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. Guptaetal.Immunity&Ageing2013,10:3 Page2of7 http://www.immunityageing.com/content/10/1/3 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. Guptaetal.Immunity&Ageing2013,10:3 Page3of7 http://www.immunityageing.com/content/10/1/3 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. Guptaetal.Immunity&Ageing2013,10:3 Page4of7 http://www.immunityageing.com/content/10/1/3 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, Guptaetal.Immunity&Ageing2013,10:3 Page5of7 http://www.immunityageing.com/content/10/1/3 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 http://www.immunityageing.com/content/10/1/3 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 blockingbufferwasaddedfor≥1hour.Plateswerewashed References 1. CavaA,MatareseG:Theweightofleptininautoimmunity.NatRev 3 times with sterile PBS. Purified B cells (12,500; 25,000; Immunol2004,4:371–379. and50,000/well)wereplacedintriplicate.Twentyfiveand 2. LoffredaS,RaiR,YangSQ,etal:Leptinregulatesproinflammatory immuneresponses.FASEBJ1998,12:57–65. 50 ng/ml leptin was added to each well and plates were 3. 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