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LA-B35 Influences the Apoptosis Rate inuman Peripheral Blood Mononucleatedells and HLA-Transfected Cells

iulia Salazar, Gualtiero Colombo, Stefania Lenna,ita Antonioli, Lorenzo Beretta,

lessandro Santaniello, and Raffaella Scorza

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ABSTRACT: Human leukocyte antigen (HLA) class Iantigens can act as signal-transducing molecules thatinfluence individual reactivity to external stimuli andthe existence of haplotype-specific cell signal regulationhas been suggested. In this article, we provide definiteexperimental evidence for the existence of a HLA-B35haplotype-specific regulation of cell apoptosis in differ-ent experimental models. First, we demonstrated thatHLA-B35, but not other HLA-class I antigens, wasassociated with an increased cell susceptibility to apo-ptosis in human peripheral mononuclear cells (PBMCs)exposed in vitro to thapsigargin. Second, we confirmedthis association in human ECV 304 cells transfected

with HLA-B35 or with HLA-B8, an antigen that did P

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0122 Milano, Italy. E-mail: raffaella.scorza@unimi.it.Received July 12, 2005; accepted November 4, 2005.

uman Immunology 68, 181–191 (2007)American Society for Histocompatibility and Immunogenetics, 2007

ublished by Elsevier Inc.

ot appear to influence the apoptosis rate in the thap-igargin-treated PBMCs. Third, we confirmed the spe-ific influence of HLA-B35 on cell apoptosis in nonuman cells (i.e., HLA-B35-transfected NIH3T3 mu-ine fibroblasts). Our data show the existence of HLA-35 haplotype-specific regulation of cell apoptosis andpen new perspectives on the role of HLA class I genesn cell activation and disease susceptibility. Humanmmunology 68, 181–191 (2007). © American Societyor Histocompatibility and Immunogenetics, 2007.ublished by Elsevier Inc.

EYWORDS: Apoptosis, HLA-B35, HLA class I,

BMC, ECV304, NIH3T3, thapsigargin, Taxol

ABBREVIATIONSHLA human leukocyte antigenPBMC peripheral mononuclear cells

sDNA single-strand DNAG thapsigargin

NTRODUCTIONuman leukocyte antigen (HLA) class I genes encode for

ighly polymorphic transmembrane glycoproteins ex-ressed by all nucleated cells involved in the presentationf endogenous peptides to CD8 T cells and thereforerigger cell-mediated immune responses. HLA polymor-hism influences the ability of different HLA moleculeso present endogenous peptides; such differences arelaimed to underlie most of the associations betweenLA class I antigens and susceptibility to diseases [1–3]

From the Unit of Clinical Immunology and Allergology and Division ofnternal Medicine, University of Milano and Fondazione IRCCS Ospedaleaggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy.

Address reprint requests to: R. Scorza, Unità di Immunologia Clinica ellergologia, Università degli Studi di Milano and Fondazione IRCCSspedale Maggiore Policlinico, Mangiagalli e Regina Elena, Via Pace 9,

r progression of infectious diseases [4–8]. However,LA polymorphism does not explain all the associations

etween HLA and diseases. Several reports showed thatLA class I molecules, besides their pivotal role in

ntigen presentation, can act as signal transducing mol-cules that influence individual reactivity to externaltimuli. Ligand or antibody binding to different poly-orphic class I determinants have been shown to trigger

ntracellular signals that lead to the release of calciumrom intracellular stores, modulating the function ofmportant cell surface receptors with prominent regula-ory effects, ranging from cell activation and prolifera-ion [9, 10] to cell apoptosis [11–16]. Moreover, someata showed that HLA class I molecules differ in theirbility to modulate cell signaling and suggested thexistence of an haplotype-specific regulation of signal

ransduction [17–21]. Apoptosis is one basic physiologic

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echanism involved in the control of cell differentiationnd tissue homeostasis; it is also considered an importantathogenetic mechanism in many human diseases, in-luding those that are degenerative, infectious, and au-oimmune. Our working hypothesis is that the expres-ion of different HLA class I antigens influences the ratef response to apoptotic stimuli, thus providing thevidence for a possible mechanism to explain the associ-tion between HLA and diseases. In particular, we fo-used on the HLA B35 allele, whose influence on theusceptibility to several diseases [22–28] and ability toodulate specific cell functions [17, 29–31] has long

een recognized. To better solve this issue, we used aultistep approach; first, we investigated the existence

f the control of cell apoptosis by HLA antigens inuman peripheral mononuclear cells (PBMCs) and webserved that HLA.B35, but not other unrelated HLA-lass I antigens, was associated with an increased cellusceptibility to apoptosis. Second, we confirmed theLAB35 proapoptotic effect in human ECV 304 cells;

hird, we confirmed the specific influence of HLA-B35n cell apoptosis in nonhuman cells (i.e., in HLA-ransfected NIH3T3 murine cells).

ATERIALS AND METHODSellsuman PBMCs were obtained from venous blood of 18LA-typed healthy individuals by density gradient cen-

rifugation and maintained in RPMI 1640 (Invitrogen;arlsbad, CA), fetal bovine serum 10% (Hyclone, Logan,T), glutamine 2 mM, penicillin 50 U/ml, and strepto-ycin 50 �g/ml.Human ECV304 endothelial cell (ECACC No.

2091712) were grown in M199 (Invitrogen) supple-ented with 10% fetal bovine serum (Hyclone), peni-

illin 50U/ml, and streptomycin 50 �g/ml at 37 °C inumidified air with 5% CO2. ECV 304 HLA type was:LA-A*01; B*18; DRB1*03011,0305; DQA1*0501;QB1*0201 Typing was performed at a genomic levely Ino-LiPA system (Innogenetics; Gent, Belgium).

Murine NIH-3T3 fibroblasts (ATCC #CRL-1658)ere grown in DMEM (Invitrogen) supplemented with0% fetal bovine serum (Hyclone), glutamine 2 mM,enicillin 50 U/ml, and streptomycin 50 �g/ml at 37 °Cn humidified air with 5% CO2.

lasmid Constructionull-length cDNAs of the HLA class I B*3501 and*0801 alleles were cloned into the eukaryotic expres-

ion vector pIRESneo2 (Clontech; Palo Alto, CA), usingreverse transcription-polymerase chain reaction strategy

32, 33]. Briefly, total RNA was isolated from PBMC of

onors previously typed for HLA class I antigens and a

everse transcribed. First-strand cDNA was amplifiedocus specifically using forward and reverse primers thatnneal to relatively conserved 5= and 3= untranslatedegions, namely a general class I 5= primer (GGGC-AATTCGGACTCAGAATCTCCCCAGACGCCGAG)

nd a HLA-B specific 3= primer (GCCCGGATCCCT-GGGAGGAAACACAGGTCAGCATGGGAAC). Re-

triction sites for EcoRI and BamHI (underlined) werencorporated in the 5= and 3= primers, respectively, tonable the unidirectional cloning of the amplified frag-ents into the expression vector. Clones containing theLA-B35 allele were identified through an ApaI site in

xon 3, which distinguishes B35 from most other HLA-Blleles. Both strands of the entire coding region of the B35nd B8 cDNAs were sequenced and 100% sequence iden-ity with the published HLA class I nucleotide sequences34] was obtained.

IH 3T3 and ECV304 Transfectionells were seeded at a density of 1 � 106cells/ml in a4-well plate and transfected with pIRESneo2 vector,hich confers neomycin resistance, containing HLA-*3501, B*0801, or no insert, using Lipofectamine 2000eagent (Invitrogen). Briefly, ECV304 and NIH-3T3 cellsere grown in appropriate medium to 90% confluence. A

otal of 100 �l of each DNA-Lipofectamine complex (1 �gf plasmid construct and 2 �g of Lipofectamine in 100 �lf medium) were added to the cells. Three days afterransfection, 500 �g/ml G418 (Invitrogen) was added tohe medium. After 3 weeks, G418-resistant cells wereicked and maintained in complete medium containing418. Cells expressing HLA-B35 or HLA-B8 were iden-

ified by allele-specific polymerase chain reaction, andembrane localization was demonstrated by fluorescenceicroscopy using HLA class I antigen-specific antibodies.hree independent transfections were performed for eachLA allele.

reatmentsilot dose-response studies were carried out to determinehe optimal concentration of each agent in our cell sys-em. PBMC were treated with 5 �M thapsigargin (TG)n complete medium, at 37 °C in 5% CO2, for 6 and4 h. NIH-3T3 fibroblasts were grown on coverslips andaintained in a quiescent state for 12 h in completeedium supplemented with 1% fetal bovine serum;

poptosis was induced by treatment with 5 �M TG forand 24 h. ECV304 cells were treated with 50 �M

axol for 6, 12, 24, and 48 h at 37 °C in 5% CO2. Taxolas used instead of thapsigargin because, in preliminary

xperiments, the latter was not capable of inducing ap-ptosis in ECV304 cells (data not shown). Moreover,axol has been shown to be the most powerful single

gent capable of inducing apoptosis in ECV 304 cells

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35], and to our knowledge no author has ever used TGo induce apoptosis in ECV304 cells.

poptosis Detectionenomic DNA internucleosomal fragmentation was visu-

lized by DNA laddering. Briefly, after TG treatment orerum deprivation, cells were incubated with 0.5 ml lysisuffer (0.5% Triton X-100, 10 mM EDTA, and 10mMris, pH 7.4) at 37 °C for 40 minutes, and then at 4 °C for0 minutes. Cell debris were removed by centrifugation,nd low molecular-weight DNA was phenol-extractedrom supernatants. DNA fragments were ethanol-precipi-ated and resolved by agarose/EtBr gel electrophoresis.

Nuclear chromatin staining with Hoechst 33258 wassed to detect condensation of the nuclei and apoptoticodies. Both NIH-3T3 cells and PMBC were fixed with.7% paraformaldehyde (20 minutes), permeabilizedith 0.5% Triton X-100 (5 minutes), and finally incu-ated with Hoechst 33258 1 �g/mL for 30 minutes at7 °C. After a final rinse with phosphate-buffered saline,he coverslips were mounted with glycerol–phosphate-uffered saline. Nuclei were visualized under fluores-ence microscopy (ultraviolet excitation at 380 nm), andhe percentage of apoptotic cells was determined byounting at least 400 cells. Apoptotic cells were recog-ized by their smaller size, condensed chromatin, andragmented nuclei in comparison with nonapoptoticells.

To detect exposure of phosphatidylserine on cell sur-ace, we used the Mebcyto Apoptosis Kit according tohe manufacturer’s instructions (MBL; Woburn, MA).riefly, cells (2 � 105) were washed in phosphate-uffered saline, and 10 �l of FITC-conjugated Annexinand 5 �l propidium iodide (PI, 5 �g/mL) were added.

fter incubation for 15 minutes at room temperature,00 �l of binding buffer was added, and the samplesere immediately analyzed on a FACScan flow cytometer

Becton Dickinson; San Jose, CA). The excitation sourceas an argon ion laser emitting at 488 nm. Electronic

ompensation of the instrument was required to excludeverlapping of the two emission spectra. Data were col-ected in list mode, stored, and analyzed with CellQuestoftware. PI was added to the samples to discriminateecrotic events (Annexin V�, PI�) from early apoptoticvents (Annexin V�, PI�) and late apoptotic phase (sec-nd necrosis cells, Annexin V�, PI�). The results arexpressed as percentage of specific apoptosis according tohe following formula: % specific apoptosis � 100 �(% apoptotic “test” cells � % of apoptotic “isotypeontrol” cells)/(100 � % apoptotic “isotype control”ells)].

Cells were fixed, permeabilized, and stained with aonoclonal antibody specific for segments of single-strand

NA (ssDNA), which discriminates apoptotic from ne- H

rotic cells by formamide as described previously [36],sing the commercial ssDNA Apoptosis ELISA KitChemicon; Temecula, CA). Briefly, cells were fixed for 30inutes in 80% methanol at room temperature and sub-

equently heated in formamide at 75 °C for 10 minutes.onspecific binding was blocked with 1% nonfat dry milk

w/v) in phosphate-buffered saline. Cells were stained withnti-ssDNA or immunoglobulin M isotype control, fol-owed by washing and incubation with a horseradish per-xidase-conjugated anti-mouse immunoglobulin M anti-ody for 30 minutes at room temperature. After washing,ells were incubated with the substrate ABTS (2,2=-azino-is [3-ethylbenziazoline-6-sulfonic acid]) for 20 minutes atoom temperature. The blue-green color produced by thenzymatic reaction was quantitated at 405 nm in a stan-ard microplate reader.

tatistical Analysisne-way analysis of variance, Student’s t-test and anal-

sis of variance for repeated measures (Wilks’ Lambda)ere used when appropriate. Probability values �0.05ere considered significant.

ESULTSLA-B35 Phenotype Favors Apoptotic Response

f Human PBMC Exposed in vitro to Thapsigarginhe ability of thapsigargin to induce genomic intranu-leosomal fragmentation in human PBMC is shown inigure 1C. After incubation with thapsigargin 5 �Mor 6 or 24 h, HLA-B35�ve PBMCs showed a signif-cantly higher percentage of apoptotic nuclei thanLA-B35-ve PBMCs (12 � 2 vs 27 � 4% [mean �

D], p� 0.005 and 13 � 3.6 vs 47.5 � 5.5%, p�.05; Figure 1D) as assessed by Hoechst 33258 fluo-escent dye staining (Figures 1A,B). The results repre-ent the mean of 12 different experiments in whichBMC from 13 HLA-B35�ve and 27 HLA-B35-veubjects were employed (Table 1).

LA-B35 Expression Favors the Apoptoticesponse of Human HLA-Class-I Transfected ECVell Lines Modulating Early and Latepoptotic Phaseshe results obtained with thapsigargin-treated humanMBCs were highly suggestive for the existence of aontrol of the cellular susceptibility to apoptosis byLA-B35. These results, however, were not conclusive

bout the direct involvement of the HLA-B35 moleculen the phenomenon because they did not exclude thatther HLA or non-HLA molecules in linkage disequilib-ium with HLA-B35 could be involved. To rule out thisossibility, we tested the rates of apoptosis in ECV 304ells transfected with either HLA-B*3501 or a different

LA-gene (HLA-B*0801 polymorphic �-chain, treated

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n vitro by Taxol). HLA-B8 was chosen because it did notnfluence the apoptosis rate in PMBC experiments.IRESneo2-transfected cells were also employed as con-rols. ECV-transfected cells are deemed a suitable tool toest whether B35 molecule plays a direct role in apopto-is or not, because the expression of B35 and B8 were the

IGURE 1 Nuclear chromatin staining with Hoechst 3325hapsigargin for 24 h (magnification 20�). (C) DNA laddncubation with thapsigargin: lane 1, B35-ve; lane 2, B35�ve35�ve human peripheral mononuclear cells incubated with

nly difference in this model. The ability of Taxol to 0

nduce genomic intranucleosomal fragmentation in hu-an ECV304 cells after incubation for 48 h is shown in

igure 2D. HLA-B35 transfected cells exposed in vitro to0 �M Taxol showed a significantly higher percentage ofpoptotic nuclei than controls (6 h: 30.6 � 2.06 vs 16 �.17%, p� 0.001; 12 h: 70.6 � 2.4 vs 53.2 � 1.6%, p�

(A) B35-ve and (B) B35�ve human PBMC incubated withdemonstrating internucleosomal fragmentation after 24 h

an PBMC. (D) Percentage of apoptotic nuclei in B35-ve andsigargin for 6 and 24 h. TG, thapsigargin.

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s 26.5 � 2.55%, p� 0.05; 12 h: 70.6 � 2.4 vs 56.6 �.4%, p� 0.001) (Figure 3). Typical fluorescence resultsbtained with B35�ve and B8�ve and control trans-ected ECV cells are shown in Figures 2A–C. The pro-poptotic effect of HLA-B35 expression was also con-irmed when we measured the amount of denatured,ingle-stranded DNA, present in the apoptotic but notecrotic cells by using a highly specific ELISA KitChemicon International). After 24 and 48 h, opticalensity values were higher in HLA-B35–transfected cellshan in HLA-B8–transfected cells (0.617 � 0.056 vs.359 � 0.014, p� 0.005 and 1.214 � 0.055 vs 0.835

0.0363, p� 0.001, respectively) and after 48 h, theyere higher in HLA-B35–transfected cells than in con-

rols (1.214 � 0.055 vs 0.901 � 0.049, p� 0.001)Figure 4, left); these results were confirmed by thenalysis of variance for repeated measures (p� 0.005)Figure 4, right). In HLA-B8–transfected cells, the in-rease in optical density values paralleled the increasebserved in controls and no statistical difference betweenhe two groups was found at any time.

The results we obtained with Hoechst 33258 staininguggest that the effect of HLA-B35 on cell apoptosis are

ABLE 1

HLA-B35 � ve patients

HLA ID nr HLA-A HLA-B HLA-C

165 68, 32 35, 60 4254 3, 9 35 4576 1, 30 35 4798 2, 29 35, 13 4312 2, 24 35 3, 4342 2, 29 35, 22 3, 4354 3 35 4391 2 35, 18 4, 7451 24, 30 35, 18 4, 5753 1, 24 35 4216 24 35, 49 4532 2, 11 35, 5 4754 32, 2 35, 58 4

bbreviation: HLA � human leukocyte antigen.

resent at the very beginning of the apoptotic process. t

ndeed, by flow cytometry (Figure 5), we confirmed thatCV304 HLA-B35�ve cells exposed in vitro to 6 h ofaxol had higher levels of early apoptotic cells (i.e., ofnnexinV�ve, PI-ve fluoresceinated cells than ECV304LA-B8�ve [25.86 � 1.35 vs 16.01 � 1.84%, p �

.001] or control transfected ECV-304 cells [25.86 �

.35 vs 18.37 � 2.26%, p � 0.001]). Still, the ssDNAuantification showed that a significantly higher per-entage of apoptotic ssDNA was present in ECV304LA-B35�ve than in ECV304 HLA-B35-ve at all

hases of the apoptotic process.

LA-B35 Expression Favors the Apoptoticesponse of Murine NIH3T3 Cellso explore whether our results were reproducible inonhuman cells, we examined the apoptotic response ofIH 3T3 cells transfected with the polymorphic chain of

ither HLA-B35 or HLA.B8; pIRESneo2-transfectedells were also employed as controls.

The ability of thapsigargin to induce genomic in-ranucleosomal fragmentation is shown in Figure 6A.ypical Hoechst 33258 fluorescent dye staining resultsbtained with HLA-B35�ve and HLA-B8�ve–

HLA-B35 � ve patients

HLA ID nr HLA-A HLA-B HLA-C

1005 2, 1 58, 61 2, 71193 1, 68 51, 54 31398 3, 25 18, 62 71408 2, 9 7, 40 3, 71534 1, 32 13, 27 21809 3, 29 7, 44 5, 71945 2 18, 8 11961 11, 26 27, 41 2, 61975 2 13, 56 12001 3, 10 572024 3, 2 51, 72117 1, 11 18, 8 62210 24, 2 18, 49 82316 9, 2 7, 13 6, 72406 2, 30 18, 492431 2, 24 7, 182531 2, 68 18, 44 43108 2, 3 55, 8 33111 1, 2 39, 27 1, 73215 1, 9 37, 49 63225 24, 2 7, 513277 2, 3 38, 51 43353 24, 31 13, 62 33451 68, 24 39, 27 6, 73522 24, 2 44, 52 83664 2, 11 18, 7 63887 2, 3 51, 62 2, 4

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igures 6A–C. As it is shown in Figure 7, HLA-B35�veT3 fibroblast exposed in vitro to 5 �M thapsigargin forand 24 h showed a significantly higher percentage of

poptotic nuclei than HLA-B8�ve cells (25.13 � 4.15s 12.52 � 4.52%, p � 0.001 and 65.33 � 5.63 vs0.25 � 7.94%, p� 0.001, respectively) and a higherercentage of apoptotic nuclei than controls (25.13 �.15 vs 2.05 � 0.39%, p� 0.001 and 65.33 � 5.63 vs7.91 � 4.94%, p� 0.001, respectively).

ISCUSSIONn our work, we have shown that HLA class I antigensifferently affect the ability of cells to undergo apoptosishen exposed in vitro to suitable stimuli and that theLA-B35 antigen confers a proapoptotic behavior to

ifferent kind of cells. Namely, we observed that inuman PBMCs collected from healthy donors and ex-osed in vitro to thapsigargin, the HLA-B35 phenotype

IGURE 2 Nuclear chromatin staining with Hoechst 33258ontrol, (B) human leukocyte antigen (HLA)-B8 transfected ceaddering demonstrating internucleosomal fragmentation afterlane 2), HLA-B35 transfected cells (lane 3), and controls (lan

of ECV304-transfected cells after 12 h incubation with Taxol: (A)lls, (C) HLA-B35 transfected cells (magnification 40�). (D) DNA48 h incubation with Taxol 50 �M in HLA-B8–transfected cells

as associated with a dramatic increase in apoptotic t

IGURE 3 Percentage of apoptotic nuclei, as detected byuclear staining, after treatment with Taxol at 6 and 12 h inCV304-transfected cells (control, HLA-B8 and HLA-B35);

he results represent the mean of 12 different experiments.

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187HLA-B35 and Apoptosis

esponses and no difference was observed between het-rozygous and homozygous B35 lymphocytes (data nothown). We cannot exclude that other HLA antigensay have a similar behavior; however, no other HLA

lass I antigen among the many we tested was able tonhance the apoptotic response of human PBMCs. More-ver, we have demonstrated that the proapoptotic effectf HLA-B35, as shown by typical nuclear condensations,NA fragmentation, and externalization of phosphati-ylserine, was not restricted to a peculiar cell type,ecause it has been reproduced not only in humanBMCs, but also in human ECV304 cell lines and inIH3T3 murine fibroblasts.Our results are not completely surprising, because it

as been previously observed that HLA class I moleculesossess far more biologic functions than the only presen-ation of antigens, including the ability to transduceegulatory signals [9, 10]. In particular, it has beenemonstrated that class I molecules transduce apoptoticignals after specific engagement of the T-cell receptorccessible �2 region [11] or the binding with HLA class–specific monoclonal antibodies recognizing nonpoly-orphic and polymorphic epitopes of the HLA-class I-chain [12–14]. Furthermore, Daniel et al. [13] dem-nstrated that apoptosis can be induced in resting humanBMCs and Jurkat lymphoblastic T cells also by mono-lonal antibodies directed against polymorphic HLAlass I epitopes. In their experiments, mAbs directedgainst different epitopes of a selected antigen showedifferent abilities to induce cell apoptosis. Interestingly,

IGURE 4 (A) Values of optical density (OD) as assessed byncubation with Taxol for 24 and 48 h. (B) Analysis of variann B35�ve cells (black line) with respect to B8�ve cells (dar

he authors observed that HLA-B35 was characterized by a

different behavior from other HLA-antigens, becauseLA-B35 mAbs did not induce apoptosis in resting

ells, whereas the authors were able to induce this kindf response at very high rates in cells exposed to activat-ng cytokines. Our data suggest that HLA-B35 is able tonduce apoptosis independently from its direct activationn the cell membrane. On the basis of our data, it is notossible to determine the mechanisms underlying theLA-B35 proapoptotic effect. A very interesting hy-

othesis to be investigated is that the proapoptotic HLA-35 effect might be related to the control of the intra-ellular cation concentrations [17, 29–31]. Indeed, weave previously observed that low levels of intracellularg�� and the high levels of Ca�� that characterize

ymphocytes carrying the HLA-B35 were negatively cor-elated to the number of viable cells in lymphocyteultures activated in vitro by phytohemagglutinin [37].nterestingly, Ca�� signaling has been involved in theegulation of cell death [38, 39] and both apoptosis fromhapsigargin and Taxol has been associated with the lossf Ca�� homeostatic controls. Namely, thapsigarginnhibits the calcium ATPase in the endoplasmic reticu-um membrane, leading to a complete depletion of in-racellular calcium stores and activating stress responsesnd apoptotic pathways within the endoplasmic reticu-um and mitochondria [40, 41]. Taxol also exerts a rapidffect on the cytosolic Ca�� signaling via the opening ofhe mitochondrial permeability transition pores [42],hus disrupting the mitochondrial transmembrane po-ential that has been shown to be reduced during early

le strand DNA-specific kit in ECV304-transfected cells afterr repeated measures showing a significant increase OD valuesay line) or controls (light gray line).

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It is possible that differences in the intracellular andransmembrane portion of the two MHC molecules weonsidered might influence susceptibility to apoptosis.ndeed, four point mutations have been identified be-ween HLA-B35 and HLA-B8 molecules [34] (G900A,916A, A988G, and G1049C). Nonetheless, this expla-ation seems unlikely because it has previously beenemonstrated that signaling via class I MHC moleculesoes not involve the cytoplasmic and the transmembrane

IGURE 5 Flow cytometry analysis of apoptosis induced byeukocyte antigen (HLA)-B8 transfected cells (B) and HLA-B3nd propidium iodide (PI) uptake. The lower left quadrant rearly apoptotic cells (Annexin V�/PI�), and the upper rightpoptotic percentage relevated in six different experimental significant increase in the apoptotic rates in HLA-B35� cells (light gray line) (p� 0.001).

ortions of the molecule [44]. a

Our data open new perspectives about the role playedy HLA antigens in cell activation and disease suscepti-ility. For instance, our results may offer an additionalechanism to explain why HLA-B35–positive human

mmunodeficiency virus–infected subjects [22–25], es-ecially B35/B35 homozygotes [26], rapidly progressoward conclamate acquired immunodeficiency syn-rome with opportunistic infections. Indeed, apoptosis isn important factor in causing lymphocyte depletion in

ol in ECV304 pIRES-transfected cells (controls) (A), humanansfected cells. The cells were examined for annexin bindingnts living cells (Annexin V�/PI�), the lower right quadrantrant second necrotic cells (Annexin V�/PI�). (D) Graphic ofn. (E) Analysis of variance for repeated measures, showing aline) with respect to B8�ve cells (dark gray line) or controls

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189HLA-B35 and Apoptosis

hich should be further enhanced by the class I overex-ression that is induced in tissues during viral infections46, 47]. More recently, we have shown that the HLA-35 phenotype is associated with the development ofulmonary hypertension in Italian scleroderma patients27, 28], greatly increasing the risk conferred by theostmenopausal condition [28]. The present results sug-est an exciting hypothesis (i.e., that this association maye explained by an increase of endothelial cell apoptosisn HLA-B35 positive patients).

CKNOWLEDGMENTS

he present work has been supported by grant E.0697 fromhe Theleton foundation and by a grant from the CARIPLOCassa di Risparmio delle Province Lombarde) foundation.

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s.

IGURE 6 Nuclear chromatin staining with Hoechst 33258 of (After 24 h incubation with thapsigargin (magnification 20�). (D) Dfter 24 h incubation with thapsigargin: lane 1, 174/HaeIII D

IGURE 7 Percentage of apoptotic nuclei, as detected byuclear staining, after treatment with thapsigargin for 6 and

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190 G. Salazar et al.

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