Activation of Adenosine2A Receptors Attenuates Allograft
Rejection and Alloantigen Recognition1
Charles P.Sevigny,2*Li Li,2*Alaa S.Awad,*Liping Huang,*Marcia McDuffie,†
Joel Linden,*‡§Peter I.Lobo,*and Mark D.Okusa3*‡§
The current studies investigated the in vitro and in vivo effect of adenosine2A receptor(A
2A
R)agonists to attenuate allogenic
immune activation.We performed MLRs with spleen T lymphocytes and APCs isolated from wild-type and A
2A
R knockout mice of both C57BL/6and BALB/c background strains.Two-way MLR-stimulated T cell proliferation was reduced by ATL313,a
lective A
2A R agonist in a do-responsive manner(ϳ70%;10nM),an effect reverd by the A
2A
R antagonist ZM241385(100
nM).By one-way MLRs,we obrved that ATL313’s inhibitory effect was due to effects on both T cells and APCs.ATL313
suppresd the activation markers CD25and CD40L and the relea of inflammatory cytokines IFN-␥,RANTES,IL-12P
70
,and IL-2.ATL313also incread negative costimulatory molecules programmed death-1and CTLA-4expresd on T cells.In lym-phocytes activated with anti-CD3e mAb,ATL313inhibited the phosphorylation of Zap70,an effect that was reverd by the protein kina A inhibitor H-89.In skin transplants,allograft survival was enhanced with ATL313,an effect blocked by
打一动物
ZM241385.The results indicate that A
2A
R agonists attenuate allogenic recognition by action on both T lymphocytes and APCs
in vitro and delayed acute rejection in vivo.We conclude that A
2A
R agonists may reprent a new class of compounds for induction therapy in organ transplantation.The Journal of Immunology,2007,178:4240–4249.
I nnate and adaptive immunity critically impacts acute and
chronic allograft rejection as well as ischemia-reperfusion injury(IRI),4and the ability to attenuate the effects could consistently enhance long-term allograft survival.T cells play a critical role in the pathogenesis of acute and chronic allograft re-jection(1).Activation of T cells requires specific signaling by the interaction between the TCR and Ag prented by APCs and is also regulated by positive and negative costimulatory signals.B7 family ligands and receptors play an important role in
controlling the activation and proliferation of T cells.Interaction of B7-1 (CD80)and B7-2(CD86)expresd on APCs with CD28ex-presd on T cells promotes T cell expansion and cytokine cre-tion(2).In contrast,interaction of B7-1and B7-2with CTLA-4 expresd on T cells,which is up-regulated after T cell activation, suppress T cell activation(3,4).Programmed death(PD)-1ex-presd on T cells is another inhibitory molecule regulated by IFN-␥,which binds to the APC ligands PD-L1(B7-H1)and PD-L2 (B7-DC).PD-L1shares20–38%aa identity with other B7family members(B7-1and B7-2)expresd on APCs and nonlymphoid tissues.Upon T cell activation,up-regulation of PD-1expression
also contributes to T cell homeostasis(5).Thus,the negative co-stimulatory pathways CTLA-4:B7and PD-1:PD-L down-regulate T cell-mediated alloimmunity,and studies in animals would sup-port the concept that PD-1and CTLA-4can induce transplantation tolerance.
Adenosine2A receptors(A
2A
Rs)are a subtype of the G protein-coupled receptor family of adenosine receptors,which also in-
cludes A
1
R,A
2B
R,and A
3
R(6,7),and they have tissue protective
properties(8).Systemically administered A
2A
R agonists have been shown to reduce tissue injury associated with ischemia-reper-fusion(9–12).Through a ries of studies,we demonstrated that
the protective effect of A
2A
R agonists was mediated through bone marrow-derived leukocytes(13).Furthermore,additional studies
indicate that A
2A
R agonists reduce kidney IRI through activation
of A
2A
Rs expresd on CD4ϩcells(14).A
2A
R agonists,including ATL313,attenuate IFN-␥and limit anti-CD3e mAb-induced T cell activation(15).Delayed graft function is a form of IRI that is thought to increa the immunogenicity of the transplanted allo-graft(8)in part due to an increa in MHC class I and II expression leading to episodes of acute rejection and chronic graft loss.The
findings suggest that A
2A
Rs may be uniquely suited to block T cell activation associated with IRI and delayed graft function.More-
over,given the diver effects of A
2A
R activation on T cell func-
tion,we considered the possibility that A
2A
R activation attenuates
allogenic recognition,thus rendering agonists of A
2A
Rs uful in attenuating rejection in transplantation.
In the current study,we sought to determine the effect of A
2A
R agonists in vitro on alloantigen-induced T cell activation and pro-liferation as well as the effect of costimulatory molecules.We
studied the effect of A
少数民族民歌2A
R activation on the MLR,an assay in which the lymphocytes from two parate mou strains are com-bined and cultured in vitro to initiate an alloimmune respon.An advantage of this approach is that T cell activation occurs under physiological conditions that involve TCR activation and Ag pre-nted by dendritic cells(DCs)and that are regulated by positive
上学祝福语*Department of Medicine,†Department of Microbiology,‡Cardiovascular Re-arch Center,and§Carter Immunology Center University of Virginia,Charlottes-ville,VA22908
Received for publication January30,2006.Accepted for publication January 10,2007.
The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertiment in accordance with18U.S.C.Section1734solely to indicate this fact.
1This work was supported by National Institutes of Health Grants DK56223, DK62324,DK58413,and HL37942.
2C.P.S.and L.L.participated equally in this study.
3Address correspondence and reprint requests to Dr.Mark D.Okusa,Division of Nephrology,Box800133,University of Virginia Health System,Charlottesville,VA 22908.E-mail address:mdo7y@virginia.edu
4Abbreviations ud in this paper:IRI,ischemia-reperfusion injury;A
2A R,adenosine
2A receptor;DC,dendritic cell;KO,knockout;MCF,mean channelfluorescence;PD,
programmed death;PKA,protein kina A;WT,wild type.
Copyright©2007by The American Association of Immunologists,Inc.0022-1767/07/$2.00
The Journal of Immunology www.jimmunol
and negative costimulatory molecules.In vitro studies demonstrate
that,in respon to T cell activation by alloantigens,A
2A
R ago-nists attenuate T cell activation and decrea cretion of proin-flammatory cytokines through direct and independent effects on
both T cells and APCs.Importantly,the A
2A
R agonist incread expression of negative costimulatory molecules PD-1and CTLA-4 that are involved in peripheral tolerance.Lastly,we demonstrated
读研条件that the in vitro inhibitory effects of A
2A
R agonists on alloantigen-induced immune respon likely contribute to the attenuation of tissue rejection following skin transplantation.The results sug-
gest that A
2A
R activation is a unique and potent strategy in atten-uating IRI and delaying allograft rejection following organ transplantation.
Materials and Methods
Animals
All animals were handled and procedures were performed in adherence to the National Institutes of Health Guide for the Care and U of Laboratory Animals and in accordance with the University of Vi
rginia Animal Care and U Committee protocols.C57BL/6(B6)and BALB/c mice were pur-chad from Charles River Laboratories.The source and derivation of con-
genic A
2A R knockout(KO)mice was described previously(13,16).To
generate Adora2a null(A
2A RKO)mice congenic to BALB/c(17–19),a
mapping panel of55microsatellite loci informative for a(C57BL/6JϫBALB/cByJ)cross was identified with coverage for every chromosome, except X and Y,at a density ofϽ30cM.The mapping panel is available on request(mjm7e@virginia.edu).A single founder male was mated to
BALB/cByJ female mice.(BALB/cϫB6.129-Adora2a tm1jfc)F
1males car-
rying the Adora2a mutation were then mated to BALB/cByJ females in two successive generations of
backcrossing with breeders lected for maximal BALB/cByJ homozygosity.The BALB/cByJ Y chromosome was intro-duced by crossing BALB/cByJ males to genotypically lected F1N3fe-males carrying the Adora2a mutation in thefinal backcross generation before initiation of inbreeding with F1N4mice of both xes.Residual B6 or129alleles in the congenic line were detected only on mou chromo-some10between Adora2a and D10Mit35(74.77–121.60Mb,respectively; NCBIm36).
Harvesting of mou splenocytes
Spleens were extracted from B6and BALB/c mice and disrupted under sterile conditions in PBS through40-m BD Falcon cell strainers(Fisher Scientific).Leukocytes were then isolated via density gradient centrifuga-tion using Histopaque1083(Sigma-Aldrich)and washed in RPMI1640 supplemented with10%heat-inactivated FBS(Invitrogen Life Technologies) and1%antibiotic/antimitotic solution(Invitrogen Life Technologies).Cells were resuspended(1ϫ106cells/ml)in the culture medium in the prence of adenosine deamina(1U/ml;Roche Diagnostics).
魅力上海
Activation of lymphocytes by alloantigens by MLRs
In two-way MLR assays,2ϫ105leukocytes from B6mice in0.1ml were cocultured with an equal numb
er of leukocytes from BALB/c mice using 96-wellflat-bottom tissue culture-treated plates.Appropriate compounds were administered at the initiation of the two-way MLR assay.All com-pounds were diluted in10l of medium before being added to the appro-priate wells,and cells were allowed to incubate at37°C in the prence of
5%CO
2.As controls,we added vehicle without compound to cell cultures.
Second,in two-way MLR assays,cells obtained from each murine strain were cultured ,without mixing,so as to calculate the added effect obtained with cocultures.
For one-way MLR studies,cell cultures were prepared as described previously(20).Stimulator cells were prepared from spleens of wild-type
(WT)(B6background)or A
2A KO(B6background)mice.Spleens were cut
into small pieces and digested with1mg/ml collagena type IA(Sigma-Aldrich)for10min at37°C,whic
h can increa the yield of APCs.Single-cell suspension was treated with mitomycin C(50g/ml;Sigma-Aldrich) for20min at room temperature and then washed twice with RPMI1640. Responder spleen T cells from spleens of WT(BALB/c background)or
A
2A KO(BALB/c background)mice were harvested by negative isolation
using magnetic beads,according to the manufacturer’s protocol(Dynal T Cell Negative Isolation kit;Invitrogen Life Technologies).The purity of the CD3T cells was measured by FACS(BD Biosciences),and T cells withϾ98%purity were ud in this study.Responder(2ϫ105)and stim-ulator(4ϫ105)cells were added to round-bottom96-well plates to afinal volume of200l of RPMI1640with10%FCS/1%antibiotic/antiamitotic solution and in the prence of adenosine deamina(1U/ml;Roche Di-agnostics).Each experiment was performed in triplicate.
Vehicle or4-[3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetra-hydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-piperidine-1-carboxylic acid methyl ester(ATL313)was ud in two-way(0.01–100nM)and one-way(10 nM)MLRs.4-(2-[7-Amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol(ZM241385)(100nM)was ud to determine speci-ficity of ATL313for the A
2A
R.
Activation of lymphocytes by anti-CD3e
In the studies,leukocytes were activated with anti-CD3e mAb.Func-tional grade hamster anti-mou CD3e mAb(clone145-2C11;eBio-science)was ud in an insoluble form(by precoating tissue culture-treated plates)or in soluble form.Plates were coated by covering wells with Ab in PBS(2g/ml)for4h at37°C,then rind twice with PBS before adding cells.Soluble Ab was ud at different concentrations at the initiation of the culture.Controls were the same as the two-way MLR assay. Proliferation assays
Lymphocytes activated in an MLR or by anti-CD3e mAb were allowed to incubate up to3days before harvesting.[3H]Thymidine(0.5Ci/well;MP Biomedicals)was added18–24h before harvesting(Skatron Instruments) using Type Afilter mats(PerkinElmer Life and Analytical Sciences)and beta plate scintillation mixture(PerkinElmer).Disintegrations per minute were determined using a liquid scintillation counter(1205Betaplate; PerkinElmer).
Multiplex bead array for quantitating cytokines
Multiplex bead array was performed on supernatants obtained on day3of culture.Microbead labeling was performed using the Bio-Plex multiplex cytokine assay kit(Bio-Rad)in accordance with manufacturer’s protocol, and cytokine levels were analyzed on the Bio-Plex system(Bio-Rad). ELISA for quantitating IFN-␥
ELISA was performed on supernatants obtained on day3of culture.The ELISA for IFN-␥(IFN-␥Ready,Set,Go!ELISA kit;eBioscience)was performed according to manufacturer’s protocol and analyzed on a Model 680microplate reader(Bio-Rad).
Multiprobe RNa protection assay
Total RNA was prepared using RNAzol B(Leedo Medical Laboratories) and analyzed by1.5%agaro gel electrophoresis to asss the integrity of RNA before solution hybridization.Cytokine mRNA expression was as-sd by BD RiboQuant Multiprobe RNa protection system(BD Pharm-ingen),according to the manufacturer’s protocol.In brief,mRNA-specific RNA probes were labeled with32[P]UTP using multiprobe template ts (mCK3b;BD Pharmingen)for cytokine genes.Total cellular RNA was subjected to solution hybridization with each probe t.Hybridization was performed at56°C before RNa treatment.Following RNa treatment, protected fragments were
parated by gel electrophoresis on5%poly-acrylamide gels and expod to Kodak X-Omat ARfilm atϪ70°C with a single intensifying screen.Band densities were quantitated using the Personal Densitometry SI(GE Healthcare),and data were analyzed by ImageQuant5.2(GE Healthcare).
Flow cytometry
Allflow cytometry was performed on a BD FACSCaliburflow cytometer (BD Biosciences),and data were analyzed with FlowJo software4.2(Tree Star).Most of the Abs were purchad from eBioscience.
Apoptosis/Necrosis
An Annexin V FITC apoptosis detection kit(BD Pharmingen)was ud to analyze MLR-activated lymphocytes after72h of culture with the control vehicle or ATL313(10nM)with or without ZM241385(100nM).Cul-tured cells(1ϫ106cells/ml)were washed with cold PBS and resuspended in annexin V binding buffer.The cell suspension(100l)was mixed with annexin V(5l)and propidium iodide(5l)in a5-ml culture tube and incubated for15min in the dark at room temperature.A total of400l of binding buffer was added to each sample before analysis byflow cytometry.
CD4/CD25and CD4/CD40L
Three-day two-way MLR-activated cells were washed with1%BSA/PBS and labeled with anti-mou CD4-allophycocyanin(GK1.5;4g/ml)and
4241
The Journal of Immunology
either anti-mou CD25-PE (PC61.5;4g/ml)or anti-mou CD40L-PE (MR1;4g/ml)in 25l of 0.2%BSA/PBS after blocking FcRs by CD16/32(2.4G2;eBioscience)for 30min on ice.Isotype controls were also performed.Samples were washed with 0.2%BSA/PBS and 2g/ml 7-aminoactinomycin D (Invitrogen Life Technologies)before analysis by flow cytometry.
CD4/P-Zap70
Spleen cells were isolated from B6mice as described above,then incubated with soluble anti-CD3e mAb (145-2C11;10g/ml)to stimulate lympho-cytes (1ϫ106)for 5min with or without ATL313(10nM)or with or without ZM241385(100nM).Due to the short reaction time in the pres-ence of soluble anti-CD3e mAb,the cells were pretreated with the com-pounds for 30min before stimulation with the Ab.Adding 4volumes of cold PBS terminated the reaction.Cells were collected by centrifugation and t
hen fixed with 2%formaldehyde for 10min at room temperature,centrifuged again,and rind once with PBS.The supernatant was dis-carded,and the tubes were chilled on ice for 1min.Ice-cold methanol (90%)was added slowly during gentle vortexing before incubating the suspension for 30min on ice.Cells were centrifuged and resuspended in 0.5%BSA/PBS and anti-mou CD16/32(2.4G2;10g/ml)for 10min at room temperature to block nonspecific binding.Rabbit anti-human P-Zap70(1/100;Cell Signaling Technology)was added and incubated for 30min at room temperature.Cells were rind as before in blocking buffer,centrifuged,and resuspended in goat anti-rabbit IgG-PE (1/2000;Southern Biotechnology Associates)in blocking buffer for 30min at room temper-ature,rind,resuspended in 200l of cold PBS,and analyzed by flow cytometry.Isotype and condary Ab controls were also performed at the same time.
PD-1and CTLA-4
Cells activated for 3days with soluble anti-CD3e mAb (145-2C11;10g/ml)were washed with 0.2%BSA/PBS,incubated with rat anti-mou CD16/32(2.4G2;10g/ml)to block FcRs,and added hamster anti-mou CD3e-Alexa 647(500A2,4g/ml;Invitrogen Life Technologies)and PD-1-PE (RMP1–30;4g/ml),CD4-allophycocyanin (GK1.5),or PD-1-PE (RMP1–30;4g/ml)for 30min on ice.Appropriate isotype control (ham-ster IgG-Alexa 647,rat IgG2b-PE,rat IgG2a;4g/ml each)was u
d.For intracellular T cell CTLA-4staining,cell surface was stained with hamster anti-CD3-Alexa 647(500A2,4g/ml;Invitrogen Life Technologies),washed,fixed,and permeabilized for 30min using the BD Cytofix/Perm kit (BD Biosciences).Cells were then washed using 1ϫpermeabilization buffer,and hamster anti-mou CTLA-4-PE (UC10-4B9;4g/ml)was added on ice for 30min.Isotype controls using hamster IgG-Alexa 647and hamster IgG-PE were also performed accordingly.Cells were washed and suspended in 0.2%BSA/PBS before running the sample on FACSCalibur.
Skin transplantation
Experiments were conducted in B6mice (recipient)and BALB/c mice (donor)(7–8wk of age;Charles River Laboratories)and were allowed free
access to food and water until the day of surgery.Mice were anesthetized with a regimen that consisted of ketamine (100mg/kg,i.p.)and xylazine (10mg/kg,i.p.)and were placed on a thermoregulated pad to maintain body temperature at 37°C.Full-thickness tail skin (1cm 2)from donor mice was grafted on the dorsal flank area of recipient mice.The skin grafts were cured with paraffin-embedded gauze and with adhesive tape for 5days.Graft survival was assd by visual inspection from day 6to day 9in a masked fashion.The measured parameters were 1)necrosis,2)loss
of viable tissue,and 3)hair growth.Rejection was diagnod when the graft loss was Ն70%.At the end of the experimental period,animals were eu-thanized,and skin graft was removed for H&E staining.Sections were viewed using a Zeiss AxioSkop microscope,and digital images were taken using a SPOT RT Camera (software version 3.3;Diagnostic Instruments).A miquantitative score was assigned bad on the masked reading for thickness of the epidermis (0–2)in which 2ϭnormal;1ϭloss of Ͻ50%thickness;and 0ϭloss of Ͼ50%thickness and the degree of inflammation (0–5)in which score 0ϭnormal;1ϭϽ20%;2ϭ20–40%;3ϭ40–60%;4ϭϾ60–80%;and 5ϭϾ80%inflammation.Skin allograft recipients were treated with vehicle or ATL313(1ng ⅐kg Ϫ1⅐min Ϫ1)alone or com-bined with ZM241385(5ng ⅐kg Ϫ1⅐min Ϫ1)via osmotic pump (ALZA)(n ϭ8for each group).
Statistical analysis
Statistical analysis of all do-respon curves was analyzed by two-way ANOVA.Tukey or Bonferroni post hoc analysis was performed to asss significance at specific concentrations in the curves as well as for mean channel fluorescence (MCF)comparisons.Least significant difference post hoc analysis and paired and unpaired Student’s t tests were also ud in some analys.All curves were fit by nonlinear regression,sigmoidal do-respon parameters.Kaplan-M
eier survival curve was ud to asss the difference between allograft survival.All statistical analys were per-formed using GraphPad Prism version 4.0(GraphPad).A value of p Ͻ0.05was ud to determine significance.
Results
ATL313attenuates T cell proliferation
In the two-way MLR assay,maximum proliferation of cells was obrved by day 3of culture.Hence,we ud this time point to evaluate the effect of various compounds on two-way MLR-in-duced cell proliferation.
Two-way MLRs were performed in the abnce or prence of ATL313(0.01–100nM).ATL313reduced proliferation in a do-dependent manner with maximum inhibition achieved at a do of 10nM (33%of vehicle control,p Ͻ0.001,n ϭ8).The addition of the A 2A R antagonist ZM241385(100nm)attenu-ated the effect of ATL313(10nm;83%of vehicle control,p Ͻ0.001,n ϭ6;Fig.1a
).
FIGURE 1.ATL313activates A 2A Rs and reduces lymphocyte proliferation.a ,The effect of ATL313on the proliferative respon in two-way MLRs was examined in the abnce (solid line,n ϭ8)and prence (dashed line,n ϭ6)of ZM241385(100nm).Values are means ϮSEM and are calculated as pe
rcent increa in proliferation of cells compared with control vehicle-treated cells.Under vehicle conditions,[3H]thymidine incorporation in two-way MLRs,a measure of cell proliferation,ranged from 10,000to 20,000dpm/well.b ,Reduction of proliferation by ATL313in anti-CD3e mAb-activated lymphocytes.The effects of ATL313on lymphocyte proliferation were examined after 24h of activation by plate-bound anti-CD3e mAb in both WT (solid line,n ϭ4)and A 2A RKO (dotted line,n ϭ4)B6background splenocytes.Values depict mean normalized percentages between control anti-CD3e mAb-induced proliferation (vehicle-treated)and nonactivated lymphocytes (0%)ϮSEM.Activated lymphocytes under vehicle treatment demonstrated a range of ϳ20,000–30,000dpm/well.
4242
A 2A R AGONISTS ATTENUATE ALLOGRAFT REJECTION
The effect of ATL313was not the result of an increa in apo-
ptosis/necrosis as revealed by annexin V and propidium iodide labeling,markers for apoptosis and necrosis,respectively.There was no significant difference in the level of apoptosis or necrosis when cells were cultured without alloantigen activation(control) or when cells were activated in a two-way
MLR.Similarly,the addition of ATL313and/or ZM241385did not increa apoptosis/ necrosis(data not shown).
To confirm that the attenuation by ATL313on lymphocyte pro-liferation was due to activation of A
局域网搭建
2A
Rs,we performed anti-CD3e mAb-induced T cell proliferation by using leukocytes obtained
from WT B6and A
2A
KO mice(B6background).In splenocytes obtained from WT B6mice and stimulated for24h with anti-CD3e mAb,ATL313(0.01–100nM)reduced lymphocyte prolif-eration.The effect was maximal at10nM(51%of control,pϽ
0.001,nϭ4).In splenocytes obtained from A
2A
RKO mice, ATL313(0.01–100nM)had no effect(Fig.1b).The results in-dicate that lymphocyte proliferative respons induced by alloan-tigens or anti-CD3e mAb are markedly attenuated by specific ac-
tivation of A
2A Rs.
ATL313inhibits lymphocyte proliferation by action on both
T lymphocytes and APCs in one-way MLRs
The ATL313-specific effect on T cell and APC A
2A
Rs was exam-
ined by using A
2A
R KO mice.In this one-way MLR system,T
cells were harvested from BALB/c background WT and/or A
2A
R
KO mice;APCs were harvested from B6background WT and/or
A
2a
R KO mice.The purity of the negative isolation T cells was
Ͼ98%.We performed four groups of one-way MLRs:responder
WTϫstimulator WT(T cell BALB/cϫAPC B6),responder
KOϫstimulator KO(T cell BALB/c KOϫAPC B6KO),re-
sponder KOϫstimulator WT(T cell BALB/c KOϫAPC B6),
and responder WTϫstimulator KO(T cell BALB/cϫAPC B6
KO).In one-way MLRs in which both responder and stimulator
cells expresd A
2A
Rs(T cell BALB/cϫAPC B6),ATL313
reduced T cell proliferation by95%(pϽ0.01;nϭ4;Fig.2a).
This result is consistent with the two-way MLR result obrved in
Fig.1a.When both responder and stimulator cells lacked A
2A
Rs
(T cell BALB/c KOϫAPC B6KO),ATL313had no significant
effect on T cell proliferation(pϭNS;nϭ4;Fig.2b).When
Table I.Effects of A
2A
R agonists on two-way MLR-induced cytokine relea a
A(vehicle)B(ATL)C(ATLϩZM)A vs Con A vs B B vs C A vs C IFN-␥17.44 3.6413.84pϽ0.001pϽ0.001pϽ0.01NS
IL-221.8713.1221.22pϽ0.001pϽ0.05pϽ0.05NS
IL-12(p70) 2.150.67 1.44pϽ0.001pϽ0.001pϽ0.05NS
IL-12(p40) 2.14 2.14 1.97pϽ0.001NS NS NS
RANTES 2.16 1.44 1.95pϽ0.001pϽ0.001pϽ0.001NS
a Data indicate fold of two-way MLR-induced cytokine and chemokine relea over unstimulated control levels.A,vehicle;B,
ATL313(ATL,10nm);and C,ATL313(10nm)ϩZM241385(ZM,100nm).Significance columns asss differences between
vehicle and control(A vs Con),vehicle and ATL313(A vs B),ATL313and ATL313ϩZM241385(B vs C),and vehicle and
ATL313ϩZM241385(A vs C),respectively.Values are means,nϭ
八卦是什么意思
3.
FIGURE2.ATL313inhibits lym-
phocyte proliferation by action on both T lymphocytes and APCs in one-way MLRs.One-way MLRs were per-formed as described in Materials and Methods.Stimulator cells were pre-pared from spleens of WT(B6back-ground)or A
2A
RKO(B6background) mice and responder spleen T cells were from WT(BALB/c background)
or A
2A
RKO(BALB/c background) mice.We performed four groups of one-way MLR:responder WTϫstim-ulator WT(T cell BALB/cϫAPC B6) (a),responder KOϫstimulator KO(T cell BALB/cKOϫAPC B6KO)(b), responder KOϫstimulator WT(T cell BALB/c KOϫAPC B6)(c),and responder WTϫstimulator KO(T cell BALB/cϫAPC B6KO)(d). One-way MLRs were performed in the prence of vehicle,ATL31
3(10nM), ATL313ϩZM241385(100nM),and ZM241385alone.Experiments were performed in triplicate,nϭ4for each group.Values are meanϮSE and are normalized to vehicle group.4243
The Journal of Immunology
responder cells (T cell BALB/c KO ϫAPC B6)or stimulator cells (T cell BALB/c ϫAPC B6KO)lacked A 2A Rs,ATL313reduced T cell proliferation by 38%(p Ͻ0.05,n ϭ4;Fig.2c )or 53%(p Ͻ0.001,n ϭ4;Fig.2d ),respectively.The effect of ATL313on T cell proliferation was blocked by the A 2A R antagonist ZM241385(Fig.2,a ,c ,and d ).It is interesting to note that the full effect of ATL313,apparent when both responder and stimulator cells expresd A 2A R (Fig.2a ),reprented an additive effect of
ATL313expresd individually on stimulator (Fig.2c )and responder (Fig.2d )cells.The results indicate that A 2A Rs ex-presd on both T cells and APCs mediate the inhibitory effect of lymphocyte proliferation following alloantigen immune activation.ATL313inhibits IFN-␥and cytokine relea
The effects of ATL313on two-way MLR-induced cytokine relea are summarized in Table I.Using ELISA,the supernatants of two-way MLRs showed a 17-fold increa of IFN-␥over unstimulated lymphocytes that were not mixed in an MLR (p Ͻ0.001,n ϭ3).The increa of IFN-␥relea was signi
ficantly reduced in a do-dependent manner by ATL313,an effect that was inhibited by the addition of ZM241385(p Ͻ0.0001,n ϭ3).Maximum inhibition of IFN-␥relea was obrved with 10nM ATL313(21%of ve-hicle,p Ͻ0.001,n ϭ3).ZM241385reduced the respon ob-rved with 10nM ATL-313to 79%of vehicle (Table I).mRNA from the cultured lymphocytes was harvested at day 3of culture and analyzed for steady-state IFN-␥mRNA expression by RNa protection assay.Two-way MLRs produced an increa in IFN-␥mRNA expression,an effect that was reduced with 10nM ATL313.The effect of ATL313on IFN-␥mRNA expression was blocked by ZM241385(Fig.3).Quantitative analysis of band den-sities,corrected for loading (GAPDH),demonstrated that IFN-␥mRNA incread in MLRs to 10.8-,5.5-,and 10.5-fold over un-stimulated cultured lymphocytes following incubation with vehi-cle,ATL313,and ATL313plus ZM241385,respectively (n ϭ2).Supernatant levels of IL-2,IL-12(p70),IL-12(p40),and RANTES were measured simultaneously by multiplex bead array (Table I).In the studies,cytokines in supernatants obtained from day 3of two-way MLR cultures were compared with baline cytokine relea from unstimulated cultured lymphocytes.
IL-2
FIGURE 3.Effect of ATL313on IFN-␥mRNA expression.Total RNA was isolated from cells of two-way MLRs and subjected to RNa protec-tion assay analysis of IFN-␥.A reprentative photograph of this result is shown.Each lane reprents solution hybridization with a radiolabeled probe for IFN-␥and RNA derived from two-way MLRs.GAPDH is shown as a control for loading.Veh,vehicle;unstim,
unstimulated;ATL,ATL313;ZM
ZM241385.
环保建议FIGURE 4.Effect of ATL313on cell surface expression of CD25on CD4ϩT cells in two-way MLRs.a–c ,Cell surface expression of CD25(y -axis)was measured by gating on CD4ϩ-expressing T cells (x -axis)after treatment with vehicle (a )or 10nM ATL313in the abnce (b )and pres-ence of 100nM ZM241385.Levels of CD25and CD4,as analyzed by flow cytometry,are expresd as MCF of PE and APC,respectively.d ,Quan-titative asssment of CD25expression.Results reprent mean of per-centage of control values for unstimulated lymphocytes (100%)ϮSEM;n ϭ4;ء,p Ͻ0.001compared with vehicle or ATL313ϩ
ZM241385.
FIGURE 5.Effect of ATL313on cell surface expression of CD40L on CD4ϩT cells in two-way MLRs.a–c ,Cell surface expression of CD40L (y -axis)was measured by gating on CD4Ϫ-expressing T cells (x -axis)follow-ing treatment with vehicle (a )or 10nM ATL313in the abnce (b )and pres-ence of 100nM ZM241385.Levels of CD40L or CD4,as analyzed by flow cytometry,are expresd as MCF of PE and APC,respectively.d ,Quantitative asssment of CD40L expression.Results reprent mean of percentage of control values for unstimulated lymphocytes (100%)ϮSEM;n ϭ3.
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A 2A R AGONISTS ATTENUATE ALLOGRAFT REJECTION
