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Original Studies |
Department of Internal Medicine, Division of Rheumatology (C.G., N.P.), Division of Endocrinology and Diabetes (M.G.D., C.A.M.), and Division of Immunology and Allergy (R.C.), University Hospital Geneva, CH-1211 Geneva, Switzerland
Address correspondence and requests for reprints to: Dr. Christoph A. Meier, Division of Endocrinology and Diabetes, University Hospital Geneva, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. E-mail: cameier{at}bluewin.ch
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-induced shock, which is associated with lower
levels of interleukin 1 receptor antagonist (IL-1Ra), we investigated
whether leptin is a direct regulator of IL-1Ra in human monocytes. In
human moncytic cells, leptin was capable of inducing a 6- to 10-fold
increase in secreted IL-1Ra in a time- and dose-dependent manner.
Moreover, leptin induced the messenger RNA for IL-1Ra within 8 h
and specifically activated the promoter for this gene. However, leptin
had no effect on the expression or secretion of IL-1 in THP-1 cells.
This effect of leptin on monocytic cells requires the presence of the
functional leptin receptor OB-Rb, which we have shown to be present in
human monocytes by RT-PCR and by measuring the activation of the
Jak/STAT pathway. In summary, we have demonstrated that leptin is capable of inducing the expression and secretion of IL-1Ra by human monocytes, an effect that is potentially mediated through the presence of functional leptin receptors on these cells. These findings suggest that leptin may have immunomodulatory functions in vivo.
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Introduction |
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, interleukin
(IL)-6 and IL-12 by peritoneal macrophages, as well as of interferon
(IFN)-
by T cells, whereas it inhibits the production of Th2
cytokine by the latter cells (7, 8).
Leptin-deficient (ob/ob) mice have recently been shown to
exhibit an increased susceptibility to lipopolysaccharide (LPS)- and
TNF--induced mortality, with a protective effect of exogenously
administrated recombinant leptin (9, 10). Measurement of
circulating levels of cytokines following LPS injection revealed that
ob/ob mice had significantly lower concentrations of IL-1
receptor antagonist (IL-1Ra) compared with their lean littermates
(ob/+), whereas the levels of other cytokines, including
IL-1ß, IFN-, macrophage inflammatory protein-1, and TNF-
were not significantly different in ob/ob and
ob/+ mice (9). These results suggested that the
relative deficiency of IL-1Ra may contribute to the enhanced mortality
in ob/ob mice following treatment with LPS. In
vitro, leptin enhanced the effect of LPS on the production of
different cytokines by murine peritoneal macrophages, including IL-1Ra,
IL-6, IL-12, and TNF- (8). In addition, leptin alone
was demonstrated to induce a 1.4-fold increase in IL-1Ra secretion by
the murine macrophage cell line RAW 264.7, suggesting that leptin has a
direct effect on the regulation of this cytokine antagonist
(9). Similarly, leptin has recently been shown to activate
human monocytes in vitro by inducing the expression of
cell-surface markers and of cytokines, including IL-6 and TNF-
(11).
IL-1Ra is a member of the IL-1 family that binds to IL-1 receptors but
fails to induce any cellular response and, hence, inhibits the effects
of IL-1 on its target cells (12). Administration of
exogenous IL-1Ra has been shown to exert anti-inflammatory effects
in vivo in several experimental disease models, including
septic shock, arthritis, and colitis (13). Recent studies
have demonstrated that the balance between agonists (IL-1 and
IL-1ß) and an antagonist (IL-1Ra) of the IL-1 receptor may play an
important role in the regulation of inflammatory responses, as
evidenced by the enhanced sensitivity of IL-1Ra knockout mice to septic
shock and their predisposition to the spontaneous development of
inflammatory disorders (14, 15, 16). Similarly, an imbalance
between IL-1ß and IL-1Ra is present in the rheumatoid synovium and in
inflammatory lesions of Crohn’s disease (17, 18). Hence,
understanding the factors selectively inducing IL-1Ra secretion and
altering the balance of IL-1 to IL-1Ra is of interest. However,
although IL-1Ra has been suggested to be weakly induced by leptin in
murine macrophages, this has not been examined in human monocytic cells
and no data regarding the specificity or mechanism of this observation
are available. Therefore, we have now examined the effect of leptin on
the production of IL-1Ra and IL-1ß in the human monocytic cell line
THP-1 and in peripheral blood mononuclear cells (PBMCs). In
addition, we investigated whether THP-1 cells express functional
OB-Rb.
Our results demonstrate that leptin has a direct effect on the production of IL-1Ra by THP-1 cells and PBMCs and that THP-1 cells express functional OB-Rb with activation of the signal transducer and activator of transcription (STAT) intracellular signaling pathway in response to leptin. Moreover, leptin is able to activate transcription from the IL-1Ra promoter in an OB-Rb-dependent manner.
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Materials and Methods |
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LPS (0111:B4) and polymyxin B sulfate were obtained from Sigma (St. Louis, MO), recombinant murine leptin from Pepro Tech (Rocky Hill, SC). RPMI 1640 and L-glutamine, as well as penicillin-streptomycin were obtained from Life Technologies, Inc. (Paisley, UK). Primers were synthesized by Microsynth (Balgach, Switzerland). EDTA and EGTA were obtained from Fluka (Buchs, Switzerland). Complete, a protease inhibitor mixture, was purchased from Boehringer Mannhein (Mannheim, Germany).
Cell culture
The THP-1 cell line was obtained from Dr. J.-M. Dayer (Division of Immunology and Allergy, University Hospital Geneva). The PBMCs were isolated by a Ficoll density gradient from blood samples obtained from healthy volunteers of the University Hospital of Geneva Transfusion Center. The cells were maintained in RPMI 1640 supplemented with 10% heat-inactived FBS, 50 IU/mL penicillin-streptomycin, and 2 mM L-glutamine in a 5% CO2-air humidified atmosphere at 37 C. THP-1 cells were stimulated at a density of 4 x 105 cells/mL with different concentrations of leptin or 100 ng/mL LPS during 1–72 h. Total RNA was prepared using Trizol (Life Technologies, Inc.), and culture supernatants were stored at -80 C for further analysis.
Measurement of cytokines
IL-1Ra concentrations were measured in the supernatants from THP-1 cells and human PBMCs using a sandwich enzyme-linked immunosorbent assay (ELISA), as described previously (19). The levels of IL-1ß were determined by the EIA IL-1ß ELISA kit (Immunotech, Marseille, France). The sensitivity of the immunoassays for IL-1Ra and IL-1ß was 80 pg/mL and 10 pg/mL, respectively.
RNase protection assay
THP-1 cells and human PBMCs were cultured in the presence or absence of leptin or LPS for 1, 6, 19, and 48 h. Total RNA was extracted at different time points, quantitated spectrophotometrically, and its integrity verified by agarose gel electrophoresis. RNase protection assays were performed using 10 µg or 2 µg total RNA from THP-1 cells or PBMCs, respectively. A specific probe for the different isoforms derived from the human IL-1Ra gene was generated by PCR and subcloned, as described previously (19). This probe specifically recognizes the different IL-1Ra isoforms as protected fragments of distinct sizes after treatment with RNases. The plasmids containing the probes for IL-1Ra and glyceraldehyde 3-phosphate deshydrogenase (GAPDH) messenger RNA (mRNA) were linearized with EcoRI and transcribed with T7 RNA polymerase and 32P-labeled UTP. Total RNA from THP-1 cells and PBMCs was hybridized simultaneously with antisense 32P-labeled riboprobes for IL-1Ra and GAPDH, and the RNase protection assay was performed as described (19). The protected fragments were analyzed on denaturing 6% acrylamide-8 M urea gels. Autoradiography was performed, and the fragments of interest were quantitated by PhosphorImager (ImageQuant; Molecular Dynamics, Inc., Sunnyvale, CA).
RT-PCR
Five micrograms of total RNA were reverse-transcribed in a total volume of 67 µL using 800 U Moloney murine leukemia virus of reverse transcriptase (Life Technologies, Inc.), 0.3 U/µL RNasin (Promega Corp., Madison, WI), 7.5 µM oligo(dN)6, 1.2 mM dNTP, and 5x buffer with 12 µM DTT. Samples were first diluted with DEPC H2O to a RNA concentration of 0.17 µg/µL and heated at 80 C for 10 min. After cooling for 5 min on ice, the other components were added and two cycles of RT were performed at 37 C for 40 min. Two microliters of Moloney murine leukemia virus were added at the start of each cycle. At the end, samples were heated at 95 C for 5 min to terminate the reaction. Two microliters of RT product were subsequently used for PCR amplification.
PCR reactions were performed in 25 µL with 10x reaction buffer and 2 µM of each dNTP, 1.5 mM MgCl2, 0.05 U/µL Taq polymerase (Life Technologies, Inc.), and sense and antisense primers (2 µM each). The sequences for the sense and antisense primers for hOB-Rb were 5'-GTAATTGTGCCAGTAATTATTTCC-3' and 5'-CAGAGAAGTTAACACTGTT-3', respectively. The sequences for the sense and antisense primers for the ribosomal 36B4 internal standard were 5'-CTCAACATCTCCCCCTTCTC-3' and 5'-CAAATCCCATATCCTCGTCC-3', respectively. PCR amplification was carried out for 40 or 35 cycles with annealing temperatures of 50 C or 60 C to detect OB-Rb or 36B4, respectively.
Preparation of nuclear extracts and gel mobility shift assays for STATs
Nuclear extracts from THP-1 cells were prepared as described
previously (3). The THP-1 cells were cultured in the
absence or presence of 625 nM leptin or 10 ng/mL IL-6 for
10 min or 30 min. The cells were harvested and centrifuged. The pellet
was homogenized in 10 mM KCl, 10 mM HEPES (pH
7.9), 1 mM DTT, 0.1 mM EDTA, 0.1 mM
EGTA, and 1 mM phenylmethylsulfonyl fluoride. The
cells were left to swell on ice for 15 min, after which 25 µL 1%
NP40 were added and the tubes vortexed vigorously. The homogenate was
centrifuged for 30 sec at full speed in a microfuge. The pellet was
resuspended in a cold hypertonic buffer containing 20 mM
HEPES (pH.7.9), 0.4 M NaCl, 1 mM EDTA, 1
mM EGTA, 1 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, and 20% glycerol. After
incubation, the tubes were centrifuged and the supernatants were stored
at -80 C. Two double-stranded oligonucleotides containing binding
sites for either STATs 1, 3, and 4 (M67-SIE), or for STATs 5 and 6 (ß
casein), were labeled by fill-in with Klenow DNA polymerase in the
presence of [-32P] dATP (3).
Eight micrograms of nuclear extracts from THP-1 cells were then
incubated with 30,000 cpm of the labeled probe and 2 µg poly
[d(I-C)] in electromobility shift assay binding buffer [50
mM KCl, 20 mM HEPES, 20% glycerol, 0.05%
NP40, and 10 mM ß-mercaptoethanol (pH 7.5)] in a final
volume of 25 µL. Incubation was performed at room temperature for 20
min, and the samples were analyzed on a 5% polyacrylamide gel in 0.5x
TBE [44.5 mM Tris, 44.5 mM boric acid, and 1
mM EDTA (pH 8)] for 75 min at 300 V. Gels were dried and
autoradiographed.
Transfection experiments
NIH 3T3 fibroblasts were cultured in DMEM supplemented with 50 IU/mL penicillin-streptomycin, 2 mM glutamin, and 10% heat-inactived FBS. Cells were cultured in 6-well plates with complete medium and 5% of heat-inactived FBS and transiently transfected using the calcium phosphate method. Luciferase reporter activity was determined and normalized for protein concentrations, as described (20). The transfected cells were then stimulated by leptin for 24 h in a medium with 2.5% heat-inactived FBS. The pCDNA3-OB-Rb expression plasmid containing the complete complementary DNA (cDNA) for the mouse cDNA was kindly provided by Dr. R. Skoda (University of Basel, Basel, Switzerland; Ref. 21). The psIL-1Ra-Luc and the picIL-1Ra-Luc reporter plasmids contain the 1680 or 4555 bp of the 5'-flanking regions relative to the first exons of sIL-1Ra or icIL-1Ra1, respectively. These fragments were previously shown in in vitro and in vivo studies to contain the cis-acting elements responsible for the cell type-specific and inducible expression of sIL-1Ra and icIL-1Ra1, respectively (22, 23).
Statistical methods
Results are expressed as means ± 1 SEM. The Student’s t test (unpaired, two-tailed) was used for comparisons between specified conditions.
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Results |
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The production of IL-1Ra was examined in the supernatants of THP-1
cells cultured in the absence or presence of increasing concentrations
of recombinant leptin. As shown in Fig. 1
, leptin stimulated the production of
IL-1Ra in a dose-dependent manner. The levels of IL-1Ra were already
significantly increased in response to 50 nM leptin
(P < 0.05). In contrast, leptin even at high doses did
not stimulate the production of IL-1ß, and LPS only marginally
stimulated the secretion of IL-1Ra.
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The concentrations of IL-1Ra and IL-1ß were measured in the
supernatants of THP-1 cells cultured in the presence of different
concentrations of leptin (50–625 nM) for different time
periods (Fig. 2, A and B). IL-1Ra
increased rapidly after 8 h of stimulation with leptin and peaked
after 24 h, remaining elevated throughout the subsequent 48 h
(Fig. 3A). However, no alterations in
IL-1ß secretion were observed (Fig. 2B), and IL-1 levels were
also undetectable before or after treatment with leptin (data not
shown).
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, C and D, sIL-1Ra steady-state mRNA levels increased
already after 8 h of stimulation with 625 nM leptin
and decreased at the later time points. In contrast, no detectable
icIL-1Ra1 mRNA was produced by THP-1 cells. The absence of icIL-1Ra1
mRNA in THP-1 cells was also confirmed by RT-PCR (data not shown).
To exclude an effect of leptin on the proliferation rate of THP-1
cells, we determined the cell number after different times of exposure
to various concentrations of leptin. The results summarized in Fig. 3A
demonstrate the absence of any significant alterations in cell number
although the concentrations of IL-1Ra increased in a time- and
dose-dependent manner. Thus, the increased levels of IL-1Ra induced by
leptin were not related to cell proliferation but to a stimulatory
effect of leptin on IL-1Ra secretion.
To determine whether the stimulatory effect of leptin could be
partially related to the presence of contaminating LPS, THP-1 cells
were cultured with leptin or LPS in the absence or presence of
polymyxin B, an inhibitor of LPS binding to its CD14 receptor. The
results in Fig. 3B showed that polymyxin B at concentrations of 1 and
10 µg/mL did not significantly alter the effect of leptin on the
production of IL-1Ra, whereas the modest response to 100 ng/mL LPS was
completely blocked by the addition of 10 µg/mL of polymyxin B
(P < 0.01).
Leptin stimulates the production of IL-1Ra by human PBMCs
To determine whether leptin was able to induce the production of
IL-1Ra by human monocytes in primary culture, human PBMCs were isolated
from healthy donors and cultured in the absence or presence of leptin
or LPS. Culture supernatants were collected after 24 h for
determination of IL-1Ra and IL-1ß levels. As shown in Fig. 4A, both 625 nM leptin and
100 ng/mL LPS stimulated the production of IL-1Ra and IL-1ß in PBMCs.
However, the ratio of IL-1Ra over IL-1ß in the culture supernatants
was 6.2 and 2.2 for leptin and LPS stimulation, respectively,
indicating that leptin had a more potent effect on IL-1Ra production.
The stronger effect of LPS on the production of IL-1Ra by PBMCs
compared with THP-1 cells is likely due to the higher expression of
CD14 on primary cells. Therefore, we also examined the effect of
polymyxin B on the production of IL-1Ra by PBMCs after stimulation with
leptin or LPS. As shown in Fig. 4B, the addition of polymyxin B did not
alter the stimulatory effect of leptin on IL-1Ra production, whereas
the stimulating effect of LPS was significantly inhibited
(P < 0.01). Hence, the stimulatory effect of
leptin on the production of IL-1Ra by PBMCs cannot be related to the
presence of a contamination by LPS.
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, C and D). The PBMCs were also
able to produce small amounts of icIL-1Ra1 mRNA, which is compatible
with recent reports (24). THP-1 cells express functional leptin receptors
To examine whether THP-1 cells express functional OB-Rb, we
performed RT-PCR and gel mobility shift experiments. Using cDNA from
THP-1 cells, we were able to detect the presence of transcripts for the
OB-Rb (data not shown). When gel-shift experiments were performed with
nuclear extracts from THP-1 cells prepared at 0, 10, or 30 min after
stimulation by vehicle, leptin (625 nM), or IL-6 (10
ng/mL), a strong band shift was observed on the M67-SIE binding site,
suggesting that leptin activated STATs 1, 3, and/or 4 in these cells
(Fig. 5). The band shift induced by IL-6
was weaker but migrated at the same position. In contrast, binding
activity to the ß-casein element was only weakly induced, suggesting
that STATs 5 and 6 play a quantitatively minor role in the signal
transduction by the OB-Rb in THP-1 cells, which is concordant with
previous reports in nonmonocytic cells (3).
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To test whether leptin and the OB-Rb directly activate
transcription from the IL-1Ra promoter, we transiently transfected NIH
3T3 fibroblasts with IL-1Ra reporter constructs and an OB-Rb expression
vector. As shown in Fig. 6, the promoter
for the secreted IL-1Ra promoter was not activated by leptin in NIH 3T3
cells, indicating that these cells do not contain functional OB-Rb.
However, on the transfection of the OB-Rb, leptin increased the
activity of the sIL-1Ra reporter 1.7-fold (Fig. 6), whereas no
leptin-dependent trans-activation occurred on the icIL-1Ra1
reporter (data not shown). These data are perfectly compatible with the
RNase protection data obtained in THP-1 cells, where leptin induced
sIL-1Ra but not icIL-1Ra1 mRNA (Fig. 2C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The term IL-1Ra refers to four different peptides, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, and 3), which are isoforms derived from the same gene. sIL-1Ra, icIL-1Ra1, and icIL-1Ra2 are the products of different mRNAs, whereas icIL-1Ra3 is synthesized by alternative translation initiation primarily from sIL-1Ra mRNA (reviewed in Ref. 13). Recent studies in human and mouse PBMCs, as well as macrophages showed that both sIL-1Ra and icIL-1Ra1 mRNA are produced in response to LPS, although sIL-1Ra is the predominant isoform (24, 26). Similarly, our data demonstrate the selective induction of sIL-1Ra in THP-1 and human PBMCs, although a marginal increase in the mRNA for icIL-1Ra1 was present in PBMCs.
Several questions arise from these observations, notably the
specificity of the effects of leptin on monocytes and their
physiological relevance. Although a maximal effect of leptin was
observed at supraphysiological levels (500 nM), a nearly
2-fold increase in IL-1Ra secretion was already observed at 50
nM. Although the leptin levels required to induce the
secretion of IL-1Ra in vitro were significantly higher than
those observed under most circumstances in vivo, it is still
possible that monocytes are more sensitive to leptin in vivo
and/or that local levels of leptin may exceed those in the circulation
(e.g. in sc inflammatory disorders). Furthermore, the leptin
levels required for the release of IL-1Ra could be achievable by the
exogenous administration of leptin, thereby providing a potential novel
anti-inflammatory therapeutic agent. However, because leptin has also
proinflammatory effects on macrophages and T cells resulting in the
enhanced secretion of TNF-, IL-6, and IFN-, it is difficult to
predict the net effect on the inflammatory response in vivo
(6, 7, 8). Although we demonstrate that the effect of leptin
on IL-1Ra is not due to any contaminating LPS, as evidenced by the lack
of an appreciable effect of LPS on the secretion of IL-1Ra by THP-1
cells as well as the persistence of the activity of leptin in the
presence of polymyxin B, it cannot be formally ruled out that leptin
acts through a monocytic non-OB-Rb receptor, such as other members of
the gp130 family. However, such a mechanism has not been demonstrated
so far, because leptin is inactive in some cells expressing gp130
receptors but lacking cell-surface OB-Rb (27). In
hepatocytes, for example, leptin is unable to induce the production of
acute-phase proteins (Gabay, C., unpublished data), whereas these cells
respond to IL-6 and other cytokines acting through gp130 receptors
(28). More importantly, our present transfection
experiments clearly show that the induction of the sIL-1Ra promoter by
leptin occurs in a strictly OB-Rb-dependent manner in fibroblasts,
demonstrating that 1) leptin induces IL-1Ra, at least in part, by the
induction of gene transcription, and 2) this effect is entirely
dependent on the presence of the long form of the leptin receptor.
One of the most intriguing findings of our study is the selective
stimulation of the secretion of IL-1Ra, but not IL-1ß, suggesting an
overall anti-inflammatory action of leptin. This is in keeping with the
enhanced susceptibility of leptin-deficient animals to LPS- or
TNF--induced mortality, which can be partially reversed by the
administration of exogenous leptin to these animals (9, 10). Similarly, the injection of a synthetic leptin receptor
antagonist enhanced the TNF--induced mortality in normal mice
(10). In addition, two recent studies showed that in
patients with sepsis circulating levels of leptin were significantly
higher in survivors than in nonsurvivors, suggesting that leptin has
also protective effects in endotoxic shock in humans (29, 30). Although many stimuli concomitantly enhance IL-1ß and
IL-1Ra secretion, leptin is not the only cytokine increasing the
balance between IL-1Ra and IL-1. Th2 cytokines such as IL-4, IL-10, and
IL-13 have been shown to enhance the stimulatory effect of LPS on
production of IL-1Ra by monocytes, whereas they decrease the synthesis
of IL-1 (31, 32, 33). Similarly, IL-4 and, to a lesser extent,
IL-10 increase the secretion of IL-1Ra on their own, whereas they
down-regulate the release of IL-1 by rheumatoid synovial explants
in vitro (34). The molecular basis for the
selective induction of IL-1Ra over IL-1 by leptin is speculative.
Because we demonstrate that monocytic cells express functional OB-Rb,
and because mice deficient in leptin receptors seem to lack an
anti-inflammatory counter regulatory response (10), it is
plausible to assume that the selective effects of leptin on IL-1Ra
production are mediated through this receptor. The OB-Rb as a member of
the gp130 IL-6 receptor family is known to activate STATs 1, 3, and 5,
depending on the cellular model (27). In addition, the
OB-Rb was recently shown to activate the ERK1/2 and potentially the p38
stress kinase pathways (35). However, it is unclear which
of these pathways is critical for the induction of IL-1Ra.
Taken together with the previously described effects of leptin on lymphocytes and hematopoiesis, our findings support a model whereby leptin has immunomodulatory functions, which may partly explain the altered immune response during starvation, as well as open potential therapeutic approaches. Hence, it will now be of interest to assess the anti-inflammatory potential of physiological and pharmacological doses of leptin in various animal models of inflammatory diseases.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
Received September 13, 2000.
Revised October 26, 2000.
Accepted October 30, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by phosphorylation of a
ligand-independent trans-activating domain. J Biol
Chem. 274:10505–10510.This article has been cited by other articles:
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M Bokarewa, D Bokarew, O Hultgren, and A Tarkowski Leptin consumption in the inflamed joints of patients with rheumatoid arthritis Ann Rheum Dis, October 1, 2003; 62(10): 952 - 956. [Abstract] [Full Text]
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 C. E. Juge-Aubry, E. Somm, V. Giusti, A. Pernin, R. Chicheportiche, C. Verdumo, F. Rohner-Jeanrenaud, D. Burger, J.-M. Dayer, and C. A. Meier Adipose Tissue Is a Major Source of Interleukin-1 Receptor Antagonist: Upregulation in Obesity and Inflammation Diabetes, May 1, 2003; 52(5): 1104 - 1110. [Abstract] [Full Text] [PDF]
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T. Ueland, C. Kristo, K. Godang, P. Aukrust, and J. Bollerslev Interleukin-1 Receptor Antagonist Is Associated with Fat Distribution in Endogenous Cushing's Syndrome: A Longitudinal Study J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1492 - 1496. [Abstract] [Full Text] [PDF]
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 R. R. Gonzalez, K. Leary, J. C. Petrozza, and P. C. Leavis Leptin regulation of the interleukin-1 system in human endometrial cells Mol. Hum. Reprod., March 1, 2003; 9(3): 151 - 158. [Abstract] [Full Text] [PDF]
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C. A. Meier, E. Bobbioni, C. Gabay, F. Assimacopoulos-Jeannet, A. Golay, and J.-M. Dayer IL-1 Receptor Antagonist Serum Levels Are Increased in Human Obesity: A Possible Link to the Resistance to Leptin? J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1184 - 1188. [Abstract] [Full Text] [PDF]
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N. Busso, A. So, V. Chobaz-Peclat, C. Morard, E. Martinez-Soria, D. Talabot-Ayer, and C. Gabay Leptin Signaling Deficiency Impairs Humoral and Cellular Immune Responses and Attenuates Experimental Arthritis J. Immunol., January 15, 2002; 168(2): 875 - 882. [Abstract] [Full Text] [PDF]
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 R. FAGGIONI, K. R. FEINGOLD, and C. GRUNFELD Leptin regulation of the immune response and the immunodeficiency of malnutrition FASEB J, December 1, 2001; 15(14): 2565 - 2571. [Abstract] [Full Text] [PDF]
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) and IL-1ß (
) were measured by ELISA
in culture supernatants (see Materials and Methods). The
values represent the mean ± SEM of triplicates (*,
P < 0.05 in comparison with unstimulated cells).
) of 625
nM recombinant leptin for different periods of time. The
levels of IL-1Ra (A) and IL-1ß (B) were determined by ELISA in
culture supernatants. The values represent the mean ±
SEM of triplicates. C, Total RNA was prepared from the same
experiment and analyzed by RNase protection assay. Ten micrograms of
total RNA from THP-1 cells were hybridized simultaneously with
32P-labeled riboprobes complementary to IL-1Ra and GAPDH
mRNA and then digested with RNase A and T1 (see Materials and
Methods). D, The gel from panel C was
quantitated by PhosphorImager. The values represent the IL-1Ra/GAPDH
mRNA ratio x 103. Total RNA from A431 cells and from
U937 cells cultured in the presence of phorbol 12-myristate
13-acetate and LPS were included as controls.
