Leptin promotes cell survival and activates Jurkat T lymphocytes by stimulation of mitogen-activated protein kinase

Abstract

Leptin (Ob) is a non-glycosylated peptide hormone that regulates energy homeostasis centrally, but also has systemic effects including the regulation of the immune function. We have reported previously that leptin activates human peripheral blood lymphocytes co-stimulated with phytohaemagglutinin (PHA) (4 μg/ml), which prevented the employment of pharmacological inhibitors of signalling pathways. In the present study, we used Jurkat T cells that responded to leptin with minimal PHA co-stimulation (0·25 μg/ml). The long isoform of leptin receptor is expressed on Jurkat T cells and upon leptin stimulation, the expression of early activation marker CD69 increases in a dose-dependent manner (0·1–10 nM). We have also found that leptin activates receptor-associated kinases of the Janus family-signal transucers and activators of transcription (JAK-STAT), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K) signalling pathways. Moreover, we sought to study the possible effect of leptin on cell survival and apoptosis of Jurkat T cells by culture in serum-free conditions. We have assayed the early phases of apoptosis by flow cytometric detection of fluorescein isothiocyanate (FITC)-labelled annexin V simultaneously with dye exclusion of propidium iodide (PI). As well, we have assayed the activation level of caspase-3 by inmunoblot with a specific antibody that recognizes active caspase-3. We have found that leptin inhibits the apoptotic process dose-dependently. By using pharmacological inhibitors, we have found that the stimulatory and anti-apoptotic effects of leptin in Jurkat T cells are dependent on MAPK activation, rather than the PI3K pathway, providing new data regarding the mechanism of action of leptin in T cells, which may be useful to understand more clearly the association between nutritional status and the immune function.

Introduction

Leptin (Ob) [1] is a 16-kDa non-glycosylated hydrophylic peptide hormone [2]. It belongs to the family of adipokines, which are generated by adipose tissue and may act in an autocrine, paracrine or endocrine manner to modify the function of different organs [3]. The major source of leptin is the subcutaneous adipocyte [4]. Leptin is released into the bloodstream [5] and plasma levels correlate with total body fat mass [6, 7]. There are other important sources of leptin in human body, such as placenta, stomach or epithelial breast cells [8–14]. Leptin is a molecule with functional pleitropy [2]. The different isoforms of leptin receptor (Ob-R) are distributed widely in the human body [8]. This widespread distribution throughout peripheral tissues supports the role of leptin in neuroendocrine, energy homeostasis and immune regulation [15].

Functional data suggest that Ob-R is a member of the class I cytokine receptor superfamily [16]. Thus, similar to other receptors of this class, Ob-R lacks intrinsic tyrosine kinase activity but requires the activation of receptor-associated kinases of the Janus family (JAKs) [17] which initiate downstream signalling, including members of the STAT (signal transducers and activators of transcription) family of transcription factors [18]. After ligand binding, JAKs autophosphorylate and tyrosine phosphorylates various STATs. Activated STATs then dimerize and translocate to the nucleus, where specific gene responses are elicited [18, 19].

The role of leptin in T lymphocyte function has been demonstrated in mice lacking leptin (ob/ob) or leptin receptor (db/db) expression [20]. We then found that leptin is able to promote activation and proliferation of human monocytes and to enhance activation and proliferation of preactivated T lymphocytes [21, 22]. These effects of leptin on peripheral blood mononuclear cells (PBMC) are mediated by the leptin receptor, which is present in peripheral blood monocytes and T lymphocytes [21, 22]. Thus, we have also found that human leptin can trigger signal transduction activating JAK-STAT, phosphatidylinositol 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in human PBMC [23, 24]. T lymphocytes from leptin-deficient ob/ob mice have a reduced sensibility to stimulatory agents, whereas monocytes increase sensibility to proinflammatory stimuli [25–27]. Ob/ob mice and leptin receptor mutant db/db mice display immune dysfunction and lymphoid organ atrophy, affecting thymic size and cellularity, similar to that observed in starved animals and malnourished humans [26, 28, 29]. Thus they have reduced levels of peripheral T and B cells, suggesting that leptin may have a role in lymphopoiesis [30]. Leptin also protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice [28]. Moreover, human leptin deficiency caused by a missense mutation also produces immune system dysfunction [31]. Conversely, it has been shown that leptin receptor deficiency affects the immune system indirectly via changes in the systemic environment [20]. Thus, leptin has a selective thymostimulatory role in settings of leptin deficiency and endotoxin administration, and may be useful for protecting the thymus from damage and augmenting T cell reconstitution in these clinical states [32]. Nutritional status acting via leptin-dependent mechanisms may alter the nature and vigour of the immune response [33]. Many cytokines have a trophic effect on immune cells promoting cell survival by inhibiting apoptotic stimuli [26, 34]. In this context, we have found previously that leptin promotes dose-dependent cell survival of monocytes after 24–96 h of serum-free culture. This effect is mediated by the activation of the p42/44 MAPK pathway [34]. In recent studies, leptin has been demonstrated to inhibit the apoptosis of thymic cells through a mechanism that is independent of the activation of JAK-2 but depends on the engagement of the insulin receptor substrate (IRS)-1/PI 3-kinase pathway [35].

In the present work, we sought to study further the role of leptin-activating T cells and the trophic effect of leptin preventing serum-deprived induced apoptosis using Jurkat T cells. In addition, we investigated the signalling cascade of leptin receptor and the relative contribution of different signalling pathways in these effects of leptin on Jurkat T cells.

Materials and methods

Materials

Human recombinant leptin was obtained from Sigma-Aldrich (St Louis, MO, USA) and phytohaemagglutinin (PHA) from Roche Diagnostics GMBH (Mannheim, Germany). All the anti-CD monoclonal antibodies (mAbs) were obtained from Beckton Dickinson Immunocytometry Systems (BDIS, San Jose, CA, USA) and were used according to the manufacturer's recommended concentrations. The mAbs used in this study were anti-CD69 fluorescein isothiocyanate (FITC) and anti-CD4 phycoerythrin (PE). Antibodies against leptin receptor (C-terminal) and JAK-2 were from Santa Cruz (Santa Cruz, CA, USA). Antibodies against protein kinase B (AKT), caspase-3, MAP/extracellular regulated kinase (ERK) (MEK)-1/2 and STAT-3 were from BD Biosciences Pharmingen™. Monoclonal antibodies to phosphotyrosine (α-PY) were purchased from Transduction Laboratories (Lexington, KY, USA). Pharmacological inhibitors PD980059 and wortmannin were from Sigma-Aldrich; the annexin V-FITC Apoptosis Detection Kit I was from BD Biosciences Pharmingen™.

Cell preparation and culture

Jurkat T cells were cultured in the appropriate medium for cell culture, RPMI-1640, supplemented with 25 mM HEPES, 100 μU/ml l-glutamine, 100 μU/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml amphotericin B and 10% heat-inactivated fetal bovine serum (all from Biological Industries, Kibbutz Beit Haemek, Israel).

Cells were treated for different times depending on the nature of the experiment at 37°C, with different leptin concentrations and in the presence and absence of 1 μM pharmacological inhibitors. Cell cultures were centrifuged to remove medium and were solubilized for 30 min at 4°C in lysis buffer containing 20 mM Tris, pH 8, 1% Nonidet P-40, 137 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM dithiothreitol (DTT), 10% glycerol, 1 mM phenylmethylsulphonylfluoride and 0·4 mM sodium orthovanadate. After centrifugation, soluble cell lysates were used for the study. Protein concentration was determined by a kit from Bio-Rad (Richmond, CA, USA).

Ob-R mRNA detection by reverse transcription–polymerase chain reaction (RT–PCR)

Total RNA from Jurkat T cells was extracted with the easy-BLUE™ Total RNA Extraction Kit (Intron Biotechnology, Inc., Jungwon-Gu, Korea). First-strand cDNA synthesis was performed using an oligo(dT) primer kit (Roche Molecular Diagnostics, Barcelona, Spain) and used for detection of the Ob-R messenger RNA (mRNA) by RT–PCR. The PCR products were analysed by 2% agarose gel with ethidium bromide staining. The primer sequences for Ob-R have been used previously for detection of Ob-R expression [36].

Immunoprecipitation and Western blotting analysis

Soluble cellular lysates (0·5 mg of protein) were precleared with 50 μl of protein A-Sepharose CL-4B or 6MB (Amersham Pharmacia Biotech, Barcelona, Spain) for 2 h at 4°C by end-over-end rotation. The precleared cellular lysates were incubated with the appropriate antibodies for 3 h at 4°C. Next, 50 ml of protein A-Sepharose was added to immune complexes and incubation was continued for 2 h at 4°C. The immunoprecipitates were washed three times with lysis buffer. We added 60 ml of sodium dodecyl sulphate (SDS) stop buffer containing 100 mmol/l of DTT to the immunoprecipitates followed by boiling for 5 min. The soluble supernatants were then resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred electrophoretically onto nitrocellulose membranes. The membranes were blocked with Tris-buffered saline−0·05% Tween 20 (TBST) containing 5% non-fat dry milk for 30 min at 23°C. The blots were then incubated with primary antibody overnight, washed three times in TBST and incubated further with secondary antibodies linked to horseradish peroxidase. Bound horseradish peroxidase was visualized by a highly sensitive chemiluminescence system (SuperSignal; Pierce, Rockford, IL, USA). The bands obtained in the blots were scanned and analysed using the Scion Image-Release Alpha, version 4·0·3·2 for Windows.

Flow cytometry activation assay of Jurkat T cells

Jurkat T cells were cultured in the presence of appropriate stimuli for 6 h to detect the early activation marker CD69 on the cell surface. At the end of the stimulation time, cells were centrifuged, washed in phosphate-buffered saline (PBS) and resuspended in 100 ml PBS containing ethylenediamine tetraacetic acid (EDTA) (FACS-Flow; BDIS). Finally, cells were incubated with the appropriate mAbs for 20 min, washed and fixed for flow cytometry analysis. We used anti-CD69 FITC monoclonal antibody for analysing the percentage of cells expressing CD69. We gated Jurkat T cells according to side scatter (SSC) and forward scatter (FSC). The positive cells were CD69⁺. A total of 20 000 cells were acquired.

Flow cytometry apoptosis assay of Jurkat T cells

Jurkat T cells were cultured routinely in complete medium. For the apoptosis experiments, cells were centrifuged previously and cultured in medium without serum, and in the presence of human leptin at different concentrations for 10 h. At the end of the stimulation time, we used the annexin V-FITC Apoptosis Detection Kit I (BD Biosciences Pharmingen™) following the manufacturer's recommended procedure for flow cytometry analysis. This test discriminates intact cells (annexin V^–/PI^–), early apoptotic cells (annexin V⁺/PI^–) and late apoptotic/necrotic cells (annexin V⁺/PI⁺). A total of 20 000 cells were acquired.

Data acquisition and analysis

Data were acquired on a fluorescence activated cell sorter (FACS)Calibur flow cytometer using CELLQuest software (BDIS). Results are displayed as percentage of cells expressing any marker (CD69, annexin V/PI).

Results

Leptin receptor expression in Jurkat T cells

The presence of leptin receptors was investigated in Jurkat T cells by analysis of Ob-R mRNA expression using RT–PCR, and the presence of the protein was confirmed by immunodetection of Ob-R. We carried out immunoprecipitation studies using anti-Ob-R antibodies that recognize the C-terminal region of the receptor and the immunoprecipitates were then analyzed by immunoblot using the same antibody. As shown in Fig. 1, the stained gel with the RT–PCR sample showed the expected band of 338 base pairs (bp) corresponding to Ob-R (Fig. 1a). The specific immunoblot of the immunoprecipitated long form of the receptor shown in Fig. 1b confirms the presence of Ob-R in Jurkat T cells.

Fig. 1.

The long isoform of leptin receptor is expressed in Jurkat T cells associated constitutively with JAK-2, which is activated upon leptin stimulation. (a) Detection of the leptin receptor in Jurkat T cells. Reverse transcription–polymerase chain reaction (RT–PCR) analysis of leptin receptor (Ob-R) mRNA expression in Jurkat T cells. Total RNA extracted from Jurkat T cells was reverse transcribed and Ob-R expression determined by RT–PCR amplification. Ethidium bromide stained gel with the PCR product is shown. The 338 base pairs (bp) PCR product of Ob-R. (b) Detection of the leptin receptor in Jurkat T cells. Jurkat T cells were incubated at 37°C for 10 min with 0–10 nM human leptin. Cells were washed, lysed and immunoprecipitated with anti-leptin receptor (long isoform) antibody. Soluble supernatant was then analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting using the anti-C-terminal (C-t) antibody, which recognizes only the long isoform of leptin receptor. IP-Ab, immunoprecipitating antibody; WB-Ab, Western blot antibody. Immunoblots are representative of five independent experiments. (c) Ob-R is associated with JAK-2 in Jurkat T cells. Jurkat T cells were incubated at 37°C for 10 min with 10 nM leptin. Cells were washed, lysed and immunoprecipitated with anti-leptin receptor (long isoform) antibody. Immunoprecipitates were analysed by specific immunoblotting with antibodies to JAK-2 (α-JAK-2). A control immunoblot of anti-Ob-R is also shown. IP-Ab, immunoprecipitating antibody; WB-Ab, Western blot antibody. Immunoblots are representative of three independent experiments. (d) Human leptin activates JAK-2 in Jurkat T cells. Jurkat T cells were incubated at 37°C for 10 min with 10 nM leptin. Cells were washed, lysed and immunoprecipitated with antibodies to JAK-2. Immunoprecipitates were analysed by specific immunoblotting with anti-phosphotyrosine antibodies (α-PY) to detect autophosphorylated JAKs. Control immunoblots were performed with anti-JAK-2. IP-Ab, immunoprecipitating antibody; WB-Ab, Western blot antibody. Immunoblots are representative of three independent experiments.

Human leptin activates the JAK pathway

To study the activation of JAK kinases by the leptin receptor in Jurkat T cells, we investigated the possible association between JAK-2 isoforms and the leptin receptor. The JAK-2 isoform was associated physically with the leptin receptor, as assessed by immunoprecipitation of the receptor and Western blot with anti-JAK-2 (Fig. 1c). The lower immunoblot shows the control anti-Ob-R blotting of the samples. The opposite approach (immunoprecipitation of JAK isoforms and Western blot with anti-leptin receptor) yielded similar results (data not shown). The association is constitutive and occurs both in the absence and presence of the ligand (Fig. 1c). Also, we stimulated Jurkat T cells with human leptin and analysed the phosphorylation of immunoprecipitated JAK proteins by Western blot using anti-phosphotyrosine antibody (Fig. 1d). For immunoprecipitation we used antibodies against the JAK-2 kinase (Fig. 1d). Immunoprecipitation was controlled by immunoblotting with the same immunoprecipitating antibody. A phosphorylated band corresponding approximately to 120 kDa was detected in both immunoprecipitates in response to 10 nM leptin. We have demonstrated that leptin increases tyrosine phosphorylation of JAK-2.

Human leptin stimulates tyrosine phosphorylation of STAT-3 in Jurkat T cells

We next sought to study the possible activation of STAT-3 by human leptin in Jurkat T cells. Cells were stimulated with leptin (0–10 nM). Solubilized lysed samples were analysed by specific immunoblot with the antibody against phosphorylated STAT-3. As shown in Fig. 2a, tyrosine phosphorylation of STAT-3 was observed in response to human leptin and the effect of leptin was dependent upon the dose. The amount of total STAT-3 in every sample was controlled by immunoblot using anti-STAT-3 antibody (Fig. 2a).

Fig. 2.

Leptin stimulation triggers the activation of signal transucers and activators of transcription (STAT), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K) pathways. (a) Human leptin stimulates tyrosine phosphorylation of STAT-3 in Jurkat T cells. Jurkat T cells were incubated at 37°C for 10 min with 0–10 nM leptin. Cells were lysed and soluble lysates were analysed by immunoblotting using the specific antibody against phosphorylated STAT-3. The same lysates were analysed by anti-STAT-3 immunoblot to control the amount of STAT-3 in each sample. Leptin stimulated STAT-3 tyrosine phosphorylation in a dose-dependent manner. WB-Ab, Western blot antibody. We show representative blots of four different experiments. (b) Leptin stimulation activates MAP/extracellular regulated kinase (ERK) (MEK)-1/2 in Jurkat T cells. Jurkat T cells were incubated in the presence of (0–10) nM leptin for 10 min. Cells were lysed and soluble lysates were analysed by immunoblotting using the specific antibody against phosphorylated MEK-1/2. The same lysates were analysed by anti-MEK-1/2 immunoblot to control the amount of MEK in the samples. Data shown are representative of four independent experiments. (c) Leptin stimulation activates protein kinase B (AKT) (PKB) in Jurkat T cells. Jurkat T cells were incubated in the presence of 0–10 nM leptin for 10 min. Cells were lysed and soluble lysates were analysed by immunoblotting using the specific antibody against phosphorylated AKT. The same lysates were analysed by anti-AKT immunoblot to control the amount of protein kinase B in the samples. Data shown are representative of three independent experiments.

Leptin stimulates MAPK pathway in Jurkat T cells

On the basis of the previously described effect of leptin on MAPK pathways in different systems, we checked whether MAPK is activated by leptin in Jurkat T cells by studying its serine/threonine phosphorylation level, which reflects the activation of MEK and therefore the whole MAPK pathway. As shown in Fig. 2b, leptin stimulated serine/threonine phosphorylation of MEK dose-dependently, as assessed by specific immunoblot with the antibodies against phosphorylated MEK-1/2. The amount of MEK in every sample was controlled by immunoblot using anti-MEK antibodies.

Leptin stimulates PI3K pathway in Jurkat T cells

In order to investigate the possible activation of the PI3K pathway in Jurkat T cells after leptin stimulation, we studied a central protein of this signalling pathway to verify the effects caused by leptin over Jurkat T cells. As reflected in Fig. 2c, leptin stimulated phosphorylation of AKT dose-dependently, as observed by specific immunoblot with the antibody against phosphorylated AKT. The amount of AKT in every sample was checked by immunoblot with antibodies that recognize total AKT.

Leptin enhances the expression of early activation markers in Jurkat T cells

We have described previously that leptin increased the expression of different activation markers (CD69, CD25, CD71 and CD38) in human monocytes and lymphocytes. In the present study we measured the expression of CD69 in Jurkat T cells by flow cytometry. Jurkat T cells were cultured for 6 h in the presence of 0–10 nM leptin and we added 0·25 μg/ml PHA as co-stimulus. As shown in Fig. 3, leptin potentiated the effect of the PHA co-stimulus on the CD69 expression by Jurkat T cells (Fig. 3a). The effect of leptin on CD69 expression was dose-dependent (Fig. 3b), and the maximal effect was achieved at 10 nM leptin. At this leptin concentration the effect of submaximal concentrations of PHA (0·25 μg/ml) was enhanced twofold (Fig. 3b).

Fig. 3.

The effect of leptin on CD69 expression (6 h) in Jurkat T cells. The expression level of CD69 was measured by flow cytometry. Total cells were analysed by a fluorescence activated cell sorter (FACS)Calibur. Viable cells were gated by side–forward-scatter characteristics. (a) One-colour immunofluorescence analysis of Jurkat T cells stained with anti-CD69-fluorescein isothiocyanate (FITC) (x-axis) after 6 h of culture in the absence or the presence of leptin (10 nM) and submaximal doses of phytohaemagglutinin (PHA) (0·25 μg/ml). The numbers in the corners of each contour plot represent the net percentage (%) of positive cells in the appropriate quadrant. Dot plots are from a typical experiment representative of six independent experiments. (b) Data show percentage of maximal effect of leptin on CD69 expression in CD4 Jurkat T cells when stimulated with increasing doses of leptin 0–10 nM and submaximal concentrations of PHA (0·25 μg/ml). Data are means of five different experiments. Standard errors of the mean are lower than 10%.

Leptin promotes cell survival of Jurkat T cells cultured in the absence of serum

The effect of leptin on apoptosis in Jurkat T cells promoted by the withdrawal of serum was assessed by two different approaches. First, early apoptotic events were evaluated by measuring the exposure of phosphatidylserine on plasma membranes by flow cytometric analysis, using annexin V-FITC/PI. Cells were incubated for 10 h in the presence and absence of 10 nM leptin in serum-free medium. As shown in Fig. 4, treatment with 10 nM leptin reduced phosphatidylserine externalization in Jurkat T cells. In the presence of 10 nM leptin, apoptotic cells were reduced by 60%.

Fig. 4.

Leptin prevents apoptosis in Jurkat T cells cultured in serum-free medium. Data are dot-plot diagrams of fluorescein isothiocyanate (FITC)–annexin V/phosphatidylinositol (PI) flow cytometry of Jurkat T cells after 10 h culture in the absence of serum (a), with (c) or without 10 nM leptin (b). In the left column,the data show gated viable cells by side–forward-scatter characteristics. In the right column, the lower left quadrants show the viable cells, which exclude PI and are negative for FITC–annexin V binding. The upper right quadrants contain the non-viable, necrotic and late apoptotic cells, positive for FITC–annexin V binding and for PI uptake. The lower right quadrants represent the apoptotic cells, FITC–annexin V-positive and PI-negative. An experiment representative of 10 independent experiments is shown.

Secondly, late apoptotic events were evaluated by immunoblot using antibodies that recognize specifically the cleaved activated form of caspase-3. Different apoptotic pathways converge to activate the key apoptotic effector caspase-3 later. Thus, we measured the caspase-3 activity in Jurkat T cells after stimulation with 0–10 nM leptin. Figure 5 shows that cells treated with leptin decreased caspase-3 activity dose-dependently compared with control cells. The amount of caspase-3 in every sample was controlled by immunoblot using anti-actin antibodies. The results obtained from the measure of phosphatidylserine externalization in Jurkat T cells are consistent with data obtained from caspase-3 activity measurement, confirming that leptin prevents the programmed cell death of Jurkat T cells induced by serum deprivation.

Fig. 5.

Human leptin reduces dose-dependently the caspase-3 activation produced by the triggering of apoptosis by the withdrawal of serum. Jurkat T cells were incubated at 37°C for 18 h with 0–10 nM leptin in serum-free medium. Jurkat T cells were cultured at 37°C for 18 h in the presence of serum and without leptin. Cells were lysed and soluble lysates were analysed by immunoblotting using the specific antibody against activated caspase-3. The same lysates were analysed by anti-actin immunoblot to control the amount of protein in the samples. Data shown are representative of three independent experiments.We show representative blots of four different experiments.

Augmented expression of early activation markers in Jurkat T cells is mediated by the MAPK pathway

To identify the pathway whereby leptin enhances the expression of early activation markers (CD69), we designed pharmacological inhibition experiments. We employed three pharmacological inhibitors: one pharmacological inhibitor of MAPK, PD980059 and two inhibitors of PI3K, wortmannin and LY294002. Jurkat T cells were cultured for 6 h in the presence and absence of 10 nM leptin and we added 0·25 μg/ml PHA as co-stimulus. The pharmacological inhibitors were added to the culture 10 min before the addition of leptin. After the stimulation time, we measured the expression of CD69 in Jurkat T cells by flow cytometry. The culture was washed with PBS and resuspended in 100 ml PBS containing EDTA (FACS-Flow; BDIS). We used anti-CD69 FITC monoclonal antibody for analysing the percentage of Jurkat T cells expressing CD69.

As shown in Fig. 6a, the stimulatory effect of 10 nM human leptin on CD69 activation marker in Jurkat T cells co-stimulated with a submaximum dose of PHA is inhibited completely by 1 μM PD98059. However, neither wortmannin nor LY294002 reverted the stimulatory effect of leptin on CD69 expression by Jurkat T cells co-stimulated with 0·25 μg/ml PHA. To determine the inhibition percentage of the effect, we considered a maximum effect of CD69 expression obtained by Jurkat T cells stimulated with 10 nM leptin and co-stimulated with PHA 0·25 μg/ml.

Fig. 6.

(a) Leptin enhances the expression of CD69 activation marker by means of mitogen-activated protein kinase (MAPK) activation. The level of expression of CD69 in gated viable Jurkat T cells was assessed by flow cytometry at 6 h of culture by using anti-CD69 fluorescein isothiocyanate (FITC)-labelled antibodies. Data show the percentage of stimulation of Jurkat T cells compared with maximal effect obtained with 10 nM leptin and submaximal concentrations of phytohaemagglutinin (PHA) (0·25 μg/ml), in the presence or absence of 1 μM PD98059, 1 μM wortmannin and 1 μM LY294002. Data are means of five independent experiments. (b) Leptin reduces the caspase-3 activation in absence of serum by means of MAPK activation. Jurkat T cells were incubated at 37°C for 18 h with 0–10 nM leptin in serum-free medium. Jurkat T cells were cultured at 37°C for 18 h in the presence of serum and without leptin. Pharmacological inhibitors (1 μM PD98059 and 1 μM wortmannin) were added to culture medium 10 min before leptin addition. Cells were lysed and soluble lysates were analysed by immunoblotting using the specific antibody against activated caspase-3. The same lysates were analysed by anti-actin immunoblot to control the amount of protein in the samples. Data shown are representative of three independent experiments. We show representative blots of eight different experiments.

Leptin promotes the cell survival of Jurkat T cells cultured in the absence of serum by means of MAP kinase activation

To identify the pathway whereby leptin prevents apoptosis of Jurkat T cells cultured in the absence of serum, we again employed the pharmacological MEK inhibitor PD980059 and the PI3K inhibitor. First, we studied phosphatidylserine externalization in plasma membranes using annexin V-FITC/PI double staining by flow cytometric analysis. Jurkat T cells were cultured for 10 h in the presence and absence of 10 nM leptin with or without pharmacological inhibitors, which were added to serum-free medium 10 min before the addition of 10 nM leptin. As shown in Fig. 6a, pretreatment of Jurkat T cells with PD980059 but not wortmannin reverted the effect of leptin, preventing annexin-V binding to the cells. Similarly, using the same pharmacological inhibitors, we found that the effect of leptin preventing the activation of caspase-3 was inhibited by blocking the MAPK pathway, whereas inhibition of the PI3K pathway did not inhibit the effect of leptin. The amount of caspase-3 in every sample was controlled by immunoblot using anti-actin antibodies (Fig. 6b).

Discussion

It has been described previously that leptin modulates the T cell immune response and reverses starvation-induced immunosuppression in ob/ob mice [28], and similar results were obtained in ob gene mutations in obese humans [37]. The role of leptin as an immunomodulatory cytokine is now accepted widely [13, 38, 39]. More precisely, leptin activates T lymphocytes in vitro [21] and prevents apoptosis of T lymphocytes, as well as thymocytes in vivo[28, 29, 40]. Recently, leptin has been found to promote survival of T lymphocytes in vitro by suppressing Fas-mediated apoptosis [33]. Nevertheless, the signalling pathways triggered by leptin receptor and their relative relevance mediating the activation and anti-apoptotic effects in lymphocytes are not understood fully.

We have found previously that leptin, by itself, activates human monocytes [41]. On the other hand, leptin needs a co-stimulatory effector in order to activate T lymphocytes from PBMC of donors, as we have shown in previous studies [21]. Other groups have also shown that the leptin effect on T lymphocytes requires a co-stimulator (PHA or concanavalin A) [5, 42]. Similar to the results observed in human T lymphocytes, leptin activation of Jurkat T cells also needs co-stimulation with suboptimal doses of PHA. Thus leptin, along with PHA, increased the expression level of the early activation marker CD69 in Jurkat T cells. However, the dose of PHA used in Jurkat T cell experiments (0·25 μg/ml) was lower than that employed previously in T lymphocytes (4 μg/ml). Nevertheless, in the present study we found a lesser activation effect of leptin in Jurkat T cells compared to that observed previously in T lymphocytes from peripheral blood [21]. These results are consistent with the hypothesis that leptin needs a co-stimulatory agent in order to activate T lymphocytes. The lower PHA concentration required to allow the leptin effect on Jurkat T cells may be explained by the increased basal activation state of this cell line, as assessed by the study of CD69 expression, and the high level of leptin receptor expression. In fact, the PHA dose employed in Jurkat T cells hardly affected the basal expression level of the activation marker.

Similar to other cytokines, leptin receptor can also be up-regulated upon T lymphocyte activation in vitro [24]. Moreover, we have confirmed previously the inmunomodulatory role of leptin in vivo in human immunodeficiency virus-positive (HIV⁺) patients [36], whose circulating lymphocytes had an increased expression level of leptin receptor, correlating with the increased activation level of these cells. Class I cytokine receptors such as Ob-Rb transmit extracellular signals by recruiting SH2 domain-containing proteins to phosphorylated tyrosine residues [43]. Members of the STAT family of transcription factors bind to phosphorylated tyrosine by these SH2 domains. Thus STAT proteins are activated, translocate to the nucleus and stimulate transcription of specific genes [18, 19]. In the present work, we have demonstrated the constitutive association of JAK-2 kinase with leptin receptor in Jurkat T cells, the tyrosine phosphorylation of JAK-2 in response to leptin, as well as the tyrosine phosphorylation of Ob-Rb after leptin stimulation. We have also described tyrosine phosphorylation of Ob-Rb in PBMC in vitro, upon activation with PHA [23]. Similarly, lymphocytes for HIV⁺ patients were also found to have phosphorylated/activated leptin receptors [36].

Leptin stimulation of Jurkat T cells leads to the tyrosine phosphorylation of STAT-3, as observed previously in the PBMC [24]. The leptin receptor can activate STAT-3, STAT-5 and STAT-6 in transfected COS cells [44], whereas STAT-3 and STAT-5 have been shown to be activated in CACO-2 cells of human intestinal epithelium in human hepatic cells WRL68 and in insulinoma rat cells BRIN-BD11 [45–47]. On the other hand, only STAT-3 can be activated by leptin in the hypothalamus [48, 49], in human gastric cancer cells [50] and in the neural cell lines [51]. We have not studied other STAT forms, and therefore the possible role of STATs other than STAT-3 in leptin receptor signalling in Jurkat T cells cannot be ruled out.

The long isoform of the leptin receptor has been shown to activate JAK-2 in human gastric cancer cells [52] in a megakaryoblast cell line (MEG-01) [53] in rat liver [54] in placental cells [55] and human T lymphocytes [21]. We have also found JAK-2 activation in Jurkat T cells in response to leptin. Moreover, we have observed that JAK-2 kinase is associated physically with the leptin receptor in this cell line, and they can be co-immunoprecipitated in both the absence and presence of the ligand. Pre-association of JAK proteins with cytokine receptors has been described for other members of the family [16] and for the leptin receptor itself with JAK-2 [24, 56]. Other JAK isoforms have been found to be activated by leptin receptor, including T cells [24]. Therefore, we cannot exclude the possible participation of other members of the JAK family.

In addition, we have found that leptin stimulates another signalling pathway in Jurkat T cells: the MAPK cascade. In this context, leptin has been found previously to activate ERK-1/2 MAPK in different systems, mediating a proliferative response [6, 7, 57, 58]. Nevertheless, the possible implication of the MAPK pathway in the activation of T cells by leptin on T cells was not demonstrated as a direct link [21, 22]. As Jurkat T cells have the characteristics of a cancer cell line, and ERK-1/2 is one of the better-known signalling pathways transducing growth and proliferation, the involvement of MAPK pathways in the proliferative response to leptin stimulation should be expected. In fact, the role of MAPK in the proliferative effect of leptin has been demonstrated in breast cancer cell lines [59]. In this line, we have found that leptin stimulation of Jurkat T cells produces MEK-1/2 phosphorylation and this phosphorylation is dependent on the dose of leptin. MEK-1 phosphorylation [60] is a necessary intermediate step for the phosphorylation and activation of ERK-1/2. Phosphorylated ERK-1/2 will act on target genes, such as c-fos or egr-1, which then influence the initiation of cellular growth and differentiation [8, 61]. Various in vitro and in vivo studies [62, 63] describe the activation of MAPK pathway after stimulation with leptin of different target tissues, i.e. hypothalamus, liver and adipose tissue [61, 64–66]. Moreover, leptin induces proliferation of the pancreatic beta cell line MIN6 through activation of MAPK [58].

Another important pathway in cell growth and carbohydrates metabolism is PI3K: a cross-talk between leptin and insulin signalling [67, 68]. Leptin regulates insulin sensitivity via phosphatidylinositol-3 kinase signalling in hypothalamic neurones from arcuatus nucleus [69]. In Jurkat T cells, leptin activates AKT by means of a phosphorylation-dependent dose. AKT is a central protein belonging to the PI3K pathway. This kinase is activated with the participation of IRS-1/2. In this manner, JAK-2 is implicated to tyrosine phosphorylate IRS after leptin stimulation in Jurkat T cells [70, 71]. We have demonstrated previously that tyrosine phosphorylated Sam68 and IRS-1 proteins were asociated with p85 [40] to recruit PI3K activity in human PBMC. The association of phosphorylated IRS-1 activates PI3K [72].

Another possibility of transducing the AKT signal to the cellular nucleus is PDE3B phosphorylation, and its translocation to the nucleus where it interacts with STAT-3. In the hypothalamus there is an interaction between two pathways of leptin: PI3K/PDE3B/AMPc and JAK-2/STAT-3 [73]. As we have described previously, in this lymphoblastic cell line STAT-3 is phosphorylated in response to leptin stimulation. Thus, we cannot rule out the involvement of PI3K/PDE3B/AMPc in the signalling of the leptin receptor in Jurkat T cells.

In order to investigate the mechanism that produces the expression of early activation marker CD69 on Jurkat T cells we have designed several pharmacological inhibition experiments. Jurkat T cells express constitutively low levels of CD69 and the expression of this early activation marker is up-regulated by 10 nM leptin. By using PD98059 a specific inhibitor of MEK, and therefore a inhibitor of the MAPK pathway, we have found that the stimulatory effect of leptin activating Jurkat T cells was abrogated completely, demonstrating that the MAPK pathway is necessary to accomplish this effect. On the other hand, inhibition of the PI3K pathway either with wortmannin or LY294002 did not prevent the leptin effect on CD69 expression by Jurkat T cells, suggesting that even though leptin activates the PI3K pathway in these cells, this signalling pathway is not necessary to produce early expression of the activation marker CD69. Similarly, we have found previously that the anti-apoptotic effect of leptin on human monocytes is dependent on the MAPK but not the PI3K pathway [34], whereas the proliferative effect of leptin in prostate cancer may be mediated by MAPK or PI3K, depending on the cell type [74].

Apoptosis is a process of cellular death which has an important role in T cell biology. The non-functional thymocytes or autoreactive T cell receptors (TCRs) are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. Dysregulation of apoptosis in the immune system results in autoimmunity, tumorigenesis and immune deficiency. Two major pathways lead to apoptosis: the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signalling. These two pathways work together to regulate T lymphocyte development and function [75]. Some studies have proposed a role of leptin in the control of apoptosis in cells from the immune system [34, 35], as well as other cell types [76, 77]. Moreover, leptin has been found to inhibit stress-induced apoptosis of T lymphocytes in vivo [39]. In this context, we wanted to test the possible effect of leptin on Jurkat T cells survival and whether this effect was based on the anti-apoptotic action of leptin, when Jurkat T cells are cultured in the absence of serum. Data presented here demonstrate clearly that leptin maintains the number of T cells after 10 h of serum-free culture, allowing the survival of about 40% of the total number of apoptotic Jurkat T cells induced by serum withdrawal. These data support the hypothesis that considers leptin as a trophic factor for Jurkat T cell survival. This hypothesis has been demonstrated by other groups. Magarinos et al. [78] have described that leptin is a trophic and mitogenic factor for trophoblastic cells by inhibiting apoptosis and promoting proliferation. Thus, leptin promotes cell proliferation and survival of trophoblastic cells [78]. In addition, leptin acts as a mitogenic and anti-apoptotic factor for colonic cancer cells, increasing cell numbers in colonic cancer cell lines in a dose-dependent manner, and reducing the number of apoptotic cells in a cell line-dependent manner [79]. Moreover, pretreatment with leptin prevents gut mucosal damage and improves intestinal rehabilitation following intestinal ischaemia–reperfusion injury in the rat. In these animals, a significantly lower intestinal injury score was registered as well as a lower apoptotic index and higher cell proliferation index in jejunum and ileum compared to the animals without treatment [80]. On the other hand, the apoptotic effect of leptin on adipose tissue has been described, which can contribute to the decrease of adiposity after either central nervous system or peripheral administration [81]. Injections of leptin into rat ventromedial hypothalamus decreases the adipocytepopulation in bone marrow, primarily through apoptosis of marrow adipocytes [82]. Indeed, leptin does not act directly to induce adipocyte apoptosis, but can act directly to inhibit maturation of pre-adipocytes [81].

In the present work we have investigated whether the leptin stimulation of Jurkat T cells would produce a decrease of any marker of the apoptotic process. Regardless of the signalling pathways that lead to apoptosis (intrinsic or extrinsic), both of them finalize in the proteolytic cleavage of pro-caspase 3 to yield the active form, caspase-3. Thus, we measured the activation level of caspase-3 in response to leptin stimulation of Jurkat T cells. We found that leptin hinders the activation of caspase-3 induced by the culture of Jurkat T cells in the absence of serum. Moreover, we have found that this impairment of the apoptotic process is dependent on the leptin dose. Similar results regarding the anti-apoptotic effect and dose-dependence of leptin have been reported in different studies. In neutrophils, it has been shown that leptin inhibits the cleavage of Bid and Bax, leading to prevention of the mitochondrial release of cytochrome c and the subsequent release of mitochondria-derived activator of caspase, therefore impairing the activation of both caspase-8 and caspase-3 [83, 84]. The anti-apoptotic effect of leptin requires the long form of the leptin receptor in hepatic stellate cells and interacts with the apoptotic pathway proximal to mitochondrial activation. Leptin protects hepatic stellate cells from in vitro and in vivo apoptosis [77]. Moreover, leptin reduces apoptosis in β cells (BRIN-BD11 cells) at physiological concentrations, possibly via its ability to up-regulate Bcl-2 and Bax expression [85].

In order to investigate the mechanism of trophic effect of leptin in Jurkat T cells, we designed several experiments of pharmacological inhibition. By using PD98059, a specific inhibitor of MEK, and therefore an inhibitor of the MAPK pathway, we have found that the trophic effect of leptin in Jurkat T cells was abrogated completely, demonstrating that the MAPK pathway is necessary to accomplish this effect. On the other hand, inhibition of the PI3K pathway with wortmannin did not prevent the leptin effect on cell death in Jurkat T cells, suggesting that this signalling pathway is not necessary to decrease the apoptosis of Jurkat T cells, even though in these cells leptin activates the PI3K pathway. Similarly, we have found previously that the anti-apoptotic effect of leptin on human monocytes is dependent on the MAPK, but not the PI3K, pathway [34], whereas the proliferative effect of leptin in prostate cancer may be mediated by MAPK or PI3K, depending on the cell type [74]. In addition, pretreatment with inhibitors of MAPK and PI3K inhibited ERK-1/2 and AKT phosphorylation caused by leptin, attenuated the mitogenic action of leptin and abolished its anti-apoptotic effects in colonic cancer cells [75]. Our results are coincident with previous evidence that MEK may regulate the caspase-3-dependent component of diethylmaleate (DEM)-induced apoptosis in Jurkat T cells. The MEK pathway has been implicated previously in protection from various apoptotic signals [86, 87]. Regulation of apoptosis by MEK-dependent signalling is not restricted to the Jurkat cell line. The d11S T cell hybridoma has relatively high levels of endogenous ERK-1/2 and is resistant to DEM-induced apoptosis. In these cells, inhibition of ERK by a dominant negative MEK construct or pharmacological inhibition revealed a DEM-induced caspase-3-dependent apoptotic pathway. These data suggest that MEK regulation of this pathway at the level of caspase activation may be a more general event, thus sustained activation of ERK abrogates the activation of caspase-3 [86].

In summary, we have found that the long isoform of leptin receptor is present in Jurkat T cells and leptin stimulation triggers the activation of different signalling pathways (JAK-STAT, PI3K and MAPK), and activation of the MAPK pathway is necessary to produce the increase in expression of the activation marker CD69. Moreover, we have shown that leptin promotes the survival of Jurkat T cells by activating the pathways that control growth and cellular proliferation and prevents triggering of the apoptotic process.