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Departments of
* Immunology and
Endocrinology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom; and
Cattedra di Immunologia, Dipartimento di Biologia e Patologia Cellualare e Molecolare, Universita di Napoli "Federico II", Napoli, Italy
Correspondence: Prof. R. I. Lechler, Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, W12 0NN, U.K. E-mail: r.lechler{at}ic.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was substantially increased. These data show that leptin can inhibit certain immune responses in vitro. However, despite this inhibition of proliferation, the production of proinflammatory cytokines is significantly enhanced by leptin. The findings demonstrated here show further complexity in the actions of leptin on the immune system.
Key Words: interferon- • immunity • ObRb • immunoglobulin • T cell receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vivo, leptin skews immune responses toward the T helper cell type 1 (Th1) phenotype and suppresses Th2 responses [16 , 17 ]. In the absence of leptin, mice are totally resistant to T cell-mediated diseases such as experimental autoimmune encephalomyelitis [20 , 21 ] and autoimmune hepatitis [22 ]. This resistance is reversed by administration of exogenous, recombinant leptin [20 , 22 ].
However, there are situations where leptin has been shown to inhibit the proliferation of lymphocytes. In particular, when leptin has been administered intracerebroventricularly (icv), subsequent polyclonal stimulation of splenocytes has been inhibited [23 ]. In these experiments, cytokine production from these stimulated splenocytes was not assessed, so no comments can be made about the polarity of the immune response.
To address further the effects of leptin on T cell immune responses, the system chosen was that of antigen-independent, antibody-mediated T cell receptor (TCR) stimulation. In this system, T cell stimulation is achieved by using monoclonal antibodies (mAb) that bind to the CD3 signaling complex of the TCR and deliver an activating signal. Only T cells express the CD3 molecule, thus allowing cell type specificity in the assays. CD3 is stably associated with the TCR 
ß heterodimer at the surface of T cells and consists of immunoglobulin (Ig)-like domains CD3, 
, and 
along with the dimeric CD3
. Given that the TCR has a very short, intracytoplasmic tail, the CD3 complex performs signal transduction. Thus, antibody binding to CD3 mimics antigen binding to the TCR and delivers a robust signal to large numbers of polyclonal T cells, inducing them to proliferate and release IL-2 and other cytokines.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All cell culture and assays were performed in RPMI 1640 (Life Technologies Ltd., Paisley, Scotland) supplemented with either 10% fetal calf serum (FCS), 10% human serum, or 2/10% autologous serum and 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin (Gibco-BRL, London). Murine cultures were further supplemented with 0.5 mM ß-mercaptoethanol.
The following mAb were used for purifying the cells for the proliferation and cytokine assays: L243 [anti-human leukocyte antigen-DR; American Type Culture Collection (ATCC), Manassas, VA], Leu-19 (anti-CD56; Becton Dickinson, San Jose, CA), UCHT4 (anti-CD8; ATCC), BU12 (anti-CD19; ATCC), UCHM1 (anti-CD14; ATCC), UCHM1 (anti-CD14; ATCC), Leu-11b (anti-CD16; ATCC), Leu-M9 (anti-CD33; ATCC), SN130 (anti-CD45RA; gift from G. Janossy, Royal Free Hospital, London), and UCHL1 (anti-CD45RO; gift from P. Beverley, Imperial Cancer Research Fund). OKT 3 (mouse anti-human CD3), 2C11 (rat anti-mouse CD3; both from ATCC), and anti-CD28 (CD28.2; gift from Prof. Daniel Olive, INSERM, Marseille, France) were used for proliferation assays and were purified by affinity-column chromatography and quantified by spectrophotometric analysis. The following directly conjugated mAb were used for flow cytometric analysis: anti-DR, anti-CD3, anti-CD4, anti CD8, anti-CD11b, and anti-CD71 (Sigma Chemical Co., Dorset, UK); anti-CD2 and anti-CD62L (Pharmingen, San Diego, CA); and anti-CD49a-f, anti-CD50, anti-CD54, anti-CD58, anti-CD45RA, and anti-CD45RO (Serotec, Oxford, UK). These antibodies were usually used after conjugation with fluorescein isothiocyanate or phycoerythrin.
The degree of T cell apoptosis was assessed by flow cytometry after staining CD4+ cells with Annexin V (Pharmingen).
Cell separation
Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation using Ficoll-Hypaque (Pharmacia, Sweden). Briefly, whole blood was diluted 1:1 with an equivalent volume of phosphate-buffered saline (PBS) and layered over Ficoll in a 2:1 ratio by volume. Tubes were then centrifuged at 2000 rpm for 20 min at 4°C with minimal centrifuge braking. PBMC were then harvested by pipetting cells from the Ficoll/serum interface, resuspended in RPMI medium (Life Technologies Ltd.), and washed twice at 1800 rpm for 5 min. RPMI was supplemented with 10% heat-inactivated FCS (Globepharm, UK) or human AB serum (North London Blood Transfusion Service, Colindale, UK), 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 IU/ml penicillin, and 50 µg/ml streptomycin. For the experiments using umbilical cord blood (UCB) cells, 2–3 ml blood was taken from the umbilical vein attached to the placenta following the spontaneous, vaginal delivery of a healthy, full-term infant and was prepared as described above. The cells were used without further manipulation or after the purification of various subsets as described below.
Purification of CD4+CD45RA+ and CD4+CD45RO+ T cells
CD4+ T cells were obtained by immunomagnetic negative selection and divided into two equal aliquots. CD4+CD45RA+ T cells were purified by incubating one of the above aliquots with a saturating concentration of anti-CD45RO mAb (UCHL1) for 30 min at 4°C. The cells were then washed at least three times in PBS or RPMI to remove the unbound antibody and then incubated with sheep anti-mouse IgG-coated Dynabeads (Dynal, UK) at a 4:1 ratio of magnetic beads-to-cells for 30 min at 4°C. The bead/mAb-coated cells were then removed by passage through a magnetic column. A second round of immunomagnetic negative selection was performed for 30 min at 4°C at a 2:1 ratio of magnetic beads-to-cells. CD4+CD45RO+ T cells were purified by incubating the other aliquot of CD4+ T cells with anti-CD45RA mAb (SN130) for 30 min at 4°C and performing the same immunomagnetic bead depletion as described above. The purity of the separated cells was assessed by flow cytometry and was usually >95%.
Depletion of T cell subpopulations from responding T cells
For the depletion experiments, PBMC were taken after two rounds of 45 min of adherence on plastic and were subjected to two further rounds of immunomagnetic negative selection as described above, after incubation with saturating concentrations of anti-CD45RA antibody (SN130) or anti-CD45RO (UCHL1) antibody for 30 min at 4°C. After depletion, the purity was assessed by two-color flow cytometry, and the contamination by each single cell population was <5%.
Preparation of murine splenocytes
Spleens were removed from euthanized mice, and a single cell suspension of splenocytes was prepared using the blunt end of a syringe and passage through nylon gauze. Red blood cells were lysed by resuspending the splenocyte suspension in ACK lysing buffer [0.15 M NH4 Cl, 1 mM KHCO3, 0.1 mM Na2 ethylenediaminetetraacetate (EDTA), pH 7.2–7.4] for 5–10 min and then washing twice in PBS. If red cells were still present macroscopically, this procedure was repeated.
Antibody stimulation assays
T cells were stimulated in a noncognate manner via the binding of mAb to CD3 in the presence of cellular transcostimulation or by anti-CD3 and anti-CD28 antibodies in its absence. In the case of purified T cell populations, costimulation was provided by anti-CD28 antibody, whereas in the other cases, anti-CD3 was used alone with costimulation being provided by the antigen-presenting cells (APC) present in the assay. Cells were prepared as appropriate and placed into flat-bottomed, 96-well plates (Nunc, Roskilde, Denmark) in a final volume of 200 µl at a concentration of 2 x 105 cells/well. Soluble anti-CD3 mAb (OKT3 for the human assays and 2C11 for the murine assays) was added to the wells at various concentrations in the presence or absence of recombinant leptin. For other experiments, a fixed dose of anti-CD3 was used, which gave 50% maximal proliferative stimulation, and various doses of leptin were added. For experiments with the purified T cell subpopulations, the 96-well plates were precoated with anti-mouse Fc antibody. The cell populations were then preincubated with anti-CD3 and anti-CD28 antibody for 30 min at 4°C before being washed three times to remove any unbound antibody. These cells were then added to the precoated wells at a concentration of 2 x 104 cells/well in the presence or absence of leptin.
All assays were incubated for 72 h at 37°C and then pulsed with 0.5 µCi 3H-thymidine (TdR) for an additional 6–18 h and harvested onto glass fibre filters. Proliferation was measured as 3H-TdR incorporation by liquid scintillation spectroscopy. Standard errors were routinely <10%.
RNA extraction and reverse transcription
Total cellular RNA was extracted using RNAzol reagent (AMS Biotechnology, Abingdon, Oxon, UK) according to the manufacturer’s instructions. This method uses a single step to remove protein and DNA from the RNA based on the guanidiumthiocyanate-phenol-choroform-extraction procedure. The integrity of the extracted RNA was checked by electrophoresis on a denaturing 3-(n-morpholino) propane sulphonic acid-formaldehyde gel containing 1% agarose. First strand cDNA was synthesized from 5 µg total RNA using 10 units avian myloblastosis virus (AMV) reverse transcriptase (RT; Promega, Madison, WI) and 200 ng (deoxythymidine)12–18 "oligo DT" primer (Pharmacia). The RNA and primer were heated to 65°C for 5 min in a buffer consisting of 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM MgCl2, 0.5 mM spermine, 10 mM dithiothreitol, 1 mM dNTPs (Pharmacia), after which the solution was allowed to cool to room temperature for 30 min. After annealing, 10 units AMV RT were added, and the reaction was incubated at 42°C for 1 h. Following the incubation, the reaction was stored at -20°C.
The CD45RO+ and CD45RA+ T cells used for RNA extraction in this experiment were extensively purified and were >99.5% pure on flow cytometric staining. The remaining 0.5% was negative for B cell, monocyte, natural killer, and CD8 surface markers.
Polymerase chain reaction (PCR)
Primer selection and PCR amplification were performed as described [24
] to generate a product specific for the ObRb (nucleotide positions 2831–3719). Briefly, the first round primers were: 5'-AAGATGTTCCGAACCCCAAG-3' (forward) and 5'-CAAATTTGGACTCTGGTTTCT-3' (reverse). The second round primers were: 5'-AATTGTTCCTGGGCACAAGG-3' (forward) and 5'-CACAAATCTGAAGGTTTCTTC-3' (reverse). Half of the reverse-transcribed RNA was added to a tube containing 1x Taq buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100), 0.2 mM dNTPs, and 25 pM each primer. This was overlaid with mineral oil, and the reaction was heated to 95°C for 5 min, after which 10 U Taq DNA polymerase was added. First round amplification was performed during 35 cycles of PCR: 95°C 30 s (denaturing), 56°C 60 s (annealing), 72°C 60 s (extension), with an additional extension step, 72°C 5 min. Second round PCR was set up identically to that above, except for 1µl of the product generated from the first round PCR reaction was used as template, and annealing was carried out at 50°C for 45 s. After completion of the reaction, products were size-separated by electrophoresis on 1x TAE (40 mM Tris-acetic acid and 1 mM EDTA, pH 8.0)-1% agarose gel containing 2 µg/ml ethidium bromide to confirm the presence of the expected 888 bp PCR product present if ObRb mRNA was in the starting sample. To control for cDNA integrity, PCR for human ß actin was performed. The reaction was set up as described above using 35 cycles of PCR: 95°C 30 s, 60°C 60 s, 72°C 60 s, with an additional extension step, 72°C 5 min. The primers used were 5'-GTGGGGCGCCCCAGGCACCA-3' (forward) and 5'-GAAATCGTGCGTGACATTAAGGAG-3' (reverse), which generated a 540 bp product.
Enzyme-linked immunosorbent assays (ELISAs)
Murine and human samples were assayed with the appropriate ELISA kits (R&D Systems) and were used according to the manufacturer’s instructions. For the human in vitro assays, supernatants of the culture medium were taken during the course of a cellular assay. The precise timing of the sample (usually 50 µl from a total volume of 200 µl in a 96-well plate) was determined from kinetic experiments where the peak of the cytokine response had been determined. IL-2 was assayed at 18 h, and IL-4 and interferon- (IFN-) were assayed at 24–36 h. All samples were assayed in duplicate, and the discrepancy between samples was routinely <10%. Actual cytokine concentrations were determined by reference to a standard curve generated using highly purified recombinant cytokine at various concentrations performed contemporaneously with each assay. The standard curve also served as an internal control over the sensitivity and range of each assay.
Statistics
Statistical analyses were performed using unpaired Student’s t-test with Statview software. P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15 ng/ml. It is interesting that leptin had a marked effect on T cell function in this assay by inducing a dose-dependent inhibition of proliferation with an ID50 of 10 nM (Fig. 1a
). All data are representative of at least three independent experiments. Proliferation was assessed at 24, 48, and 72 h by TdR incorporation, and the results of this experiment are shown in Figure 1b
. This illustrates that the inhibition of the OKT3 response of unseparated PBMC induced by leptin occurred at all time points and was maximal at 72 h.
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Leptin differentially affects memory and naïve T cell responses
Naïve and memory T cells differ significantly in their activation requirements and their kinetics of activation. Memory cells tend to reach their peak of proliferation in vitro significantly earlier than naïve T cells. Naïve T cells can be distinguished by their expression of the RA isoform of CD45; memory cells express the RO isoform. The first system used to examine whether leptin had differential effects on naïve and memory cells was analysis of the responses of peripheral blood lymphocytes (PBL) after depletion of CD45RA+ cells or CD45RO+ cells. CD45RA+ or CD45RO+ cells were depleted from this population by incubation with anti-CD45RO or anti-CD45RA antibody at 4C, respectively, followed by two roundsof immunomagnetic negative selection. There was routinely <2–3% contamination of each population with the depleted cells. These cells were then used as responders in a dose-response OKT3 assay with a fixed dose of leptin (10 nM). Interestingly, depletion of CD45RO+ cells reversed the effect of leptin from inhibition to stimulation of proliferation (Fig. 2a
). Conversely, the proliferation of the population depleted of CD45RA+ cells was significantly inhibited by leptin (Fig. 2b)
. This would indicate that the proliferation of naïve (CD45RA+) T cells is enhanced by leptin, whereas the proliferation of memory (CD45RO+) T cells is inhibited by leptin. This seems to demonstrate differential activation requirements of these two populations of cells with respect to leptin signaling.
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Anti-CD3 responses of highly purified CD4+CD45RO+ and CD4+CD45RA+ T cells
For confirmation that the target cell of leptin action was mainly the CD4+CD45RA+ and CD4+CD45RO+ T cells, experiments were performed with highly purified T cell subsets being stimulated by mAb with no accessory cells present. The observation that the proliferation of memory T cells was inhibited by leptin was confirmed by the use of highly purified CD4+CD45RO+ T cells (Fig. 2d)
. In the presence of leptin, the proliferation of these cells was significantly inhibited, suggesting that leptin was acting directly on the memory T cells, as there is no other cell population present. Figure 3
shows the results of an experiment with naïve CD4+ T cells stimulated with increasing doses of OKT3, which had been additionally preincubated with anti-CD28 or medium and then had medium or soluble leptin added to the assay. Leptin induced a marked increase in proliferation of naïve T cells when they are simultaneously stimulated with OKT3 and anti-CD28. The reason that the purified, naïve T cells proliferated poorly compared with UCB cells may have been because costimulatory requirements in the former situation had not been optimized adequately.
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production from human PBMC after anti-CD3 stimulation. When the experiment was performed in the presence of 10 nM leptin, IL-2 production was suppressed, analogous to the inhibition of proliferation. Surprisingly, despite the inhibition of proliferation, leptin induced a marked increase in IFN- production. This pattern of cytokine production was observed from the PBMC of most donors, and their relative ratio of CD4+CD45RA+ to CD4+CD45RO+ is shown in Figure 4b
. As can be seen, this ratio was approximately 1:2 with a preponderance of memory cells. It proved impossible to detect any IL-4 in the culture medium at any time point tested during OKT3 stimulation, in the presence or absence of leptin.
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10% of people tested) showed minimal inhibition of proliferation induced by exogenous leptin. When the cytokine profile of these cells was examined, it revealed that despite lack of proliferative inhibition induced by leptin, production of IL-2 was markedly suppressed by leptin, and IFN- production was enhanced (Fig. 4c)
. Analysis of the relative proportion of naïve-to-memory CD4+ T cells demonstrated a preponderance of naïve cells by a ratio of 2:1 (Fig. 4d) . This was an inversion of the ratio seen in most PBMC samples and may help to explain the relative lack of proliferative changes induced by leptin in this situation.
Cytokine profiles of separated CD45RA+ and CD45RO+ T cells
The production of IL-2 and IFN- was assessed in PBL depleted of CD45RA+ or CD45RO+ cells. Figure 5a
shows that in a responding population containing only naïve T cells (i.e., depleted of CD45RO+ T cells), leptin induced a marked increase in IL-2 production, which parallels the increase in proliferation observed in this population. As would be predicted, in the absence of memory T cells, minimal IFN- was produced in the presence or absence of leptin. When the responding population was depleted of naïve T cells, leptin induced marked suppression of IL-2 production and an increase in IFN- production (Fig. 5b)
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, as they do not contain differentiated Th1-type cells. The presence of leptin was unable to induce any significant production of IFN- (Fig. 5c)
. However, the production of IL-2 was significantly increased, confirming that in this system, leptin selectively enhances naïve CD4+ T cell proliferation and markedly increases the production of IFN- from memory cells despite inhibiting their proliferation.
Leptin receptor expression
The leptin receptor (ObRb) is expressed in naïve and memory T cells
It was important to confirm that both subpopulations of CD4+ T cells expressed the ObRb. mRNA was extracted from highly purified populations of CD4+CD45RA+ and CD4+CD45RO+ cells separated from the PBL of two independent donors. Figure 6
shows the results of the RT-PCR from these two donors. The molecular weight marker is shown on the left-hand side of the figure followed by RT-PCR performed with water instead of mRNA (lane 1). Lanes 2 and 5 represent PBMC mRNA from each donor; lanes 3 and 6 represent resting CD4+CD45RA+ T cells from each donor; and lanes 4 and 7 represent resting CD4+CD45RO+ T cells from each donor. Lane 8 is a further negative control (PBMC mRNA with no RT step). The band of 888 bp shown on the gel indicates that the ObRb transcript was expressed by all of these populations of T cells, which is consistent with the functional data discussed above.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was markedly enhanced. The inhibition of proliferation in the unseparated cells was reversed by the depletion of memory T cells from the responding population. Furthermore, using UCB cells as responders, leptin induced a dose-dependent increase in proliferation and IL-2 production. Experiments with highly purified memory and naïve T cells confirmed the inhibition of proliferation of the CD4+CD45RO+ population and the augmentation of CD4+CD45RA+ proliferation. The degree of inhibition of the anti-TCR-stimulated proliferation was related to the proportion of naïve-to-memory CD4+ T cells in the unseparated, responding population. Th1-type cytokine production from memory T cells was markedly enhanced, as evidenced by the production of IFN- in the presence of leptin. In vivo, leptin stimulates T cell and proinflammatory immune responses [16
17
18
19
], unless it is given icv, when it seems to cause inhibition [23
]. This may be explained by the fact that icv administration induces activation of the corticosteroid and sympathetic axes, which would act to suppress subsequent T cell responses.
These data indicate that leptin has its effect on T cells via ObRb. Whether the observed effects are direct or operate via an indirect mechanism through accessory cells is unclear. However, the data with the purified T cells stimulated in the absence of APC would suggest that it is a direct effect of leptin. The action of leptin in stimulating IL-2 production and proliferation of naïve T cells and the up-regulation of IFN- secretion by memory T cells may well be related to its activation of T cell signaling intermediates, such as various molecules of the JAK/STAT system. Additionally, leptin may induce or augment IL-2 gene transcription in naïve T cells. The marked increase in IFN- induced in the memory population may explain the inhibition seen in this population and in unseparated populations of cells that contain a significant proportion of memory T cells. This was considered possible because it is known that high levels of IFN- can inhibit T cell proliferation [25
, 26
]. However, the proliferation of PBMC was not inhibited by the addition of recombinant IFN- to the cultures at similar doses to those measured by ELISA (data not shown). This does not completely rule out the possibility that the inhibition was a result of high levels of IFN-, as exogenously added recombinant cytokines do not always reproduce the effects of endogenously produced cytokines. This is being investigated with the use of neutralizing anti-IFN- antibodies. Alternatively, the mechanism for inhibition of IL-2 secretion and proliferation at the same time as a marked increase in IFN- secretion may be mediated via the novel T cell transcription factor, T-bet, as T-bet is known to transactivate the IFN- gene and repress IL-2 gene transcription [27
]. If leptin activates signaling molecules upstream of T-bet, then this would explain the observations presented here, and this possibility is the subject of further studies. It is unclear why leptin inhibits T cell proliferation at higher concentrations. Perhaps it is because of supramaximal stimulation of T cells causing inhibition of proliferation. Alternatively, leptin may induce the production of suppressors of cytokine signaling at higher doses of leptin in T cells as it does in other cells [28
].
Naïve and memory T cells clearly have different activation requirements [29 ]. We have shown that ObRb is expressed in both subsets of T cells, but we are not able to comment about the levels or kinetics of expression and whether these differ between naïve and memory T cells and may underlie some of the differential effects of leptin on naïve and memory T cells. There is evidence that receptor signaling can have different effects on different subsets of T cells [30 31 32 ], and it is becoming apparent that leptin can signal via different pathways such as phosphatidylinositol 3 kinase and mitogen-activated protein kinase [33 ]. Leptin may transduce different signals in memory and naïve T cells, which could be responsible for the effects observed here.
Initially, leptin was implicated in the regulation of bodyweight and reproductive function [34 ]. However, reports of its effect on the pancreas and haemopoiesis, together with the detection of peripherally distributed receptors, suggest a wider role [35 ]. The data presented here suggest that leptin is able to modulate specific aspects of T cell function, including proliferation and cytokine production, with differential effects on naive and memory T cells.
Other groups have been able to demonstrate effects of leptin on activated macrophages and potentiation of proinflammatory immune responses [18 ,19 ]. Furthermore, examination of an extended pedigree of a highly consanguineous Turkish family with a missense leptin mutation revealed that more than 60% of the homozygotes died from infectious disease during childhood [15 ]. This would indicate that leptin does indeed play an important role in the normal homeostasis of immune responses.
That leptin can modulate immune responses in vitro and in vivo now seems clear [36 , 37 ]. Its role would appear to be as a regulator of the bioenergetic aspects of the immune response, such that in the relative absence of leptin, immune responses are suboptimal. Immune responses are very energy-expensive [38 ], and we propose that leptin serves as the link to modulate immune responses in times of energy or substrate deficiency [39 ].
In conclusion, we have shown that leptin is able to modulate certain aspects of T cell function in vitro, inhibiting the anti-CD3-driven proliferation of unseparated lymphocytes and purified memory T cells but enhancing their production of proinflammatory cytokines. These data provide further insight into the complexity of immune homeostasis and activation influenced by leptin, which may prove to be a useful axis for the manipulation of immune responses.
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ACKNOWLEDGEMENTS |
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Received May 21, 2001; revised December 17, 2001; accepted March 4, 2002.
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