted from either 107 PMN or seeded monocytes using TRIzol reagent (Life Technologies, Paisley, U.K.), and 1 μg was reverse transcribed. mRNA was amplified using PCR primers specific for the Ob-Ra (short) and Ob-Rb (long) leptin receptor isoforms (23).
Effect of leptin on CD11b expression
24). After incubation, tubes were placed on ice, and 2 μl of FITC-conjugated murine anti-human CD11b mAb or a FITC-conjugated isotype-matched, nonreactive negative control mAb (Serotec) was added. Samples were incubated on ice for 30 min before washing twice with PBS (250 × g, 5 min). For whole blood samples, erythrocytes were lysed using Erythrolyse lysis buffer (Serotec). Samples were analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences) and CellQuest data acquisition and analysis software. PMN exhibit characteristic light scatter properties that can be identified on a forward vs side light scatter plot. The PMN population was located using these parameters, and a live analysis gate was set around this population. Data were acquired from 10,000 cells (events), and the fluorescent intensity of Ag expression on the gated cells was determined and expressed as the mean fluorescence intensity (MFI).Escherichia coli, serotype 0111:B4), TNF-α (1 U = 10 pg; Roche, Mannheim, Germany), or TNF-α inhibitor at 37°C, 5% CO2 for the times indicated in Results. The TNF-α inhibitor (human TNF-α p55 receptor-IgG fusion protein) was a kind gift from Centocor (Malvern, PA) and was supplied by M. Feldmann (Imperial College, London, U.K.) (
The paired t test was used to compare the effects of leptin and the TNF-α inhibitor on the intensity of CD11b expression. For analysis of dose-response and time-course studies, repeated measures ANOVA was used with Bonferroni correction, as appropriate. Data are expressed as the mean ± SEM, and a p < 0.05 was considered to indicate statistically significant differences.
Leptin receptor expression on isolated PMN
Flow cytometric analyses of isolated PMN from three subjects confirmed that the leptin receptor is expressed at the protein level by isolated PMN (Fig. 1⇓). A total of 46 ± 2.8% of the isolated PMN showed specific binding to Ob-R Ab (MFI: 53 ± 11 for Ob-R Ab vs 12.4 ± 3.2 for isotype control Ab).
Leptin receptor isoform gene expression on peripheral blood PMN and monocytes
To determine the isoform of leptin receptor, Ob-R gene expression by PMN and monocytes was assessed using RT-PCR. PCR products for both short (Ob-Ra) and long (Ob-Rb) isoforms of leptin receptor were detectable in monocytes, although Ob-Ra was always more highly expressed and Ob-Rb expression was more variable. In contrast, only the gene for the short isoform of the leptin receptor (Ob-Ra) was expressed in PMN (Fig. 2⇓).
Effect of leptin on CD11b expression by human PMN in whole blood
A 90-min incubation with leptin increased the expression of CD11b by PMN in a dose-dependent manner (p < 0.001; Fig. 3⇓). TNF-α induced a progressive increase in the expression of CD11b by PMN, which was first apparent after 10 min; however, the induction of CD11b expression by leptin was delayed in that significant increases were only apparent after 90 min (Fig. 4⇓). As leptin has been shown to induce the release of TNF-α from peripheral blood monocytes (13), the difference in the kinetics of CD11b induction was consistent with a hypothesis that leptin indirectly activates PMN through the release of TNF-α from monocytes. To test this hypothesis, the effects of leptin on purified PMN and on whole blood PMN in the presence of a TNF-α inhibitor were evaluated. The TNF-α inhibitor completely blocked the effect of TNF-α on CD11b expression and inhibited leptin-induced up-regulation of CD11b expression by 71% (p < 0.002), suggesting that the effect of leptin on CD11b expression is primarily mediated by TNF-α (Fig. 5⇓).
The capacity of leptin to augment the neutrophil response to other stimuli was also determined. For these experiments, dose-response effects of PMA and TNF-α on CD11b expression of PMN were performed, and submaximal doses of TNF-α (0.5 ng/ml) and PMA (3.3 × 10−8 M) for activation of PMN in whole blood were defined (data not shown). Whole blood was incubated with leptin (250 ng/ml) for 1 h before the addition of submaximal doses of PMA or TNF-α. Prior incubation with leptin augmented the subsequent response of PMN to suboptimal doses of PMA (p < 0.05) and TNF-α (p < 0.02; Fig. 6⇓).
Effect of leptin on CD11b expression by human isolated peripheral blood PMN
The preceding data demonstrated that the leptin-induced CD11b appeared later than that induced by LPS and TNF-α (90 vs 10 min, respectively) and suggested that the influence of leptin on the expression of CD11b by PMN might be an indirect effect mediated by peripheral blood monocytes. To test this hypothesis, the effect of leptin on isolated PMN was determined. Leptin (1–2000 ng/ml) had no effect on the expression of CD11b by isolated PMN, whereas TNF-α (2 ng/ml) increased the intensity of CD11b expression 2- to 3-fold (data not shown). Increasing the incubation times (up to 180 min) had no effect (data not shown). To exclude the possibility that a subset of PMN expressing the leptin receptor responds, double-staining experiments were performed and the effect of leptin on PMN-expressing leptin receptor was examined. No effect of leptin on CD11b was observed (data not shown). Preincubating isolated PMN with leptin (250 ng/ml) for 1 h did not alter the subsequent response of PMN to submaximal doses of PMA and TNF-α (Fig. 7⇓).
Effect of leptin on isolated PMN in the presence of purified monocytes
Addition of isolated monocytes to isolated PMN alone did not significantly increase CD11b expression; however, addition of 250 ng/ml leptin to the mixed population of isolated PMN and isolated monocytes increased CD11b expression by 31 ± 5.1% (p < 0.001). Thus, addition of purified monocytes to isolated PMN restored the ability of leptin to increase the expression of CD11b (Fig. 8⇓).
Leptin levels are regulated by nutritional status and fall rapidly during starvation. Leptin deficiency is associated with impaired cell-mediated immunity, an increased incidence of infectious disease, and an associated increase in mortality (6, 10, 11). To date, studies into the influence of leptin on immunity have primarily focused on the adaptive immune response, especially that elicited by CD4+ T cells (6, 7, 12, 25). The administration of recombinant human leptin increases circulating T cell numbers and reverses the T cell hyporesponsiveness (proliferation and cytokine release) that is observed in children congenitally deficient in leptin (5). However, despite its clear influence on adaptive immunity, little is currently known about the effects of leptin on the innate immune system, particularly neutrophil function.
Previous studies have reported the presence of a leptin receptor on peripheral blood PMN (26), and we have confirmed this on isolated PMN. The proportion of PMN expressing the leptin receptor on isolated PMN was greater than that we have previously reported in whole blood (14), and this most likely results from the density-gradient centrifugation procedure that has been shown to change receptor expression on PMN (27). Gender, body mass index, and age are known to influence levels of soluble leptin receptor that may reflect cell surface receptor (28, 29), and thus, these factors might also affect leptin receptor expression on PMN. The use of an Ab directed against the extracellular constant domain of the leptin receptor has rendered it impossible to differentiate the short and long forms of the receptor. We have extended these findings, and report in this work that whereas monocytes express mRNA for both the Ob-Ra and Ob-Rb isoforms of the leptin receptor, PMN express only mRNA for the Ob-Ra isoform. The leptin receptor (Ob-R) is the product of a single gene, but alternative mRNA splicing results in various isoforms (Ob-Ra to Ob-Rf) having a common extracellular domain. Ob-Rb, which is expressed in lymphoid tissues, has a long cytoplasmic tail (long form) and is considered to be of prime importance for leptin signaling, having full signaling capability via activation of the mitogen-activated protein (MAP) kinase and Janus kinase/STAT signaling pathways (30, 31). Ob-Ra is the predominant truncated form of the receptor that lacks most of the cytoplasmic domain of the receptor. The functional capacity of Ob-Ra is not yet fully established. It may have signaling capability through MAP kinase, but not through STAT3 (30), and generates soluble leptin receptor (32).
We have demonstrated that leptin can activate human PMN, but that this is an indirect action mediated to a large extent by TNF-α. The inability of the TNF-α inhibitor to completely inhibit the effects of leptin on PMN activation most likely results from other proinflammatory cytokines such as IL-6, which are released by monocytes in response to leptin (13). The observation that adding purified monocytes to purified PMN restores the activation of PMN by leptin confirms that the likely source of TNF-α is monocytes. The finding that leptin cannot activate purified PMN contrasts with a previous study that demonstrated that leptin was able to induce the production of reactive oxygen species from purified PMN (26). Differential signaling capacities of the Ob-Ra and Ob-Rb receptors and the different outcome measures of PMN activation status might explain the discrepancies between our own and previous data (26). The MAP kinase pathway is important for cytoskeletal processes, such as those involved in the transfer of CD11b from cytoplasmic granules to the plasma membrane, and for the generation of reactive oxygen species; these responses are differentially sensitive to other pathways such as the phosphatidylinositol-3-(OH) kinase pathway (33). In addition, inhibition studies have revealed that protein-phosphorylating kinase and lipid-phosphorylating kinase are of significance for adhesion and respiratory-burst function, respectively (34, 35). Our results demonstrate that expression of CD11b on PMN is not mediated by activation of Ob-Ra on PMN.
Our repeated experiments were performed with a leptin dose of 250 ng/ml. This dose was chosen because our dose-response studies clearly indicated that this dose could activate PBMCs and PMN in whole blood. Similar, and in some cases higher, serum leptin levels are found in some nonphysiological conditions. For example, leptin levels up to 400 ng/ml have been reported in children with chronic renal failure (36) and also in individuals undergoing leptin treatment (37). Treatment of a patient with genetic leptin deficiency with low levels of leptin (0.028 mg/kg lean body mass) increased the serum leptin up to 107 ng/ml (38). In obese, but otherwise healthy subjects treated with leptin (1 mg/kg/day), serum leptin levels up to 736 ng/ml have been reported (37). Thus, the high leptin doses that we used in our study were similar to those found in some nonphysiological conditions, and it might be expected that levels of leptin will be higher in adipose tissue, the source of leptin, compared with peripheral venous blood.
This study has also shown that the purity of the PMN population is critical for the interpretation of data from studies such as these, as our findings suggest that any contamination of the PMN with monocytes would contribute to PMN activation. In conclusion, leptin can influence the activation state of human peripheral blood neutrophils; however, this effect is mediated via its capacity to induce TNF-α secretion by monocytes.
↵1 H.Z.-E. is a member of the academic staff of the Biology Department, University of Isfahan, Isfahan, Iran, and supported by a grant from the Special Trustees of Sheffield Teaching Hospitals. This study was also supported, in part, by Wellcome Trust Equipment Grant 043571 (to A.G.P. and A.P.W.).
↵2 Address correspondence and reprint requests to Prof. Richard J. M. Ross, Clinical Sciences Centre, Northern General Hospital, Sheffield, S5 7AU, U.K. E-mail address:
↵3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; MAP, mitogen-activated protein; MFI, mean fluorescence intensity.
- Received August 5, 2003.
- Accepted November 17, 2003.
- Copyright © 2004 by The American Association of Immunologists