Increased activation and cytokine secretion in B cells stimulated with leptin in aged humans.

0.3 MB·English

by  Gupta, Sudhir

Guptaetal.Immunity&Ageing2013,10:3
IMMUNITY & AGEING
http://www.immunityageing.com/content/10/1/3
RESEARCH Open Access
Increased activation and cytokine secretion in
B cells stimulated with leptin in aged humans
Sudhir Gupta1,2*, Sudhanshu Agrawal1 and Sastry Gollapudi1
Abstract
Aging is associated chronic inflammation and autoimmunity, and increased levels ofleptin. Increased levels of
leptinare associated with inflammation and autoimmunity.We have recently reported that leptin activates B cells
to induce secretion ofproinflammatory and anti-inflammatorycytokines. Role of Bcellsand leptin ininflammation
associated with aging has notbeen explored. In this study we demonstrate that leptin activates and induces
significantly greater amount ofIL-6, TNF-α,and IL-10 by Bcells from agedhumans as compared to young controls.
Thisis associated withincreased leptin-induced phosphorylation of STAT3 (signal transducerand activator of
transcription-3) inBcells from agedhumans as compared to young subjects. These data suggest that
leptin-induced Bcell-derived proinflammatory cytokines may play a rolein chronic inflammation associated with
human aging.
Keywords: TNF-α,IL-6, IL-10, Bcells
Background proinflammatory cytokines IL-6,TNF-α, and immunore-
Leptin, one of adipokines induces secretion of a number gulatory and anti-inflammatory cytokines, such TGF-β,
of proinflammatory cytokines, including IL-6, IL-1, and and IL-10 [21-24]. We have recently reported that leptin
TNF-α [1-5]. Serum levels of leptin,TNF-α, IL-6, and activates human B cells to induce secretion of IL-6 and
other proinflammatory cytokines are increased during TNF-α and IL-10, and this effect of leptin on B cells is
human aging and age-associated inflammatory disorders mediated via JAK2/STAT3 and p38MAPK/ERK1/2 sig-
[6-15]. However, the cellular source of these cytokines in naling pathways [25].
aging is poorly understood. These cytokines are pro- In this study, we show that leptin activates B cells and
duced by Tcells, macrophages, and B cells. However, T B cell subsets from aged subjects to a significantly
cell-derived and macrophage-derived proinflammatory greater extent, and induces significantly higher levels of
cytokines are decreased in aging [16-20]. Therefore, B secretion of IL-6, TNF-α, and IL-10 as compared to B
cells are likely the source of increased proinflammatory cells and B cell subsets from young subjects. This
cytokines inaging. B cellderived inflammatory cytokines appears to be due to an increased leptin-induced phos-
havenotbeenstudiedinaging.Duringagingadiposetis- phorylation of STAT-3 in B cells from aged as compared
sue is increased in the lymph nodes and the bone mar- toyoung controls.
row, which may result in leptin levels that may be 10 to
100 fold greater than those in plasma. In addition to
Results
their well-defined role in antibody production, B cells
LeptininducesgreateractivationofBcellsfromagedas
may also regulate immune responses to microbes and
comparedtoyounghumans
participate in inflammation through production of cyto-
Purified B cells were activated with 50 ng/ml of leptin
kines, chemokines, and growth factors [21-24]. Human
(optimum concentration-reference# 25) for 24 hours.
B cells have shown to produce cytokines, including
Cells were washed and stained with anti-CD19 antibody,
anti-CD5 antibody, and antibodies directed against CD25,
*Correspondence:[email protected] and CD69 as activation markers. Figure 1 shows a repre-
1ProgramsinPrimaryImmunodeficiencyandAging,DivisionofBasicand
sentative cytofluorograph. In aging, leptin induced (red
ClinicalImmunology,UniversityofCalifornia,Irvine,CA,USA
2MedicalSciencesI,C-240,UniversityofCalifornia,Irvine,CA92697,USA line)greateractivation(increasedexpressionofCD25and
©2013Guptaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Activation Marker
CD19 CD5- CD5+
Aging
CD25
Young
Aging
CD69
Young
Basal Leptin
Figure1EffectofleptinonactivationofBcellsandBcellsubsets.PurifiedBcellsfromyoungandagedsubjectswerestimulatedwith
leptin(50ng/ml)for24hoursandexpressionofCD25andCD69asactivationmarkers,usingspecificantibodiesandisotypecontrols.Bluelines
areforunactivatedBcells,andredlineforleptin-activatedBcells.
CD69)overthebaselinelevels(bluecontrol)ascompared Leptin-inducedproductionofIL-6,TNF-α,andIL-10byB
to young control. Furthermore, increased activation in cellsfromyoungandagedsubjects
aging was observed in both CD5+ and CD5- B cells. Purified B cells were stimulated with 25 ng/ml and
Tables1and2showdatafrom10agedand10youngsub- 50 ng/ml of leptin for 24 hours and supernatants were
jects. A significantly greater upregulation of both CD25 collected and stored at −20°C until assayed for IL-6,
and CD69 antigens over the baseline levels was observed TNF-α, and IL-10 by ELISA. Cytokine secreting B cells
on CD19+, and CD5+ and CD5- B cells in aged humans were also analyzed by ELISPOT. Figure 2 show data
as compared to B cells and B cell subsets in young from ELISA assay. Leptin at both 25 ng/ml and 50 ng/ml
(Table1).Furthermore,greaterupregulationwasobserved induced significantly (P<0.05) greater amounts of IL-6,
inCD5+B cells as compared toCD5- B cells.Whendata TNF-α, and IL-10 by B cells from aged as compared to
were analyzed for the density of these molecules as mea- young subjects. Similar data were obtained for IL-6,
suredbymeanfluorescenceintensity(MFI),significantin- TNF-α, and IL-10 secreting B cells as determined by
crease in aging was observed for CD25 expression ELISPOT assay (Figure 3). We observed no difference
(Table2). in these cytokines production between B cells purified
Table1EffectofLeptinontheexpressionofactivationmarkersonBcellsubsets(%)
Marker Young Aged
CD19 CD5+ CD5- CD19 CD5+ CD5-
CD25 Control 2.8±0.4 3.5±0.4 3.0±0.3 3.5±0.2 5.0±0.7 1.8±0.4
Activated 8.3±1.1 4.0±−.3 10.0±1.3 26.3±2.8 15.0±2.3 12.0±1.9
CD69 Control 1.8±0.2 2.4±0.5 1.9±0.5 5.5±1.0 3.1±0.4 2.5±0.3
Activated 4.3±0.6 2.6±0.3 4.8±0.3 8.2±0.5 7.6±0.8 6.9±0.4
Pvalue:
CD25,Agingvs.Young:CD19=P<0.01,CD5+=P<0.001,CD5-=P<0.05.
CD69,Agingvs.Young:CD19=P<0.05,CD5+=P<0.01,CD5-=P<0.05.
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Table2EffectofLeptinontheexpressionofactivationmarkersonBcellsubsets(MFI)
Marker Young Aged
CD19 CD5+ CD5- CD19 CD5+ CD5-
CD25 Control 3.6±.4 5.6±2 5.6±2 10±4 7±1.25 10±7
Activated 9.6±1.7 9.5±1.4 9.5±1.4 26±2 15±2.5 51±4.6
CD69 Control 4.5±2 8.8±5 4.1±1.6 4.5±.4 7±3.5 3.6±8
Activated 7.6±2.6 11±5.9 7.3±1.7 8.6±.47 20±5.7 8±.8
Pvalue:
CD25,Agingvs.Young:CD19=P<0.01,CD5+=P<0.001,CD5-=P<0.045.
CD69,Agingvs.Young:CD19=ns,CD5+=P<0.02,CD5-=ns.
by positive selection versus those purified by negative of STAT3 in aging B cells as compared to B cells from
selection, therefore, excluding any possibility of activa- young subjects suggesting an increased activation of posi-
tion of B cells during positive selection (data not tivedownstreamsignalinginaging.
shown).
Discussion
IncreasedsensitivityofBcellstoleptininagingisdueto In this study we have demonstrated that leptin activates
increasedpositivedownstreamsignalingevent B cells from aged humans and induces significantly
Since leptin induces cytokines by activation of down- higher amounts of TNF-α, IL-6, and IL-10 as compared
stream signaling pathways, including phosphorylation of to B cells from young subjects. This is in part due to an
STAT3 [25], we examined leptin-induced phosphoryl- increasedphosphorylationofSTAT3.
ation of STAT3 in purified B cells from aged and young Aging is considered a state of chronic inflammation.
subjects. Purified B cells were stimulated with 50 ng/ml Plasma levels of proinflammatory cytokines, including
of leptin for 10 min, and phosphorylation of STAT3 was IL-6 and TNF-α are increased in human aging [6-11];
determined by Western blotting using specific anti- however, the cellular source of increased proinflamma-
bodies. Actin was used as a loading internal control. tory cytokines is poorly understood. These cytokines are
Quantitative analysis was performed by densitometry. produced by Tcells, macrophages, and B cells. However,
Figure4showsthatleptininducesgreaterphosphorylation macrophage-derived IL-1β, TNF-α and IL-6 and Th1
400 TNF α IL-6
2500
350
300 2000
250
ml) 1500
g/ 200
(P 150 1000
100
500
50
0 0
0 25 50 0 25 50
2000 IL-10
1800
1600
1400
ml) 11020000
g/ Young
P 800
(
600 Aged
400
200
0
0 25 50
Leptin (ng/ml)
Figure2Leptin-inducedsecretionofcytokines.PurifiedBcellsfromyoungandagedwereculturedintheabsenceorpresenceofleptin
(25ng/mland50ng/ml)for24hoursandsupernatantswereanalyzedforTNF-α,IL-6,andIL-10usingELISAassay.Dataareexpressedasmean±
sd.Eachexperimentwasdoneintriplicates.
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s
cell 300 TNF α 3500 IL-6
B
0 250 3000
0
0,0 200 2500
5
cell / 150 12500000
g 100
n 1000
ormi 50 500
pot f 0 0 25 50 0 0 25 50
s
s
ell
B c 160 IL-10
0 140
00 120
50, 100
ell / 80 Young
c 60 Aged
g
n 40
mi
or 20
ot f 0 0 25 50
p
s Leptin (ng/ml )
Figure3Leptin-stimulatedcytokinesecretingBcells.PurifiedBcells(12,500-50,000/well)fromyoungandagedwereculturedintheabsence
orpresenceofleptin(25ng/mland50ng/ml)for24hoursandTNF-α,IL-6,andIL-10secretingBcellswereanalyzedbyELISPOTassay.Dataare
expressedasmean±sd.Eachexperimentwasdoneintriplicates.
derived cytokines are decreased in aging [16-20]. There- cytokines, chemokines, and growth factors with proin-
fore, B cells is likely one of the sources of increased flammatory properties [21-25].
proinflammatory cytokines associated with human aging, Leptin is one of adipokines, which is powerful inducer
though dendritic cells in aging may also contribute to of inflammatory cytokines by Th1 T cells and macro-
increased levels of proinflammatory cytokines [26]. Re- phages, including IFN-γ, IL-1β, TNF-α, and IL-6
cently, B cells have shown to produce a number of [1-5,27,28]. More recently, we have reported that leptin
also activates human B cells to induce inflammatory
cytokines, including IL-6 and TNF-α, and anti-inflam-
matory/ immunoregulatory cytokine IL-10 [29-31]. We
Young Aged
have reported that leptin activates human B cells [25],
Leptin 0 10 0 10 (min)
and serum levels of leptin are increased in aging [17-20].
p-Stat3 Since leptin and proinflammatory cytokines are increased
in aging, and T cell-and macrophage-derived proinflam-
Stat3
matory cytokines are decreased in aging, we investigated
β-Actin the effects of leptin on purified B cells. In the present
study we show that leptin activates human aged B cells
actin normalized ratio to a greater extent than B cells from young subjects as
evidenced by upregulation of CD25 and CD69, which
Young Young Aged Aged
was observed on both CD19+CD5+ B cells and CD19+
Control Leptin Control Leptin
CD5-B cells, greater upregulation was observed in CD5+
p-Stat3 0.33 0.70 0.67 2.43
B cells as compared to CD5- B cells. CD5+ B cells are
Stat3 1.04 0.98 1.25 1.66
β-Actin 1.00 1.00 1.00 1.00 reported to produce IL-10 with immunoregulatory prop-
erty [32], which may explain a role of leptin in auto-
Figure4Leptin-inducedphosphorylationofSTAT3inBcells.
immunity[1,33].
PurifiedBcellsfromyoungandagedwerecutluredinthepresence
andabsenceof50ng/mlleptinfor10minandnativeand ActivationofB cells wasalsoassociated withincreased
phosphorylatedSTAT3wereanalyzedbyWesternblotusingspecific production of cytokines by B cells. Leptin induced sig-
antibodies.Actinwasusedananinternalloadingcontrol. nificantly higher (P<0.05) secretion of IL-6,TNF-α, and
Quantitationofbandswasperformedbydensitometryandshown
IL-10 by B cells (ELISA assay) from aged humans as
intablebelow.
compared to B cells from young subjects. Furthermore,
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we demonstrate that leptin not only induced increased antioxidants (if they were taking) for at least one year
amounts of secreted cytokines by aged B cells, but lep- prior to donation of blood sample. Protocol was
tin also increased significantly higher number B cells approved by Human Subject Committee of the Institu-
(ELISPOTassay) to secrete these cytokines. Increase in tion Review Board of the University of California,
leptin-induced IL-10 in aging may suggest a compensa- Irvine. Consent form approved by the Institutional Re-
tory mechanism to counterbalance increased IL-6 and view Board of the University of California was signed
TNF-α production. Since B regulatory cells produce by each subject. Consent form states that publications
IL-10 [34] it is possible that increased IL-10 production and/or presentations that result from this study will not
might be due to increased Breg in aging. This possibil- include identifiable information about you.
ity is under investigation. IL-10 rescues CD5+ cells
from apoptosis and augments the proliferation of CD5-
B cells [35-39]. Since IL-6 and TNF-α are produced by Antibodiesandreagents
both CD5+ and CD5- B cells, increased leptin-induced The following anti-human immunoglobulins were used:
B cell-derived IL-10 in aging may be playing a role in CD19 PerCP, CD69 FITC, CD25 APC, and CD5 PE; all
aging-associated chronic inflammation by surviving B fromBDParmingen(SanJose,CA).B cell enrichmentkit
cells from apoptosis to produce IL-6 and TNF-α. Buffa was purchased from Stem cell Technologies (Vancouver,
et al. [40] have reported an expansion of CD19+CD38- BC, Canada), and recombinant human leptin from
CD24- B cells in aged humans and these cells produce PeproTech (Rocky Hill, New Jersey).
bothTNF-α and IL-10.
Leptin signal via three distinct pathways, including
JAK-STAT, PI3K, and MAPK pathways [41-43]. Follow- Immunophenotyping
ing binding of leptin with leptin receptor, JAK2 (Janus B cells were stained with, APC-conjugated anti-CD25,
kinase 2) is activated by auto-or cross phosphorylation. FITC-conjugated anti-CD69, PE-conjugated anti-CD5
Activated JAK2 tyrosine phosphorylates intracellular do- monoclonal antibodies. After staining, cells were washed
main of leptin receptor. The phosphorylated receptor extensively with phosphate-buffered saline and analyzed
provides a docking site for STAT (signal transducer and byFlowcytometryusingFACScalibur(Becton-Dickenson,
activator of transcription) factors, particularly STAT3, San Jose, CA) equipped with argon ion laser emitting at
which is substrate of JAK2. STAT3 itself is a substrate 488 nm (for FITC, PE, and PerCP excitation) and a
for JAK2 and homo- or hetero-dimerization upon phos- spatially separate diode laser emitting at 631 nm (for
phorylation, translocates to the nucleus, and modulates APC excitation). Forward and side scatters were used
transcription of genes, including cytokine genes. We to gate and exclude cellular debris. Ten thousand cells
show that Leptin induced significantly greater phosphor- were acquired and analyzed using Flowjo software
ylation of STAT3 in B cells from aged subjects as com- (Treestar, Ashland, OR). Data were analyzed for both
pared to B cells from young controls, suggesting that percentages of cells expressing CD25 and CD69 as well
leptin-induced increased cytokine production by aged B as density of molecules as defined by mean fluoresence
cells isviaincreasedactivation ofSTAT3. intensity (MFI).
Conclusions
Leptin activates B cells from aged humans via increased PurificationofBcells
intracellular signaling to secrete IL-6, TNF-α, and IL-10 B lymphocytes were purified by immunomagnetic human
to a greater extent than B cells in young subjects, which Bcellenrichmentkitaccordingtomanufacturer’sinstruc-
may contribute to chronic inflammation associated with tions (STEMCELL Technologies, Vancouver, Canada). In
human aging. brief, peripheral blood mononuclear cells were suspended
at no more than 1×108 cells/ml in PBS containing 2%
Materials and methods FBS. Negative selection cocktail (100 μl/ml) was added
Subjects andincubatedatroomtemperaturefor15min.Themag-
Peripheralbloodmononuclearcells(MNCs)wereisolated neticnanoparticleswereaddedat50μl/mlcellsandincu-
frombloodof10healthyyoung(18–35years,mean23±4) bated for 10 min. Cells were placed in a 12×75-mm
and 10 healthy aged (66–84 years, mean 74±6) subjects polystyrene tube at a volume of 2.5 ml/tube and placed
by Ficoll–hypaque density gradient. Aged subjects are intothe magnet for 5 min. Themagnet was inverted,and
healthy people of middle and upper middle social sta- the supernatant was poured off. The magnetically labeled
tus, and are living active independent lives in the city of unwantedcellsremainboundinsidetheoriginaltube.The
Laguna Woods, California (senior community). All sub- purity of negatively selected cells was assessed by flow
jects were required to discontinue all vitamins and cytometry(>97%)asdetectedbythepresenceofCD20.
Guptaetal.Immunity&Ageing2013,10:3 Page6of7
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Measurementofcytokines Competinginterests
CytokinesecretionwasmeasuredbyELISA,andELISPOT Theauthorsdeclarethattheyhavenocompetinginterests.
assays. Purified B cells were activated by 25 ng/ml and
50 ng/ml of leptin for 24 h. Supernatants were collected Authors’contributions
SGmadethehypothesis,designedtheexperimentalapproach,andwritten
andstoredat−20°Cuntilassayedfordetectionofcytokines
themanuscript.SAperformedcytokineELISAandELISSPOTassaysfor
byELISA(ELISAkitsfromBDPharmingen,SanJose,CA) cytokines.SGset-uptheculturesanddidtheflowcytometryforactivation
as per manufacturer ’s protocol. Cytokine secreting cells markers.Manuscriptwasreadandeditedbyallauthors.
were measured by ELISPOTassay according to manufac-
turer ’s protocol. In brief, PVDF membrane ELISPOT Acknowledgement
AuthorswishtothankHoufenSuforhertechnicalexpertise.
plates (Millipore, Billerica, MA) were coated with 100 μl
(conc.2μg/ml)ofcaptureantibodyperwell(BDPharmin- Received:30November2012Accepted:13January2013
Published:23January2013
gen,SanJose),andwerekeptovernightat4°C.Plateswere
washed 3 times with sterile PBS and 200 μl/well of sterile
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