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J Am Soc Nephrol 15: 2366-2372, 2004
© 2004 American Society of Nephrology
doi: 10.1097/01.ASN.0000139321.98029.40
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J Am Soc Nephrol 15:2366-2372, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Leptin as a Uremic Toxin Interferes with Neutrophil Chemotaxis

Luciano Ottonello*, Paola Gnerre*, Maria Bertolotto*, Marina Mancini*, Patrizia Dapino*, Rodolfo Russo{dagger}, Giacomo Garibotto{dagger}, Tommaso Barreca* and Franco Dallegri*

*Division of Internal Medicine and {dagger}Division of Nephrology, Department of Internal Medicine and Medical Specialties, University of Genoa Medical School, Genoa, Italy

Correspondence to Dr. Luciano Ottonello, Dipartimento di Medicina Interna e Specialità Mediche, Viale Benedetto XV n. 6, I-16132 Genova, Italy. Phone: +39-010-3538686; Fax: +39-010-3538686; E-mail: otto{at}csita.unige.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Leptin is a pleiotropic molecule involved in energy homeostasis, hematopoiesis, inflammation, and immunity. Hypoleptinemia characterizing starvation has been strictly related to increased susceptibility to infection secondary to malnutrition. Nevertheless, ESRD is characterized by high susceptibility to bacterial infection despite hyperleptinemia. Defects in neutrophils play a crucial role in the infectious morbidity, and several uremic toxins that are capable of depressing neutrophil functions have been identified. Only a few and contrasting reports about leptin and neutrophils are available. This study provides evidence that leptin inhibits neutrophil migration in response to classical chemoattractants. Moreover, serum from patients with ESRD inhibits migration of normal neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine with a strict correlation between serum leptin levels and serum ability to suppress neutrophil locomotion. Finally, the serum inhibitory activity can be effectively prevented by immune depletion of leptin. The results also show, however, that leptin by itself is endowed with chemotactic activity toward neutrophils. The two activities—inhibition of the cell response to chemokines and stimulation of neutrophil migration—could be detected at similar concentrations. On the contrary, neutrophils exposed to leptin did not display detectable [Ca2+]i mobilization, oxidant production, or {beta}2-integrin upregulation. The results demonstrate that leptin is a pure chemoattractant devoid of secretagogue properties that are capable of inhibiting neutrophil chemotaxis to classical neutrophilic chemoattractants. Taking into account the crucial role of neutrophils in host defense, the leptin-mediated ability of ERSD serum to inhibit neutrophil chemotaxis appears as a potential mechanism that contributes to the establishment of infections in ERSD.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The obese (ob) gene (1) product, named leptin from the Greek term leptos, meaning thin, is a 16-kD nonglycosylated peptide hormone involved in the control of food intake (2). It is predominantly synthesized by adipocytes (3) to limit the intake of food, promote the breakdown of fat, and increase energy expenditure (4,5). Indeed, spontaneous mutations in leptin or its receptor result in marked obesity (6,7). Evidence is accumulating that leptin also plays a role in innate and acquired immunity (8,9). In fact, leptin and its receptor share structural similarities with members of the long-chain helical cytokine family, which includes IL-6, IL-11, and IL-12 (10,11). Consistent with this view, leptin regulates T lymphocyte responses, and, in particular, it polarizes T helper (Th) cells toward a Th1 phenotype by enhancing proliferation and IL-2 production of naive T cells (12). Furthermore, leptin increases the secretion of TNF-{alpha}, IL-6, and IL-12 by endotoxin-stimulated murine peritoneal macrophages (13). In addition, leptin induces the expression and secretion of IL-1 receptor antagonist (IL-1Ra) by human monocytes (14) as well as the production of TNF and IL-6 (15). Recent findings show that leptin positively modulates mononuclear cell survival by interfering with the apoptotic process (16). More striking, hypoleptinemia characterizing starvation is strictly related to increased susceptibility to infection secondary to malnutrition (12,17). Thus, leptin can be considered part of the recently categorized family of molecules produced by adipose tissue called adipokines, which are capable of linking metabolism and immune homeostasis (17,18). Adipokines include cytokines such as IL-1, IL-6, IFN-{gamma}, and TNF-{alpha} and chemokines such as IL-8, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) (17).

Nevertheless, there are some remarkable exceptions to this paradigm: In particular, ESRD is characterized by high susceptibility to bacterial infection (19) despite high levels of leptinemia (20,21). In agreement with this observation, another hyperleptinemic condition, obesity (22), is also associated with an increased incidence of infections (23). As far as ESRD is concerned, it is generally assumed that the defects in phagocytic polymorphonuclear neutrophilic leukocytes (neutrophils) plays a crucial role in the infectious morbidity (24), and, indeed, several uremic toxins (e.g., molecules that are capable of depressing neutrophil functions) have been identified (25,26). Despite active investigations regarding other immune cells, only a few reports about leptin and neutrophils are available. Furthermore, these works originated contrasting data about the capacity of leptin of modulating neutrophil activities. In particular, two papers from the group of Caldefie-Chezet (27,28) show that leptin is capable of triggering the oxidative and locomotory capacities of neutrophils without affecting phagocytosis. Conversely, Zarkesh-Esfahani et al. (29) did not observe any direct effect of leptin on neutrophil activation. The aim of the present work was to study the actual capability of leptin to modulate neutrophil functional activities and, in case of positive results, to investigate a possible role of this hormone in the pathogenesis of neutrophil dysfunctions characterizing ESRD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture Medium and Reagents
Hanks’ balanced salt solution (HBSS; EuroCLone, Wetherby West, Yorkshire, UK) mixed with Dulbecco’s PBS (EuroClone; HBBS:PBS = 3:1) containing 1 mg/ml BSA (Sigma Chemical Co., St. Louis, MO) was used as incubation medium throughout the study. Ficoll-Hypaque (Lympholyte-I), Giemsa stain, and heparin were purchased from Cedarlane Laboratories Ltd. (Hornby, Ontario, Canada), Merck (Darmstadt, Germany), and Roche (Milan, Italy), respectively. Fluorescein diacetate, HEPES, N-formyl-methionyl-leucyl-phenylalanine (FMLP), human recombinant C5a, and human recombinant leptin were purchased from Sigma. FITC-conjugated anti-CD11b mAb 44 (IgG1) and recombinant human IL-8 were purchased from Biosource International (Camarillo, CA). Mouse anti-human leptin 44802 mAb was from R&D System Europe (Abingdon, UK). Fura-2 AM and 2',7'-dichlorofluorescin-diacetate (DCFH-DA) were from Molecular Probes (Eugene, OR). Endotoxin contamination of the reagents used was tested by manufacturers or directly by QLC-1000 Assay (Cambrex Bio Science Walkersville, Inc., Walkersville, MD).

Patients
The study population consisted of 18 patients with ESRD (7 men and 11 women; mean age, 62.3 ± 17.8, x ± 1 SD) under hemodialytic (n = 13) or peritoneal dialytic (n = 5) treatment. All were outpatients, and the diagnoses were as follows: chronic glomerulonephritis (n = 6), hypertensive nephrosclerosis (n = 9), and vasculitis (n = 3). No patient had either history or clinical evidence of hepatic or gastrointestinal disease, infection, congestive heart failure, diabetes, or other endocrinopathies. Patients with active inflammation (C-reactive protein >10 mg/L) were excluded from the study. Eight patients displayed clinical signs of cardiovascular disease; six of them were previous smokers. Peritoneal dialysis patients had been peritonitis-free for at least 3 mo before the study. All patients received vitamin supplements that contained B vitamins and folic acid. None of the patients was receiving immune suppressive treatments, insulin, or androgenic steroids. Reverse osmosis was used to purify water for hemodialysis treatment. Serum was obtained from patients and from eight normal control subjects after informed consent. Leptin serum concentrations were determined by a RIA method using reagents supplied as a kit by DRG Instruments GmbH (Marburg, Germany). The lowest amount of leptin detectable in serum was 0.5 ng/ml. The within- and between-assay mean coefficients of variation were 3.9 and 4.3%, respectively. Three selected sera from patients were immunodepleted with anti-leptin mAb (10 µg/ml). The antibody concentration was chosen to achieve >90% neutralization of cytokine activity, based on neutralization assays performed by the manufacturer. After incubation (overnight, 4°C), the sera were ultracentrifuged (10,000 x g, 45 min) and immediately tested in the chemotactic assays and in leptin assays.

Neutrophil Preparation
Heparinized venous blood (10 U/ml heparin) was obtained from healthy male volunteers after informed consent. Neutrophils were isolated by dextran sedimentation and subsequent centrifugation on a Ficoll-Hypaque density gradient, as described previously (30). Contaminating erythrocytes were removed by hypotonic lysis (30). Then, neutrophils were washed three times with HBSS and resuspended in incubation medium at appropriate concentrations. Final cell suspension was >97% pure and >98% viable, as determined by usual assays (30).

Neutrophil Locomotion
Neutrophil locomotion was studied using the leading front method, as described previously (30). Tests were conducted in duplicate using blind well chambers (NeuroProbe, Cabin John, MA) with a 3-µm pore size cellulose ester filter (Millipore, Milan, Italy) separating the cells (4 x 105) from the chemoattractant. After incubation at 37°C for 45 min, the filters were removed, fixed in ethanol, stained with Harris hematoxylin, dehydrated, cleared with xylene, and mounted in Eukitt (Kindler, GmbH, Freiburg, Germany). Duplicate chambers were run in each case, and the distance (µm) traveled by the leading front of cells was measured at x400 magnifications; five randomly chosen fields were read for each filters.

Intracellular [Ca2+]i Determination
Neutrophils (2.5 x 106) were loaded with 2 µM fura-2 AM in HBSS-HEPES 10 mM (pH 7.4; 30 min, 37°C, final volume 0.5 ml). Then, cell suspension was diluted 10-fold with HBSS-HEPES, incubated for 30 min at 37C°, washed twice, and resuspended in HBSS-HEPES. Fluorescence changes before and after addition of leptin or FMLP were monitored with Perkin-Elmer LS3 spectrofluorometer at an excitation wavelength of 338 nm and an emission wavelength of 510 nm (30).

Superoxide Anion Release Assay
The release of superoxide anion was studied by using a modification of the method of Babior et al. (31) as described previously (30). Briefly, neutrophils (5 x 105) were incubated (20 min, 37°C, final volume 0.5 ml) with 80 µM ferricytochrome c, in the absence or presence of 300 U/ml superoxide dismutase (SOD). The reactions were then stopped by adding 2 ml of ice-cold 1 mM N-ethyl-maleimide, and the superoxide production was determined in the supernatants from the OD550 of samples without SOD minus OD550 of samples with SOD using a extinction coefficient of 2.1 x 10–4 M/cm.

Flow Cytometric Assessment of Neutrophil Oxidative Metabolism
Flow cytometric analysis of neutrophil oxidative metabolism was carried out according to Bass et al. (32), as described previously (33). Briefly, neutrophils were preincubated (15 min, 37°C) with DCFH-DA (5 µM). During the incubation time, DCFH-DA permeated the cells, wherein it was cleaved by intracellular esterases to give nonfluorescence DCFH trapped within the cells. After washing in PBS, the cells were incubated for 15 min at room temperature in the presence or absence of leptin (200 ng/ml). Then, the cells were incubated for additional 30 min at 37°C in the absence and presence of 100 nM FMLP. During this period, intracellular hydrogen peroxide oxidized DCFH to give a green fluorescence DCFH. At the end of the incubation, the reaction was stopped by keeping the samples on ice until flow cytometric analysis was carried out using an EPICS XL flow cytometer (Coulter).

Flow Cytofluorimetric Analysis of CD11b Surface Expression
The flow cytometric analysis of CD11b expression by neutrophils that were or were not exposed to 100 nM FMLP and/or 200 ng/ml leptin were performed as previously reported (33). FITC-conjugated anti-CD11b mAb 44 and appropriate isotype-matched mAb of irrelevant specificity were used for the analysis. All of the flow cytometry experiments were carried with the use of an EPICS XL flow cytometer (Coulter).

Statistical Analyses
Data were expressed as mean ± SD. Differences between two groups were analyzed by Mann-Whitney U test. Differences among three or more groups were analyzed by Friedman ANOVA test with Dunn post test. Correlations were calculated by Spearman test. Statistical analyses were performed using GraphPad InStat version 4.01 for Windows (GraphPad Software, San Diego, CA). Differences were accepted as significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin Inhibits Neutrophil Chemotaxis
Using 10 nM FMLP as chemoattractant, the distance traveled by normal human neutrophils into the filters in 45 min was 125.6 ± 14.4 µm (x ± 1 SD, n = 12). As shown in Figure 1, the chemotactic response of neutrophils was inhibited by leptin placed in the upper compartment of the migration chambers, in a dose-dependent manner. Similarly, leptin also inhibited the chemotactic response of neutrophils to other chemoattractants such as IL-8 and C5a (Figure 2). In these conditions, endotoxin concentration was <0.0005 ng/ml. Thus, the observed phenomenon is unlikely related to endotoxin contamination. Nevertheless, control experiments were performed in the presence of 1 µg/ml polymyxin B, which inactivates endotoxin. When preincubated in medium alone, neutrophil migration to 10 nM FMLP in the absence or presence of 50 ng/ml leptin was 125.0 ± 15.5 and 81.7 ± 14.4 µm (x ± 1 SD, n = 3), respectively. When preincubated with 1 µg/ml polymyxin B, neutrophil migration to 10 nM FMLP in the absence or presence of 50 ng/ml leptin was 122.0 ± 16.5 and 79.4 ± 10.0 µm (x ± 1 SD, n = 3), respectively.



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  		Figure 1. Dose-dependent inhibition of N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil migration by human recombinant leptin. Neutrophils were incubated in the upper compartment of the chemotaxis chamber in the absence or presence of various doses of leptin, and their locomotory response to 10 nM FMLP in the lower compartment was tested after 45 min of incubation. Results are expressed as mean ± 1 SD of three experiments with neutrophils from different donors. 0 versus 1, P < 0.01; 0 versus 10, P < 0.01; 0 versus 50, P < 0.001.

 


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  		Figure 2. Inhibition of C5a- and IL-8–induced neutrophil migration by human recombinant leptin. Neutrophils were incubated in the upper compartment of the chemotaxis chamber in the absence or presence of various doses of leptin, and their locomotory response to 1 nM C5a or 1 nM IL-8 in the lower compartment was tested after 45 min of incubation. Results are expressed as mean ± 1 SD of four experiments with neutrophils from different donors. 0 versus 50, P < 0.05.

 
Inhibition of Neutrophil Chemotaxis by Sera from ERDS Patients Is Related to Serum Levels of Leptin
It is known that serum levels of leptin are increased in patients with chronic renal failure (20,21) and that sera from renal failure patients inhibits neutrophil locomotion (34,35). Therefore, the inhibition of neutrophil migration by sera from ERDS patients might be related to serum levels of leptin. In fact, serum from 18 ERDS patients inhibited the neutrophil chemotactic response to FMLP: Neutrophil migration in the presence of 25% ESRD serum in the upper compartment of migration chambers: 82.0 ± 30.0 µm/45 min, x ± 1 SD, n = 18 (median, 80.8 µm/45 min; 25%, 58.4 µm/45 min; 75%, 111.0 µm/45 min); neutrophil migration in the presence of 25% control serum in the upper compartment of migration chambers: 119.3 ± 15.4 µm/45 min, x ± 1 SD, n = 8 (median, 119.3 µm/45 min; 25%, 110.8 µm/45 min; 75%, 129.3 µm/45 min). Neutrophil migration to FMLP in the presence of ERDS serum versus neutrophil migration to FMLP in the presence of normal serum: P = 0.005. The concentrations of leptin in sera from ESRD patients were from 1.7 to 137.1 ng/ml (mean, 70.10 ± 50.41 ng/ml, x ± 1 SD, n = 18; median, 79.1 ng/mg; 25%, 10.2 ng/ml; 75%, 115.5 ng/ml). As depicted in Figure 3, neutrophil migration to FMLP in the presence of ESRD sera and leptin concentrations in the same samples is inversely correlated (Spearman r = –0.7110, P = 0.0009).



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  		Figure 3. Relationship between the concentration of leptin in 18 ESRD sera and neutrophil migration to FMLP in the presence of the same samples. The means of two determinations of leptin concentrations in sera were plotted against the levels of migration to 10 nM FMLP of normal neutrophils in the presence of 25% sera expressed as means of two different experiments.

 
Leptin-Depleted Sera from Chronic Renal Failure Patients Do not Affect the Chemotactic Response of Normal Neutrophils
Three inhibitory ESRD sera were selected to be immunodepleted of leptin. After this procedure, the cytokine in each of the three sera was undetectable. Then, neutrophil migration to FMLP in the presence of undepleted and leptin-depleted sera was tested in parallel assays. As shown in Figure 4, each of the three leptin-depleted sera did not inhibit neutrophil locomotion in response to FMLP, whereas the undepleted serum samples were effective. This finding strongly supports the idea that the inhibitory effects exerted by serum from renal failure patients on neutrophil locomotory responses are actually mediated by leptin.



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  		Figure 4. Effect of three leptin-depleted ESRD sera on the neutrophil response to FMLP. Locomotory activity of neutrophils incubated in the upper compartment of the chemotaxis chamber with undepleted ({blacksquare}) or leptin-depleted ({square}) serum from three ESRD patients (8, 9, and 10) and exposed in the lower compartment for 45 min to 10 nM FMLP. Results are expressed as mean ± 1 SD of four experiments with neutrophils from different donors. Migration in presence of undepleted versus leptin-depleted serum, P = 0.0286.

 
Leptin as a "Pure" Chemoattractant for Neutrophils
When normal neutrophils are incubated in the upper compartment of migration chambers, without the addition of chemoattractant in the lower compartment, they migrate spontaneously. Under our experimental conditions, neutrophil spontaneous migration was 59.6 ± 10.2 µm (mean ± 1 SD, n = 12). As shown in Figure 5, neutrophils that were exposed to leptin that was added to the lower compartment of migration chambers displayed a bell-shaped dose-response curve characteristic of chemoattractants, reflecting the well-known inhibition of neutrophil orientation and directed migration in response to high-dose chemoattractant stimulation (36). It is noteworthy that the stimulatory activity of leptin was detected at concentrations comparable to those found to inhibit the chemotactic response to FMLP. Control experiments that were performed in presence of polymyxin B ruled out possible interferences from contaminant endotoxin (data not shown). A series of experiments were then performed to test the capability of leptin to trigger neutrophil activation and/or to interfere with FMLP-induced functional responses. No [Ca2+]i mobilization was observed in Fura-2–loaded neutrophils that were exposed to leptin, and leptin-treated neutrophils maintained their capacity to mount a rapid increase of [Ca2+]i in response to FMLP (data not shown). Consistent with data in [Ca2+]i assay, leptin did not stimulate the respiratory burst or impair the FMLP-triggered oxidative response by neutrophils determined as spectrophotometric analysis of superoxide anion production and as flow cytometric analysis of intracellular oxidation of 2'-7'-dichloroflourescein in DCFH-DA–loaded neutrophils (data not shown). Finally, in accordance with previous report, leptin did not influence the expression of CD11b on neutrophils and did not affect the CD11b upregulation induced by FMLP (data not shown).



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  		Figure 5. Dose-dependent induction of neutrophil migration by various doses of leptin. Neutrophils were incubated in the upper compartment of the chemotaxis chamber in medium, and their locomotory response in the absence or presence of various doses of leptin in the lower compartment was tested after 45 min of incubation. Results are expressed as mean ± 1 SD of five experiments with neutrophils from different donors. 0 versus 1, P < 0.05; 0 versus 10, P < 0.05; 0 versus 50, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bacterial infections are a major cause of morbidity and mortality among patients with ESRD (37). The high incidence of bacterial infections must be considered a multifactorial event related to diverse pathogenetic factors, such as advanced age, comorbidities, and exposure to infectious risk factors during dialysis (19,38,39). A critical role in the pathogenesis of increased susceptibility to infections is exerted by functional abnormalities of neutrophils (24,40). These dysfunctions are considered secondary to diverse causes, such as accumulation of calcium within the cells, iron overload, interactions with biocompatible dialyzers and dialysis solutions, and several uremic molecules including low molecular weight proteins (LMWP) (25,26,40). LMWP are a recently categorized class of proteins that have a MW spectrum ranging from 1,000 to 50,000 Da and are thought to be crucial for the pathogenesis of uremic syndrome, including malnutrition and increased susceptibility to infections (21,41). As far as neutrophil function is regarded, six so-called granulocyte-inhibiting proteins (GIP) have been purified from ultrafiltrate of dialyzers and peritoneal effluent (26). GIP exert their inhibitory activity toward different functional activities of neutrophils, including oxidative metabolism, chemotaxis, degranulation, and phagocytosis, with consequent impairment of neutrophil-dependent antibacterial defense (21,26). Also, leptin has been categorized as a LMWP, and, indeed, a role for this protein in some uremic manifestations, such as anorexia and weight loss, has been suggested (21,41). Here we provide evidence that leptin is capable of inhibiting in a dose-dependent manner neutrophil migration in response to classical chemoattractants (FMLP, C5a, and IL-8). Moreover, serum from patients with ESRD inhibits migration of normal neutrophils in response to FMLP with a strict correlation between serum leptin levels and serum ability to suppress neutrophil locomotion. Finally, the serum inhibitory activity can be effectively prevented by previous immune depletion of leptin. Taking into account the crucial role of neutrophils in host immune competence (42), the leptin-mediated ability of serum from ESRD patients to inhibit neutrophil chemotaxis appears as a potential mechanism contributing to the establishment of infections in ESRD. In other words, leptin must be considered a bona fide GIP detectable in serum of ERSD patients. Nevertheless, further retrospective and/or perspective studies are necessary to establish a link between hyperleptinemia and increased susceptibility to infections in ESRD patients. Our results confirm that leptin is also endowed with chemotactic activity toward neutrophils. It is of note that the two activities—inhibition of the cell response to FMLP or other chemokines and stimulation of neutrophil migration—could be detected at similar concentrations. Taking into account that certain chemotaxins not only desensitize the cells toward a further stimulation with the same chemotaxin but also prevent the cell locomotory response toward other chemotaxins (43,44), our results suggest that by means of its chemokine-like activity, leptin is capable of inhibiting neutrophil chemotaxis in response to other neutrophilic chemoattractants.

Stimulation of neutrophils by classical chemoattractants, such as FMLP, IL-8, and C5a, results in a rapid and transient rise in [Ca2+]I, and, indeed, this metabolic response has been considered for a long time a hallmark of chemoattractant-triggered neutrophil locomotion (45). Nevertheless, several pieces of evidence do not support this vision anymore. First, protrusive surface activity, gelsolin-actin complexes, and net actin assembly can occur in the absence of Ca2+ transient (46). Furthermore, extracellular and intracellular Ca2+ chelators do not block neutrophil migration in response to chemotactic factors (47,48), and chemotaxis, unlike superoxide anion production, does not depend on [Ca2+]i enhancement in human neutrophils (49). More striking, a fast-growing family of ligands, such as Substance P, TGF-{beta}1, fibrinopeptide B, and Fas ligand, induce neutrophil chemotaxis without increasing [Ca2+]i levels and without activating oxidative metabolism or granule exocytosis (30,50,51). Consequently, chemoattractants can be classified in two functional groups: classical chemoattractants (fMLP, C5a, and IL-8), which also evoke secretory responses such as superoxide anion release or lysosomal degranulation, and pure chemoattractants, which are devoid of secretagogue properties. Our results show that neutrophils that were exposed to leptin did not display detectable [Ca2+]i mobilization or oxidant production. Consistent with our findings, Zarkesh-Esfahani and et al. (29) recently showed that neutrophils do not express the long form Ob-Rb, the receptor isoform mainly involved in the regulation of multiple intracellular signaling cascades, including the classic janus-activating kinase signal transducer and activator of transcription (JAK-STAT) pathway (5,52), which in turn is critical for phospholipase C–dependent [Ca2+]i rise induced by chemokine stimulation (53). On the contrary, neutrophils express the short form of the leptin receptor Ob-Ra capable of transducing activating signal to the mitogen-activate protein kinase (MAPK) pathway without JAK-STAT activation (54). Accordingly, TGF-{beta}1, a pure chemoattractant that is capable of stimulating neutrophil migration in a Ca2+-independent manner, required MAPK activation (55). In other words, it is suggestive that classical chemoattractants, such as FMLP, induce neutrophil activation via both JAK-STAT and MAPK pathways, whereas pure chemoattractants, including leptin, require for triggering cell locomotion only MAPK activation, which in the case of leptin is mediated by Ob-Ra constitutively expressed by neutrophils.

It has been reported and herein confirmed that neutrophil stimulation by leptin does not induce the upregulation of CD11b expression (29). Once again these data are in agreement with the well known incapacity of pure chemoattractants, such as Substance P, TGF-{beta}1, to affect the expression of {beta}2 integrins (56). On the contrary, an aforementioned report regarding CD11b expression and our data are in disagreement with data of Caldefie-Chezet et al. (27,28), which show that leptin is capable of activating an oxidative burst of neutrophils. We do not have a clear explanation for this discrepancy, but at least one more consideration can be made. Our data suggest that hyperleptinemia induces deactivation of circulating neutrophils, which became incapable of responding to a subsequent chemotactic stimulus. On the contrary, leptin-mediated activation of circulating neutrophils should result in clinical pictures resembling neutrophilic vasculitis. In fact, hyperleptinemic clinical syndromes (e.g., chronic renal failure), as well as hyperleptinemic animal models, do not show signs of neutrophilic hyperactivation.

In conclusion, the results suggest that leptin behaves like a chemokine, capable of stimulating neutrophil locomotion and desensitizing the cells to stimulation by another chemoattractant, and that leptin is responsible for chemotactic desensitization of neutrophils by sera from patients with chronic renal failure, taken as a disease model of hyperleptinemia.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication April 20, 2004. Accepted for publication June 28, 2004.




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