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*
Division of Clinical Sciences (North), University of Sheffield, Sheffield, United Kingdom; and
Department of Medicine, Ludwig-Maximilians University, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-6 mRNA and a
dose-dependent production of TNF- and IL-6 by monocytes. Incubation
of resting PBMCs with high-dose leptin (250 ng/ml, 3–5 days) induced
proliferation of resting cultured PBMCs and their secretion of TNF-
(5-fold), IL-6 (19-fold), and IFN-
(2.5-fold), but had no effect on
IL-4 secretion. The effect of leptin was distinct from, and additive
to, that seen after exposure to endotoxin or activation by the mixed
lymphocyte reaction. In conclusion, Ob-R is expressed on human
circulating leukocytes, predominantly on monocytes. At high doses,
leptin induces proinflammatory cytokine production by resting human
PBMCs and augments the release of these cytokines from activated PBMCs
in a pattern compatible with the induction of Th1 cytokines. These
results demonstrate that leptin has a direct effect on the generation
of an inflammatory response. This is of relevance when considering
leptin therapy and may partly explain the relationship among leptin,
proinflammatory cytokines, insulin resistance, and
obesity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of the leptin receptor by PBMCs and
CD4+ T lymphocytes has been reported at the mRNA
level (5) and direct effects of leptin have been
demonstrated in a number of studies. Human PBMCs and murine splenocytes
show marked cellular clumping when incubated with leptin
(7), and leptin increases Th1 (IFN- and IL-2) and
suppresses Th2 (IL-4) cytokine production from activated human
lymphocytes (5). Circulating concentrations of TNF- and
IL-6 strongly correlate with leptin levels in humans (8),
and leptin levels rise after surgical stress (9). Leptin
receptors are expressed on human umbilical cord leukocytes
(10), and leptin activates umbilical cord monocytes,
polymorphonuclear cells, and lymphocytes (11). Rodents
with genetic abnormalities in leptin and leptin receptor expression
have deficits in macrophage phagocytosis and endotoxin-mediated
induction of TNF- and IL-6 (12).
High-dose leptin is now being used as a potential therapy for obesity (13), and high levels of leptin are found in extreme obesity due to leptin resistance and also in renal failure (14, 15). Injection of leptin elicits a mild localized inflammation (16), but the physiological basis for this reaction has not been established. To date, studies have focused on the effects of physiological levels (as determined by circulating plasma levels) of leptin on activated lymphocytes. In this study, we confirm the specificity of a mAb for the leptin receptor and demonstrate that monocytes are the predominant cells expressing Ob-R in the peripheral blood. Our results demonstrate that high-dose leptin induces the production of proinflammatory cytokines in resting PBMCs, which is compatible with the observed expression of Ob-R on peripheral blood monocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood samples
Peripheral blood (50 ml) was collected into heparinized tubes from healthy adult volunteers (aged 20–45 years).
Measurement of endotoxin activity in recombinant human leptin and blocking of endotoxin activity
Endotoxin activity in recombinant human leptin was measured using a QCL-1000 pyrogen testing kit (BioWhittaker, Walkersville, MD) according to the manufacturer’s instructions. The potential influence of endotoxin contamination on observed effects was excluded by the addition of polymyxin-B in control experiments (17). This was added at the same time as endotoxin and leptin.
Leptin functional assay
Biological activity of recombinant leptin was tested in transcription assays performed in human epithelial kidney (HEK293) cells transiently transfected with the leptin receptor long form and a reporter construct containing a Stat3-binding element fused to a minimal thymidine kinase promoter and luciferase. Luciferase activity was measured as previously reported (18).
RT-PCR
For the analysis of leptin receptor and cytokine gene
expression, total RNA was extracted from 4 x
107 PBMCs using TRIzol reagent (Life
Technologies, Paisley, U.K.). Total RNA (1 µg) was
reverse-transcribed and PCR amplification was performed using
previously published primers for Ob-Ra and Ob-Rb (4). For
cytokine gene analysis, mRNA was extracted at the indicated time
points, cDNA was prepared, and PCR amplifications were performed using
previously characterized primers for IL-6, TNF-, IFN-, and GAPDH
(19).
Anti-leptin receptor mAb
A mAb (9F8) raised against the recombinant human leptin receptor extracellular domain (20) was used for the study. Specificity of mAb 9F8 was shown by the lack of cross-reactivity with extracellular domains from other closely related receptors of the same class I cytokine receptor superfamily (growth hormone and prolactin receptors).
Transfection of HEK293 cells with Ob-R
The leptin receptor expression plasmid was a kind gift from A. Welcher (Amgen, Thousand Oaks, CA). HEK293 cells were transfected with 9 µg of leptin receptor cDNA using a calcium phosphate transfection kit (Life Technologies). Receptor expression was confirmed by assessing the binding of 125I-labeled leptin (Biogenesis, Poole, U.K.).
Validation of 9F8 by flow cytometric analysis of transfected and nontransfected HEK293 cells
Transfected and nontransfected HEK293 cells were suspended in PBS/BSA (washing buffer) at a density of 1 x 107 cells/ml. Cells (106; 100 µl) were transferred to 12 x 75 mm polycarbonate tubes (BD Biosciences, Oxford, U.K.) and incubated with 5 µg of an anti-leptin receptorAb (9F8) or isotype-matched negative control Ab (R&D Systems, Abingdon, U.K.) for 30 min on ice. Primary Ab binding was detected by incubation with biotinylated goat anti-mouse IgG polyclonal Ab (1 µg; Calbiochem, Nottingham, U.K.), followed by incubation with streptavidin-R-PE (SAV-R-PE)3 conjugate (10 µl; Serotec, Oxford, U.K.) for 30 min on ice. Cells were washed before flow cytometric analysis. The specificity of 9F8 was tested by preincubation with a recombinant form of the extracellular domain of the human leptin receptor rhOb-R (R&D Systems).
Detection of Ob-R on peripheral blood leukocytes by whole blood flow cytometry
Heparinized whole blood (150 µl) and 10 µl of goat serum (Serotec) were incubated for 15 min on ice, after which 1 µg of 9F8 or isotype-matched negative control Ab were added. Cells were washed in PBS containing 7.5% v/v goat serum, incubated with 0.2 µg of biotinylated goat anti-mouse IgG, and finally incubated with 10 µl of the SAV-R-PE conjugate. Incubations were conducted for 30 min on ice and cells were washed twice with washing buffer. Erythrocytes were lysed with 2 ml of Erythrolyse (Serotec) before flow cytometry on a FACScan flow cytometer (BD Biosciences) using CellQuest data acquisition and analysis software. Polymorphonuclear leukocytes (neutrophils), lymphocytes, and monocytes exhibit characteristic light scatter properties that can be identified on a forward vs side light scatter plot. Leukocyte populations were located using these parameters and a live analysis gate was set around this population. Data were acquired from 10,000 cells (events) and the proportion of given cell populations expressing the leptin receptor was determined. For two-color flow cytometry, after staining with 9F8, cells were incubated with PBS containing 2% v/v normal mouse serum (Serotec) for 30 min on ice, washed, and incubated with 10 µl of FITC-conjugated anti-human CD (CD14, CD15, CD3, CD4, CD8, and CD20) Abs (Serotec) for another 30 min on ice. Staining with mAbs specific for cell subsets revealed that the monocyte region contained 94.8 ± 1.8% CD14+ cells and the polymorphonuclear cell region contained 92.2 ± 1.7% CD15+ cells. The lymphocytes region contained 70.4 ± 1.7% CD3+, 34.5 ± 0.8% CD4+, 42.4 ± 1.06% CD8+, and 4.4 ± 0.3% CD20+ cells.
Flow cytometry cytokine production assay
Heparinized whole blood (100 µl) was treated with 10 µl of
either endotoxin, PBS, or leptin. Brefeldin A (BD Biosciences), a
pharmacological inhibitor of secretion, was added, and cells were
incubated for 6 h followed by surface staining of monocytes using
FITC-conjugated mouse anti-human CD14 (Serotec). Cells were fixed
and permeabilized, and intracellular staining of accumulated cytokines
was performed using either PE-anti-human TNF- mAb or PE
-anti-human IL-6 mAb (BD Biosciences).
Isolation and culture of PBMCs
PBMCs were isolated by density gradient (Lymphoprep Nyegaard, Oslo, Norway), washed twice in normal saline and once in medium, and suspended at 1 x 106 cells/ml in medium RPMI 1640 growth medium supplemented with L-glutamine (2 mM), penicillin/streptomycin (100 U/ml penicillin, 100 µg/ml streptomycin), and 2% v/v heat-inactivated normal human AB serum. The leptin concentration in this medium was below the sensitivity of the leptin ELISA kit (7.6 pg/ml; R&D Systems). The cell suspension (2 ml) was transferred to each well of a 24-well plate for the cytokine secretion studies. Cells were incubated with endotoxin (LPS from Escherichia coli, serotype 0111:B4) or leptin (recombinant human leptin, R&D Systems), at the concentrations indicated, for 72 h in 100% humidity, 5% CO2, after which media were centrifuged at 400 g for 10 min. Supernatants were collected and stored at -70°C until assay. Control cultures were incubated with PBS or medium alone.
Proliferation assay
The PBMCs suspension (200 µl) was transferred to each well of a 96-well plate (Costar, Cambridge, U.K.), treated according to the protocol, and incubated for 5 days. During the last 24 h, 0.1 µCi [3H]thymidine was added and proliferation, on the basis of thymidine incorporation, was assessed by liquid scintillation counting. The influence of leptin on MLR-induced proliferation and cytokine production was also assessed. For MLR studies, cultures were established using equal numbers of responder and irradiated (30 Gy) allogeneic PBMCs as stimulator cells. Cells were incubated in the presence or absence of leptin for 3 (cytokine production) or 5 (proliferation) days.
Enzyme-linked immunosorbent assays
Cytokines (IL-6, TNF-, IL-4, and IFN-) and leptin in media
were measured using ELISA kits (R&D Systems). Intra-assay coefficient
of variation values were 4.4, 8.7, 0.59, 3.7, and 3.3%,
respectively.
MAPK inhibitors
To determine signaling pathways, PBMCs in whole blood were
incubated with or without 0.01 nM of either extracellular-regulated
protein kinase (ERK) inhibitor PD98059 (Calbiochem) or p38
kinase inhibitor SB2035580 (Calbiochem) 1 h before the addition of
leptin or endotoxin. After an additional 6-h incubation, production of
TNF- on monocytes was assessed using flow cytometry.
Statistics
The paired t test was used to compare the effects of leptin on lymphocyte proliferation and cytokine production and ANOVA for the dose response. A value of p < 0.05 was considered to indicate statistically significant differences.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PCR products for both short (Ob-Ra) and long (Ob-Rb) isoforms of
leptin receptor were detectable in human PBMCs (Fig. 1
).
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HEK293 cells were transfected with Ob-R and expression of leptin
receptor was confirmed by incubation with
125I-labeled leptin in the presence or absence of
unlabeled leptin. Ob-R-transfected HEK293 cells had high levels of
leptin-specific binding (8 ± 1%), but nontransfected cells also
showed a low level of specific binding (0.7 ± 0.1%).
Ob-R-transfected cells were used in flow cytometry to test the
anti-leptin receptor Ab (9F8). Transfected and nontransfected
HEK293 cells were incubated with either 9F8 or isotype-matched control
Ab, and Ab binding was detected by flow cytometry. Binding of 9F8 to
transfected cells was apparent, but 9F8 also bound to nontransfected
cells, confirming that HEK293 cells express low levels of the leptin
receptor (Fig. 2). The specificity of 9F8
for the leptin receptor was further tested by preincubation of Ab with
recombinant human (rh)Ob-R, which completely abolished binding of 9F8
Ab to HEK293 cells (data not shown).
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Whole blood flow cytometric analysis of samples from 10 subjects
using 9F8 revealed that 25 ± 5% of monocytes, 12 ± 4% of
polymorphonuclear cells, and 5 ± 1% of lymphocytes express the
Ob-R (Fig. 3). Two-color flow cytometry
revealed that lymphocyte expression was primarily related to
CD20+ cells (B lymphocytes, 50 ± 6%) with
T lymphocytes showing no expression. Activation of PBMCs by incubation
with endotoxin (10 ng/ml) for 2 h resulted in no change in Ob-R
expression on monocytes, and incubation up to 3 days with endotoxin,
PHA, or MLR did not alter Ob-R expression on lymphocyte
populations.
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The biological activity of the recombinant leptin was tested in a
function assay using a Stat3-luciferase reporter (Fig. 4a). Leptin showed a dose
response between 1 and 100 ng/ml.
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Whole blood was incubated with leptin in doses of 10–1000 ng/ml
and production of IL-6 and TNF- by CD14+
monocytes was determined using flow cytometry. Leptin at doses of
50–1000 ng/ml induced the production of both IL-6 and TNF-
(p < 0.01; Fig. 4
b). The observed
effects were not due to endotoxin contamination of the leptin
preparation, as levels of endotoxin (<0.00036 ng/ml) were below those
that we have previously shown to be the minimum dose (0.01 ng/ml)
required to induce IL-6 (21). The possibility of endotoxin
contamination was further excluded by the demonstration that
polymyxin-B, an endotoxin inhibitor (17), completely
inhibited the induction of TNF- by 0.1 ng/ml endotoxin, yet it had
no effect on leptin-induced TNF- production (Fig. 5).
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PBMCs were incubated in the presence or absence of leptin for
between 1 and 24 h and cytokine gene expression was determined by
RT-PCR using cytokine-specific primers. Leptin induced IL-6 and TNF-
gene expression within 1 h (Fig. 6).
IFN- was barely detectable in control samples and showed little
induction.
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To confirm the activation of PBMCs by leptin, the effects of
leptin (250 ng/ml) and endotoxin on cytokine secretion by PBMCs from
six subjects were determined by ELISA. Medium from cells incubated
without leptin showed very low levels of TNF-, IL-6, IL-4, and
IFN-. Leptin caused a 5-fold induction of TNF-, a 19-fold
increase in IL-6, and a 2.5-fold increase in IFN- (Fig. 7), whereas it had no effect on
induction of IL-4. The IL-4 levels in samples from both resting
and leptin-activated PBMCs were below the detection limit of the assay,
although PHA-stimulated PBMCs produced high levels of IL-4 (122 ±
14 pg/ml). Leptin also significantly increased the proliferation of
PBMCs (PBMCs only, 923 ± 178; PBMCs plus leptin, 1253 ± 254
cpm [3H]thymidine uptake, p <
0.01). When coincubated with endotoxin, leptin augmented the
cytokine response to endotoxin with a pattern similar to the induction
of cytokines by leptin alone. Endotoxin alone at the dose of 0.01 ng/ml
did not induce IFN- (Fig. 7).
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Leptin augmented the production of TNF-, IL-6, and IFN-, but
not IL-4 from MLR-activated PBMCs in a similar pattern to that seen in
nonactivated PBMCs (Fig. 8). In addition,
the MLR-induced proliferation was augmented by leptin (PBMCs only,
923 ± 178; MLR only, 3867 ± 372; MLR plus leptin, 6350
± 584 cpm [3H]thymidine uptake,
p < 0.001).
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PBMCs were preincubated with two mitogen-activated protein kinase
(MAPK) inhibitors, PD98059 (inhibits ERK) and SB203580 (inhibits p38),
and then exposed to either endotoxin or leptin. The p38 inhibitor,
SB203580, consistently inhibited both the endotoxin and leptin
induction of TNF- by 
30%, whereas PD98059 had no effect (Fig. 9). The biological activity of the ERK
inhibitor PD98059 had previously been demonstrated in our functional
bioassay.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-6, this occurs in nonactivated as well as activated
PBMCs, and the effect of leptin was seen to be additive to the actions
of endotoxin. The expression of Ob-R on human umbilical cord blood leukocytes has been reported using flow cytometry (10). In this study the proportion of monocytes expressing the Ob-R was comparable to that reported in this work (23 ± 4%); however, the proportion of lymphocytes expressing the receptor was greater (16 ± 4%) (10). The difference in results may be relate to the different source of leukocytes. In our data, activation of monocytes and lymphocytes did not alter Ob-R expression.
Leptin treatment of activated PBMCs (MLR), in an experimental design
similar to that reported in this study, has been shown to increase Th1
(IFN-) and suppress Th2 (IL-4) cytokine production (5).
In the previous study, IL-6 and TNF- were not measured and there was
no effect of leptin on resting PBMCs. Our finding that leptin can
activate resting PBMCs may relate to the higher dose of leptin used and
confirms a study demonstrating that leptin can activate and stimulate
the proliferation of resting human PBMCs (22). Leptin has
been shown to have a direct effect on activated T lymphocytes
(5), suggesting that leptin directly acts on T
lymphocytes. We found no evidence of Ob-R on resting T lymphocytes by
whole blood flow cytometry, and our data suggest that the predominant
action on resting PBMCs is through activation of monocytes.
Our RT-PCR experiments demonstrate that there is an acute stimulation
of cytokines by leptin, and the longer incubation studies confirm
cytokine production by nonactivated PBMCs. Leptin treatment also
enhanced the cytokine response to endotoxin and the MLR. Results from
rodent studies confirm our observations in the human. In mice,
thioglycollate-elicited peritoneal macrophages produce no detectable
TNF- or IL-6, whereas leptin pretreatment augments proinflammatory
cytokine release from endotoxin-stimulated macrophages
(12). In the RAW murine macrophage cell, leptin at a high
dose of 1 µg/ml activates resting cells to produce significant levels
of IL-1R antagonist after 24 h and also augmented the response to
endotoxin in an additive manner (23).
Intracerebroventricular and i.p. injection of leptin into rats induces
the production of IL-1
and increases body temperature
(24).
Contamination of any component of the cell incubation by endotoxin
could activate cells and induce the production of inflammatory
cytokines. This seems unlikely in our experiments because endotoxin
levels in the leptin preparation were well below that which is required
to activate PBMCs (21). In addition, polymyxin-B (an
inhibitor of endotoxin) treatment inhibited the response to endotoxin
but had no effect on leptin. The profile of cytokine induction by
leptin was different from that of endotoxin, with leptin inducing
significant levels of IFN-, an effect not seen with low levels of
endotoxin.
We performed a dose response for the effect of leptin on TNF- and
IL-6 production. The biological activity of the recombinant leptin was
tested in a functional bioassay measuring leptin signaling. This
confirmed the biological activity of leptin at doses as low as 1 ng/ml.
Higher doses of leptin were required to induce cytokine production
(50–1000 ng/ml) than are usually found in venous blood under
physiological conditions (1–100 ng/ml). Our repeated experiments on
PBMCs were performed with a leptin dose of 250 ng/ml. This dose was
chosen because it was clear from our dose response studies that this
dose of leptin could activate PBMCs. Similar higher serum leptin levels
are found in some nonphysiological conditions, such as in children with
chronic renal failure (leptin levels up to 400 ng/ml are reported in
Ref. 15) and following leptin treatment (25).
Treatment of a patient with genetic leptin deficiency with low levels
of leptin (0.028 mg/kg of lean body mass) increased the serum leptin up
to 107 ng/ml (26). In obese but otherwise healthy subjects
treated with leptin (1 mg/kg/day), serum leptin levels up to 736 ng/ml
have been reported (25). Thus the high leptin doses we
used in our study were similar to those found in some nonphysiological
conditions and may lead to activation of PBMCs.
The high dose response of leptin in our study may reflect the insensitivity of our method for measuring cytokine production from incubated PBMCs, although in activated PBMCs a lower dose of leptin was reported as effective (5). Another possibility is that incubation of PBMCs with leptin over time may result in degradation of leptin and/or loss of cytokines. The higher dose of leptin may be relevant, because leptin levels could vary between tissues and leptin levels in fat tissue may be greater than those found in serum. There are other possibilities for the high dose-dependent activation of PBMCs by leptin. It is recognized that short forms of the leptin receptor can activate the MAPK signaling pathway, and it may be that either the dose response of these receptors is different from that of the long form of the receptor (27) or that leptin is activating another receptor. Recombinant leptin is known to have a lower potency than native leptin, which may be due to a different pattern of glycosylation (28).
The class I cytokine receptors are known to act through the Janus
kinase/STAT and MAPK pathways (29). Both long and
short leptin receptor isoforms can activate the MAPK pathway
(30). The major MAPK pathways are: ERK, c-Jun
NH2-terminal kinase, and p38 MAPK. A wide variety
of inflammatory mediators, such as cytokines, activate p38 MAPK in
several cell types. In neutrophils, TNF- activates p38 MAPK
specifically, whereas others, such as GM-CSF, fMLP, IL-8, and PMA
preferentially phosphorylate ERK 1/2 (31). To determine
which MAPK signaling pathway is recruited by leptin to produce TNF-
in monocytes, PD98059 and SB203580 were used to block ERK and p38
respectively. PD98059 did not show any effect on either
endotoxin-stimulated or leptin-activated monocytes, despite its
inhibitory effects on leptin induction of serum response element
transfected cells (data not shown). SB203580 reduced the percentage of
activated monocytes by 30% in leptin-activated cells, showing
involvement of p38 MAPK signaling in leptin induction of TNF- in
monocytes. Currently there is no specific inhibitor of the Janus
kinase/STAT signaling pathway, so we were unable to examine the
potential role of this pathway in PBMCs.
The physiological significance of leptin-induced proinflammatory cytokine production requires investigation. Leptin-deficient or -resistant rodents are more susceptible to the toxic effects of TNF (32). Although one might therefore expect that leptin would reduce levels of TNF, the reverse appears to be true. It is possible that leptin induces a negative regulator of TNF, and that the rise in TNF levels occurs in response to partial TNF resistance and is therefore compensatory. It may be that the local induction of proinflammatory cytokines provides a pathway for some metabolic actions of leptin. The production of IL-6 in the hypothalamus is known to activate the hypothalamo-pituitary axis (33), and TNF alone can produce an anorectic action (34). Another possibility is that the high leptin levels seen in obesity could be responsible for some of the adverse effects of central obesity. The local activation of TNF could play a role in atherogenesis or insulin resistance (35). Our data suggest that leptin directly activates monocytes and that the activation of T lymphocytes may be in response to the release of inflammatory cytokines. To date, the presence of Ob-R on human B lymphocytes has not been demonstrated. It has been proposed that leptin treatment may augment the immune response in malnourished individuals (6).
The fact that leptin can increase the response to endotoxin and promote proinflammatory cytokine release from apparently nonactivated PBMCs should be further investigated in humans. Particular care should be taken in human therapeutic trials that leptin therapy does not activate an unwanted inflammatory response, and leptin-treated patients may be more susceptible to an inflammatory response following infection.
In conclusion, human blood leukocytes express leptin receptor, and the predominant leptin receptor-expressing cell type is the monocyte. In vitro, leptin modulates the immune response, inducing the production of inflammatory cytokines, and has an additive action on cytokine production from PBMCs exposed to endotoxin.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard J. M. Ross, Clinical Sciences, Northern General Hospital, Sheffield S5 7AU, U.K. E-mail address: r.j.ross{at}sheffield.ac.uk
3 Abbreviations used in this paper: SAV-R-PE, streptavidin-R-PE; ERK, extracellular-regulated protein kinase; rh, recombinant human; MAPK, mitogen-activated protein kinase; Ob-Ra, short isoform of Ob-R; Ob-Rb, long isoform of Ob-R.
Received for publication May 24, 2001. Accepted for publication August 13, 2001.
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J. L. Chan, S. J. Moschos, J. Bullen, K. Heist, X. Li, Y.-B. Kim, B. B. Kahn, and C. S. Mantzoros Recombinant Methionyl Human Leptin Administration Activates Signal Transducer and Activator of Transcription 3 Signaling in Peripheral Blood Mononuclear Cells in Vivo and Regulates Soluble Tumor Necrosis Factor-{alpha} Receptor Levels in Humans with Relative Leptin Deficiency J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1625 - 1631. [Abstract] [Full Text] [PDF]
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 S. H. Schirmer, I. R. Buschmann, M. M. Jost, I. E. Hoefer, S. Grundmann, J.-P. Andert, S. Ulusans, C. Bode, J. J. Piek, and N. van Royen Differential effects of MCP-1 and leptin on collateral flow and arteriogenesis Cardiovasc Res, November 1, 2004; 64(2): 356 - 364. [Abstract] [Full Text] [PDF]
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 B Siegmund, J A Sennello, J Jones-Carson, F Gamboni-Robertson, H A Lehr, A Batra, I Fedke, M Zeitz, and G Fantuzzi Leptin receptor expression on T lymphocytes modulates chronic intestinal inflammation in mice Gut, July 1, 2004; 53(7): 965 - 972. [Abstract] [Full Text] [PDF]
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 T. E. Weber and M. E. Spurlock Leptin alters antibody isotype in the pig in vivo, but does not regulate cytokine expression or stimulate STAT3 signaling in peripheral blood monocytes in vitro J Anim Sci, June 1, 2004; 82(6): 1630 - 1640. [Abstract] [Full Text] [PDF]
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C. J. Hukshorn, J. H. N. Lindeman, K. H. Toet, W. H. M. Saris, P. H. C. Eilers, M. S. Westerterp-Plantenga, and T. Kooistra Leptin and the Proinflammatory State Associated with Human Obesity J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1773 - 1778. [Abstract] [Full Text] [PDF]
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H. Zarkesh-Esfahani, A. G. Pockley, Z. Wu, P. G. Hellewell, A. P. Weetman, and R. J. M. Ross Leptin Indirectly Activates Human Neutrophils via Induction of TNF-{alpha} J. Immunol., February 1, 2004; 172(3): 1809 - 1814. [Abstract] [Full Text] [PDF]
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V. D. Dixit, M. Mielenz, D. D. Taub, and N. Parvizi Leptin Induces Growth Hormone Secretion from Peripheral Blood Mononuclear Cells via a Protein Kinase C- and Nitric Oxide-Dependent Mechanism Endocrinology, December 1, 2003; 144(12): 5595 - 5603. [Abstract] [Full Text] [PDF]
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 G Palmer and C Gabay A role for leptin in rheumatic diseases? Ann Rheum Dis, October 1, 2003; 62(10): 913 - 915. [Full Text]
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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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