CXC Chemokine Ligand 13 and CC Chemokine Ligand 19 Cooperatively Render Resistance to Apoptosis in B...

CXC Chemokine Ligand 13 and CC Chemokine Ligand 19Cooperatively Render Resistance to Apoptosis in B Cell LineageAcute and Chronic Lymphocytic Leukemia CD23�CD5� B Cells1

Hu Chunsong,2*† He Yuling,2† Wang Li,2† Xiong Jie,†‡ Zhou Gang,† Zhang Qiuping,†

Gao Qingping,§ Zhang Kejian,§ Qiao Li,†¶ Alfred E. Chang,¶ Jin Youxin,‡ and Tan Jinquan3*†

CXCL13/CXCR5 and CCL19/CCR7 play a quite important role in normal physiological conditions, but the functions of bothchemokine/receptor pairs in pathophysiological events are not well-investigated. We have investigated expression and functions ofCXCL13/CXCR5 and CCL19/CCR7 in CD23�CD5� and CD23�CD5� B cells from cord blood (CB) and patients with B celllineage acute or chronic lymphocytic leukemia (B-ALL or B-CLL). CXCR5 and CCR7 are selectively expressed on B-ALL,B-CLL, and CB CD23�CD5� B cells at high frequency, but not on CD23�CD5� B cells. Although no significant chemotacticresponsiveness was observed, CXCL13 and CCL19 cooperatively induce significant resistance to TNF-�-mediated apoptosis inB-ALL and B-CLL CD23�CD5� B cells, but not in the cells from CB. B-ALL and B-CLL CD23�CD5� B cells express elevatedlevels of paternally expressed gene 10 (PEG10). CXCL13 and CCL19 together significantly up-regulate PEG10 expression in thesame cells. We have found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulate PEG10expression and function, subsequently stabilize caspase-3 and caspase-8 in B-ALL and B-CLL CD23�CD5� B cells, and furtherrescue the cells from TNF-�-mediated apoptosis. Therefore, we suggest that normal lymphocytes, especially naive B and T cells,use CXCL13/CXCR5 and CCL19/CCR7 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoidtissues organogenesis. In addition, certain malignant cells take advantages of CXCL13/CXCR5 and CCL19/CCR7 for infiltration,resistance to apoptosis, and inappropriate proliferation. The Journal of Immunology, 2006, 177: 6713–6722.

A lthough they decrease with age, CD5� B cells are themajor population in fetal life Most human IgM� cordblood (CB)4 B cells express CD5; however, in the adult,

they represent 10–25% of B cells in blood and �15–30% in tonsil,

and CD5� B cells are indicated as a self-replenishing subpopula-tion, showing an increased propensity to malignant transformation(1). CD23�CD5� B cells comprise the largest group of the ma-lignant cells in B cell chronic lymphocytic leukemia (B-CLL) andan important component in B cell lineage acute (B-ALL).

CCL19 (EBV-induced gene-1 ligand chemokine (ELC)) andCCL21 (secondary lymphoid tissue chemokine/6Ckine) are li-gands for the chemokine receptor CCR7 (Burkitt lymphoma re-ceptor-2), whereas CXCL13 (B cell-attracting chemokine 1 (BCA-1)) is the only ligand for CXCR5 (Burkitt lymphoma receptor-1)(2). Homeostatic chemokines, such as CXCL13, CCL21, andCCL19, as well as their corresponding receptors, CXCR5 andCCR7, have been shown to closely cooperate in the developmentof lymphoid organs and the maintenance of lymphoid tissue mi-croarchitecture (2). Expression of CXCR5 can be detected on ma-ture recirculating B cells, small subsets of normal CD4� andCD8� T cells, and skin-derived migratory dendritic cells (3–6).CXCR5 is essentially responsible for guiding B cells into the Bcell zones of secondary lymphoid organs (7–9). However, the ex-pression of CXCR5 on a subset of T cells strongly suggests anadditional role for this receptor in T cell migration (9, 10). CCR7is highly expressed on naive T cells, but expressed at lower levelson peripheral B cells. T cells and B cells show a transient increasein receptor expression following their activation (11), whereas Tcell differentiation toward effector cells is accompanied by a down-regulation of CCR7 on the cell surface (12–14).

The synergy of CXCL13/CXCR5 and CCL19/CCR7 has beenshown in both physiological and pathological situations. CXCR5cooperated with CCR7 control T cells and dendritic cell homing to

*Department of Immunology, School of Basic Medical Sciences, Anhui MedicalUniversity, Hefei, China; †Department of Immunology, and Laboratory of Allergyand Clinical Immunology, Institute of Allergy and Immune-Related Diseases andCenter for Medical Research, Wuhan University School of Medicine, Wuhan, China;‡The State Key Laboratory of Molecular Biology, Institute of Biochemistry and CellBiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science,Shanghai, China; §Department of Hematology, The Renmin and Zongnan UniversityHospital, Wuhan University, Wuhan, China; and ¶Department of Surgery, Universityof Michigan Medical Center, Ann Arbor, MI 48109

Received for publication April 6, 2006. Accepted for publication August 22, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the National Key Basic Research Program of Chinafrom the Ministry of Science and Technology of People’s Republic of China (Nos.2001CB510004 and 2001CB510008), and by the National Natural Science Founda-tion of China (Nos. 39870674, 30572119, 30030130, and 30471509), Science Foun-dation of Anhui Province, China (No. 98436630), and Education and Research Foun-dation of Anhui Province, China (No. 98JL063) and Research Foundation fromHealth Department of Hubei Provincial Government, China (No. 301140344), and aspecial grant from the Personnel Department of Wuhan University, China. T.J. is aChang Jiang Scholar supported by Chang Jiang Scholars Program from Ministry ofEducation, People’s Republic of China and Li Ka Shing Foundation, Hong Kong,People’s Republic of China.2 H.C., H.Y., and W.L. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Tan Jinquan, Department ofImmunology, School of Basic Medical Sciences, Anhui Medical University, 230032Hefei, China or Department of Immunology, Wuhan University School of Medicine,Wuhan University, Dong Hu Road 115, 430071 Wuchang, Wuhan, People’s Republicof China. E-mail address: [email protected] Abbreviations used in this paper: CB, cord blood; B-CLL, B cell chronic lympho-cytic leukemia; B-ALL, B cell lineage acute; ELC, EBV-induced gene-1 ligand che-mokine; BCA, B cell-attracting chemokine; PEG10, paternally expressed gene 10;HCC, human hepatocellular carcinoma; PI, propidium iodide; IAP, inhibitor of

apoptosis protein; MCNC, migrating cells on negative control; sh, short hairpin; PKC,protein kinase C; CI, chemotactic index; SIAH1, human homolog of Drosophilaseven in absenia.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

secondary lymphoid organs (12–14). The balanced expression ofCCR7 and CXCR5 determines the positioning and proper functionof follicular Th cells (11). CXCR5 and CCR7 double-deficientmice lack lymphoid follicles due to an impaired migration of Bcells (15). Overexpression of CXCR5 and CCR7 on tumor lym-phocytes closely related to cells apoptosis as well as their migra-tion and infiltration (16–19). The coexpression of CXCR5 andCCR7 were also found in B cell leukemia. However, the functionalimportance of CXCR5-CXCL13/BCA-1 and CCR7-CCL19/ELCreceptor-ligand pairs in the pathophysiological events of malignantB cells trafficking, homing, and survival is not fully understood.

A novel paternally expressed imprinted gene, paternally ex-pressed gene 10 (PEG10), is identified as a paternally expressedgene from a newly defined imprinted region at human chromo-some 7q21 (20). PEG10 shows parent-of-origin-specific expres-sion in monochromosomal hybrids (21). PEG10 knockout miceshow early embryonic lethality owing to defects in the placenta,indicating a critical role for mouse parthenogenetic development(21). An elevated level of expression has been found in the ma-jority of the human hepatocellular carcinoma (HCC) cells (22, 23).Exogenous expression of PEG10 confers oncogenic activity andtransfection of hepatoma cells with PEG10 antisense suppressing itsexpression results in cancer cell growth inhibition (22). In addition,PEG10 protein associates with human homolog of Drosophila sevenin absenia (SIAH1), a mediator of apoptosis. Overexpression ofPEG10 reduces the cell from death mediated by SIAH1 (22). Knock-down of PEG10 inhibits the proliferation of pancreatic carcinoma andHepG2 hepatocellular carcinoma cells (23).

In this study, we have found that CXCR5 and CCR7 are selec-tively expressed on B-ALL, B-CLL, and CB CD23�CD5� B cellsat high frequency. CXCL13 and CCL19 cooperate selectively toinduce resistance to apoptosis in B-ALL and B-CLL CD23�CD5�

B cells by means of activation of PEG10.

Materials and MethodsPatients and cell purification

All patients with B-ALL and B-CLL fulfilled the French-American-British(FAB) Cooperative Group criteria (24) and the guidelines of the NationalCancer Institute Working Group (25, 26). All patients gave informed con-sent according to institutional guidelines. CD19� (from normal periphery),CD23�, or CD23�CD5� cells were purified from PBMCs from peripheralblood of normal subjects, CB of uncomplicated births (IgM undetectable),or patients with B-ALL or B-CLL using a FACStarPlus sorting (27, 28). Theviability of all cultured cells �95% was tested by trypan blue exclusion.The malignancy of purified B-ALL or B-CLL CD23�CD5� cells was con-firmed by expression of CD20 and FMC-7. The cell line was Raji cell (B cellBurkitt lymphoma cell line) obtained from the American Type Culture Col-lection. The anti-CXCR5 and anti-CCR7 mAbs and chemokines (CXCL13,CCL19, CCL25, and CXCL12) were purchased from R&D Systems.

Flow cytometry

For detection of CXCR5 and CCR7, the cells were triple stained PE-la-beled CD23, FITC-labeled CD5 (DakoCytomation) and PerCP-labeledchemokine receptor Ab (R&D Systems), or matched isotype Ab (Dako-Cytomation) at 5 �g/ml in PBS containing 2% BSA and 0.1% sodiumazide for 20 min, followed washing twice with staining buffer (28). Theanalyses were performed with flow cytometer (Coulter XL; Coulter). Fordetection of apoptosis, cells were stained in staining medium (RPMI 1640,2% FBS, and 0.1% sodium azide) with 1 �g/ml propidium iodide (PI) for30 min at 4°C, then stained with FITC-conjugated annexin V with bindingbuffer (BD Pharmingen) as previously described (29, 30). Coulter XL wasused for analyses. For detection of intracellular active caspases, Cytofix/Cytoperm buffer (BD Pharmingen) was used according to the manufactur-er’s instructions to permeabilize cells, and cells were subsequently stainedwith anti-active-capsase-3 or anti-active-caspase-8 mAb (BD Pharmingen).After washing, active caspase-3 or caspase-8 fluorescence intensity wasmeasured by flow cytometry. Data were analyzed by means of the WinListprogram (The Scripps Research Institute).

Real-time quantitative RT-PCR

All real-time quantitative RT-PCR were performed as described elsewhere(28, 31). Briefly, the real-time quantitative PCR was performed in specialoptical tubes in a 96-well microtiter plate (Applied Biosystems) with anABI PRISM 7700 Sequence Detector Systems (Applied Biosystems). Byusing the SYBR Green PCR Core Reagents kit, fluorescence signals weregenerated during each PCR cycle via the 5� to 3� endonuclease activity ofAmpliTaq Gold to provide real-time quantitative PCR information. Thesequences of the specific primers are: CXCR5 sense, 5�-GGTCTTCATCTTGCCCTTTG-3�; CXCR5 antisense, 5�-ATGCGTTTCTGCTTGGTTCT-3�; CCR7 sense, 5�-GCTCCAGGCACGCAACTTT-3�; CCR7 antisense,5�-ACCACGACCACAGCGATGA-3�; PEG10 sense, 5�-ATGATGACATCGAGCTCCG-3�; PEG10 antisense, 5�-GCTGGGTAGTTGTGCATCA-3�.

All unknown cDNAs were diluted to contain equal amounts of �-actincDNA. PCR retain conditions were 2 min at 50°C, 10 min at 95°C, 40cycles with 15 s at 95°C, 60 s at 60°C for amplifications. Potential PCRproduct contamination was digested by uracil-N-glycosylase because dTTPis substituted by dUTP.

Northern and Western blot assays

For mRNA detection (Northern blot), as previously described (27, 32),each 5 �g of total RNA was electrophoresed under denaturing conditions,followed by blotting onto Nytran membranes, and cross-linked by UVirradiation. CXCR5 and CCR7 cDNA probes, labeled by [�-32P]dCTP,were obtained by PCR amplification of the sequence mentioned abovefrom total RNA from PBMC from normal adults (for CXCR5) or thymo-cytes from the specimen of thymusectomy (for CCR7), or human hepatomacell line HepG2 (for PEG10). The membranes were hybridized overnightwith 1 � 106 cpm/ml 32P-labeled probe, followed by intensively washingwith 0.2� SSC and 0.1% SDS before being autoradiographed. For proteindetection (Western blot), the cells were lysed in lysis buffer. Cell lysis wasperformed for 30 min at 4°C with lysis buffer. Expression of inhibitor ofapoptosis protein (IAP) family proteins (or other proteins indicated) wassemiquantified after Western blot analysis (33). Lysates were centrifuged at10,000 rpm for 5 min at 4°C. Protein concentration was measured by Bio-Rad protein assay. Protein (around 40 �g) was loaded onto 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes after elec-trophoresis, and incubated with the appropriate Abs at 0.5 �g/ml. Analyseswere conducted using ECL detection (Amersham Biosciences). All Abs(Bcl-2, Bcl-x, c-FLIPL, c-IAP1, c-IAP2, X-linked mammalian, and survivin)were obtained from Santa Cruz Biotechnology, except anti-livin which wasobtained from Imgenex, anti-�-actin which was obtained from Sigma-Aldrich,and CXCR5 and CCR7 mAbs which were obtained from R&D Systems.

Chemotaxis assay

The chemotaxis assay was performed in a 48-well microchamber (NeuroProbe) technique (27, 29). Briefly, chemokines in RPMI 1640 with 0.5%BSA were placed in the lower wells (25 �l). Twenty-five microliters of cellsuspension (2 � 106 cells/ml) was added to the upper well of the chamber,which was separated from the lower well by a 5-�m pore size, polycar-bonate, polyvinylpyrolidone-free membrane (Nucleopore). The chamberwas incubated for 60 min at 37°C and 5% CO2. The membrane was thencarefully removed, fixed in 70% methanol, and stained for 5 min in 1%Coomassie brilliant blue. The migrating cells were counted using a lightmicroscopy. Approximately 6% of the cells will migrate spontaneously(known as migrating cells on negative control (MCNC)). The chemotacticindex (CI) � migrating cell number at tested well of chemoattractant sam-ple/MCNC. The results were expressed as CI with SD.

Gene silencing assay

Short hairpin (sh) RNAs were produced in vitro as described (34) usingchemically synthesized DNA oligonucleotide templates (Sigma-Aldrich).Transcription templates were designed such that they contained U6 pro-moter sequences at the 5� end. shRNA transcripts subjected to in vitroDicer processing were synthesized using a Riboprobe kit (Promega).dsDNA oligonucleotides encoding shRNAs with homology to the targetedPEG10 gene were ligated into the EcoRV site to produce expression con-structs. The PEG10 sense sequence inserted immediately downstream ofthe U6 promoters was as follows: GAGCTCTCTGAAGAGATCAACtt.Negative control was DNAPEG10 with the same sequence. Cells werecultured in DMEM containing 10% heat-inactivated FBS, penicillin, andstreptomycin. Cells were harvested 2 days after the transfection.

Plasmids and cell transfection

Plasmids encoding PEG10 and CCR7 used in this study have been previ-ously described (23, 35). The cells were transiently transfected with vectors

6714 APOPTOTIC RESISTANCE BY CXCL13 AND CCL19

encoding target genes as described elsewhere (23, 35). Briefly, the cellswere cultured with DMEM containing 10% FCS, penicillin, and strepto-mycin. Cells were grown to �70% confluence in 60-mm dishes for 24 hbefore transfection. The DNA constructs of expression vectors (0.4 �gunless indicated) or vector only were mixed with 12 �l of LipofectAMINE(Invitrogen Life Technologies) in 2 ml of opti-DMEM serum-free mediumand added to cells, and incubated for 6 h. The cells were further culturedin 2.5 ml of DMEM containing 10% FCS in 5% CO2.

Statistical analysis

Statistical significance was assessed by the paired or unpaired Student ttest. Values of p � 0.05 were considered statistically significant.

ResultsCXCR5 and CCR7 are selectively expressed on B-ALLCD23�CD5� B cells at high frequency

We screened some CXC and CC chemokine receptors on differentCD23�CD5� and CD23�CD5� B cells (as well as normal pe-ripheral CD19�CD5� and CD19�CD5� cells). Flow cytometricanalysis revealed that the data of CXCR3, CXCR6, CCR4, andCCR9 were either in agreement with previous reports or that therewere no differences among four types of cell sources in a total of41 cases of typical B-ALL and B-CLL patients (Table I). Inter-estingly, CXCR5 and CCR7 were selectively expressed on B-ALLand B-CLL CD23�CD5� B cells at high frequency (86, 81, 42,and 51%, respectively) (Fig. 1, A and B), whereas, they were ex-pressed at significantly lower frequency (14 and 15%, 12 and 11%,respectively) on B-ALL and B-CLL CD23�CD5� B cells.CXCR5 and CCR7 were expressed at rather low level on CD19�

B cells from normal peripheral blood. In comparison, they wereexpressed at similar levels on CD23�CD5� and CD23�CD5� Bcells from CB.

CXCR5 mRNA were detected in similar patterns as mentionedabove in different types of CD23�CD5� B cells (Fig. 1C, left),which was confirmed by Northern blot (Fig. 1C, right, upper pan-els), as well as in Western blot (Fig. 1C, right, lower panels). Thesame results of CCR7 were shown in Fig. 1D. CXCR5 and CCR7expression patterns in CD23�CD5� B cells from distinct subjectswere also confirmed by Northern and Western blots (data notshown).

Functionally, CXCL13/BCA-1 (CXCR5 ligand) and/or CCL19/ELC (CCR7 ligand) failed to induce significant chemotaxis in B-ALL and B-CLL CD23�CD5� B cells (Fig. 2). Interestingly, both

CXCL13 and CCL19 induced very strong chemotaxis in CBCD23�CD5� B cells (Fig. 2).

CXCL13 and CCL19 together rescue B-ALL CD23�CD5�

B cells from apoptosis

We examined the protective effects of CXCL13 and CCL19 ondifferent types of cells on TNF-�-mediated apoptosis. Flow cy-tometric analysis (Fig. 3) revealed that the number of apoptoticand necrotic cells was significantly decreased in cultured B-ALL and B-CLL CD23�CD5� B cells in presence of bothCXCL13 and CCL19 (Fig. 3A, o and p), in comparison withthose in the absence of CXCL13 and CCL19 (Fig. 3A, c and d).Interestingly, CXCL13 or CCL19 alone had no such effect inB-ALL or B-CLL CD23�CD5� B cells (Fig. 3A, g, h, k, and l).CXCL13 and/or CCL19 did not render resistance to apoptosis innormal peripheral CD19� B cells and CB CD23�CD5� B cells(Fig. 3A, e, f, i, j, m, and n), in comparison with those in theabsence of CXCL13 and CCL19 (Fig. 3A, a and b). NeitherCCL13 nor CXCL19 had such an effect as to rescue different typesof B cells (normal peripheral CD19� B cells, and CB, B-ALLB-CLL CD23�CD5� B cells) from TNF-�-mediated apoptotic re-sponse (data not shown). Abs against CXCR5 and CCR7 couldcompletely block the combined protective effect of CXCL13 andCCL19, indicating that the rescuing effect was indeed induced bymeans of interaction of CXCL13/CCL19 and CXCR5/CCR7 (datashown in Fig. 4). The total numbers of dead cells (including ap-optotic and necrotic) in different types of the cells exhibited asimilar pattern as shown in Fig. 3. Thus, CXCL13 and CCL19cooperatively rescued B-ALL and B-CLL CD23�CD5� B cellsfrom TNF-�-mediated apoptosis, but not either normal peripheralCD19� B cells or CB CD23�CD5� B cells, confirming antiapop-totic effects mediated by CXCR5 and CCR7 in B-ALL and B-CLLCD23�CD5� B cells.

Many signaling events of binding of certain chemokines to che-mokine receptors were included such as PI3K, MAPK, or proteinkinase C (PKC), that appear to be involved in chemokine-mediatedchemotaxis in certain cell types (36–39). To determine whetherthese kinases were responsible for resistance to apoptosis inducedby costimulation of CXCL13 and CCL19, B-ALL and B-CLLCD23�CD5� B cells were pretreated with varying concentrationsof pertussis toxin (1 �g/ml), an inhibitor of PKC; wortmannin (50

Table I. Some chemokine receptor expression on CD23�CD5�, CD23�CD5� B cellsa

Cell Typeb CXCR3 CXCR5 CXCR6 CCR4 CCR7 CCR9

PeripheryCD19�CD5� Ndc Nd Nd Nd Nd NdCD19�CD5� 5 1d 6 2 14 8 11 1 7 4 –e

CBCD23�CD5� 21 11 53 15 25 16 11 3 59 17 21 13CD23�CD5� 11 9 21 12 15 6 – 23 11 –

B-ALLCD23�CD5� 56 20 86 10 84 12 50 14 89 12 –CD23�CD5� 35 17 29 15 15 8 20 11 27 12 –

B-CLLCD23�CD5� 51 15 47 14 24 13 21 12 46 19 3 1CD23�CD5� 6 4 12 4 5 3 13 8 11 10 5 2

a The CD23�CD5�, CD23�CD5� B cells were freshly isolated and stained in triple colors of CD23 (PE), CD5 (FITC), andindicated chemokine receptor Abs (PerCP) as described in Materials and Methods.

b The CD23� B cells were isolated from peripheral blood from normal subjects (CD19�), cord blood (CB) of uncomplicatedbirths, B-ALL, or B-CLL patients.

c Nd, No determination since few CD23�CD5� B cells in normal preipheral blood.d The listed numbers in the table were percentages of chemokine receptor-positive B cells. The data were mean values SD

at least of eight similar experiments conducted. For detection of CXCR5 or CCR7, showing data were mean values SD of 25B-ALL and 16 B-CLL patients.

e Under detectable level.

6715The Journal of Immunology

nM), a potent PI3K inhibitor; PD98059 (50 nM), a p44/42 MAPKinhibitor; or vehicle, DMSO, further costimulated with CXCL13and CCL19. As seen in Fig. 4, only pertussis toxin significantlydiminished the protective effect of CXCL13 and CCL19 costimu-lation in B-ALL CD23�CD5� B cells from apoptosis. NeitherPD98059 nor wortmannin inhibited a protective effect in B-ALLCD23�CD5� B cells from apoptosis, suggesting that CXCL13and CCL19 cooperated via the PKC pathway to protect B-ALLCD23�CD5� B cells from apoptosis. As expected, Z-VAD-FMK(20 nM) rescued B-ALL CD23�CD5� B cells from TNF-�-me-diated apoptosis. We observed similar results in B-CLLCD23�CD5� B cells (data not shown). As expected, mAbs againstCXCR5 and CCR7 together significantly inhibited the protectiveeffect of costimulation with CXCL13 and CCL19 in B-ALLCD23�CD5� B cells from apoptosis, whereas isotypes had no

FIGURE 1. CXCR5 and CCR7 selectively express on CD23�CD5� Bcells. Triple-color flow cytometric analysis of the distribution of CXCR5and CCR7 on CD23�CD5� and CD23�CD5� B cells from B-ALL (A) andB-CLL (B) patients. The CD23� B cells were freshly isolated and stainedin triple colors of CD23 (PE), CD5 (FITC), and CXCR5 or CCR7 (PerCP)as described in Materials and Methods. The indicated numbers in thegraphs were percentages of CD23�CD5� and CD23�CD5� B cells. Theindicated percentages in the graphs were numbers of CXCR5� or CCR7�

B cells. The data were from a single experiment, which was representativeof 23 (B-ALL) and 18 (B-CLL) similar experiments performed. Isotype Abcontrols were expressed as dished curves. CXCR5 expression onCD23�CD5� B cells were examined by real-time quantitative RT-PCR (C;left), Northern blot (C; upper panels), Western blot (C; lower panels), and

the same results of CCR7 (D). In C and D, the procedure for quantitativeRT-PCR amplification was described in Materials and Methods. The show-ing bars were mean values SD of eight similar experiments conducted.�, p � 0.001, normal peripheral B cells vs CD23�CD5� B cells from CB,B-ALL, or B-CLL. In C and D (upper panels), the detections of mRNA ofCXCR5 and CCR7 were by Northern blot for freshly isolatedCD23�CD5� B cells from normal PBMC (only CD19�), CB of uncom-plicated births, B-ALL, or B-CLL patients. Total RNA from different cellswas treated as described in Materials and Methods. The hybridization sig-nals for CXCR5 or CCR7 mRNA from different cells were shown in eachupper picture. The 28S rRNAs in lower pictures confirmed that comparableamounts of total RNA were used. In C and D (lower panels), the CXCR5or CCR7 protein was examined using Western blot analyses. Actins in eachlower picture indicated the quantity of total cellular protein from the testedsamples loaded in each lane. Arrows indicate markers used to verify equiv-alent molecular weights of appropriate proteins in each lane.

FIGURE 2. The chemotaxis of CD23�CD5� B cells. The chemotaxisof freshly isolated in CD23�CD5� T cells from CB of uncomplicatedbirths and B-ALL patients toward CXCL13/BCA-1 (A) (f), CCL19/ELC(B) (f), CCL3/MIP-1� (u) (100 ng/ml), or PBS control (�). All resultswere expressed as CI or percentage of adhesive cells with SD (SD), andbased on triplicate determination of chemotaxis and adhesion on each con-centration of chemokine indicated as nanograms per milliliter. Theshowing bars were mean values SD of eight experiments conducted.Statistical significant differences as compared with controls are indi-cated as �, p � 0.001. Values of p � 0.05 are considered nonsignificant.CI at optimal concentration of vs CI at medium control. MCNCCB �13,437 1,785; MCNCB-ALL � 15,239 2,021.


such effect (Fig. 4), documenting that the protective effect ofCXCL13 and CCL19 in the cells from apoptosis was indeed bymeans of CXCR5 and CCR7 pathways. As shown in Fig. 4, thedata of total fractions of dead cells (including apoptotic and ne-crotic) were similar to patterns in the results in Fig. 3A.

PEG10 expression is selectively increased in B-ALLCD23�CD5� B cells by CXCL13 and CCL19 costimulation

Western blot showed that the protein levels of one group of anti-apoptotic members (Bcl-2, Bcl-x, and c-FLIPL) in different typesof freshly isolated CD23�CD5� B cells (CB, B-CLL, and B-ALL)

FIGURE 3. Analysis of apoptotic and total dead (necrotic and apo-ptotic) cells for inhibition of TNF-�-mediated apoptosis ofCD23�CD5� B cells. Flow cytometric analysis of apoptotic (A) andtotal dead cells (B), the CD23�CD5� B cells were freshly isolated fromnormal PBMC (only CD19�), CB of uncomplicated births, B-ALL orB-CLL patients and were pretreated at absence or presence of chemo-kine as indicated (all at 100 ng/ml) for 24 h at 37°C, following stim-ulation with TNF-� (100 ng/ml) for 24 h at 37°C. The cells were an-alyzed by flow cytometry for PI (y-axis) and FITC-conjugated annexinV (x-axis) as described in Materials and Methods. The gating in theforward scatter and side scatter histograms were adhered to the lym-phocyte region. The percentages of PI� annexin V� cells and PI� an-nexin V� cells were indicated in the figure. The data (A) were from asingle experiment, which was representative of six experiments per-formed. The data for total dead cells (PI� annexin V� plus PI� annexinV�) (B) were mean values SD of six experiments performed. Sta-tistically significant differences as compared with untreated cells wereindicated (�, p � 0.001, untreated vs CXCL13�CXCL19-treatedCD23�CD5� B cells from B-ALL or B-CLL).

FIGURE 4. Analysis of the inhibition of TNF-�-mediated apoptosis inCD23�CD5� B cells. Flow cytometric analysis of apoptotic (A) and totaldead cells (B), B-ALL CD23�CD5� B cells were freshly isolated fromB-ALL patients as described in Materials and Methods, then serum starvedovernight and treated with anti-CXCR5 plus anti-CCR7 (each 5 �g/ml,Abs), isotype Abs (each 5 �g/ml, Isotypes), pertussis toxin (PT) (1 �g/ml),wortmannin (WT, 50 nM), PD98059 (50 nM), or vehicle DMSO for 1 h,followed culture in the presence of CXCL13 and CCL19/ELC (�, each100 ng/ml) for 6 h before apoptotic assay. As a control, Z-VAD-FMK (20nM), a caspase inhibitor, was used for blocking apoptosis. The cells wereanalyzed by flow cytometry as described in the legend for Fig. 3. The datawere from a single experiment, which was representative of six experi-ments performed. The data for total dead cells (PI� annexin V� plus PI�

annexin V�) (B) were mean values SD of six experiments performed.Statistically significant differences as compared with different treated cellswere indicated (�, p � 0.001, Ab- or PT-treated vs PD98059-, WT- orZ-VAD-FMK-treated CD23�CD5� B cells from B-ALL).


were identical (data not shown). Interestingly, after stimulationwith CXCL13 or/and CCL19 and further apoptotic induction withTNF-�, their expression levels were still not significantly altered indifferent types of CD23�CD5� B cells (data not shown). The ex-pression levels of another group of antiapoptotic proteins in theIAP family (XIAP, c-IAP1, c-IAP2, survivin, and livin) in differenttypes of freshly isolated CD23�CD5� B cells (CB, B-CLL andB-ALL) were identical as well (data not shown). After stimulationwith CXCL13 or/and CCL19 and apoptotic induction with TNF-�,their expression levels were still not significantly changed in dif-ferent types of B cell CD23�CD5� B cells (data not shown).

We further examined the expression levels of PEG10 in distinctCD23�CD5� B cells during stimulation with CXCL13/CCL19and TNF-�. Data obtained from real-time quantitative RT-PCRand Northern blot analyses (Fig. 5) revealed that freshly isolatednormal peripheral CD19� B cells and CB CD23�CD5� B cellsexpressed almost no PEG10 (Fig. 5A) or at very low levels (Fig.5B). PEG10 expression levels in the cells were not significantlyaltered after 24 h culture with CXCL13 and/or CCL19. Costimu-lation with TNF-� did not change PEG10 expression levels (datanot shown). In contrast, freshly isolated B-ALL and B-CLLCD23�CD5� B cells expressed elevated level of PEG10 (Fig. 5,C and D). The PEG10 expression levels were significantly up-regulated in the cells cultured for 24 h with CXCL13 and CCL19costimulation, CXCL13 or CCL19 alone did not show such aneffect (Fig. 5, C and D). Costimulation with TNF-� did not changePEG10 expression levels (data not shown). The time-course study(Fig. 5E) showed a significant up-regulation of PEG10 mRNAlevel by CXCL13 and CCL19 costimulation was already observedwithin 8 h and peaked at 24 h. The data suggested that PEG10expression and functionality might contribute to might involved inthe mechanism of resistance to TNF-�-mediated apoptosis in B-ALL and B-CLL CD23�CD5� B cells as afforded by CXCL13and CCL19 costimulation.

PEG10 expression in B-ALL CD23�CD5� B cells is essentialfor resistance to apoptosis

We applied shRNA of PEG10 (shRNAPEG10) to knockdown theendogenous PEG10 expression to verify whether it contributes toCXCL13/CCL19 afforded resistance to TNF-�-mediated apopto-sis. Northern blot results showed that culture with shRNAPEG10at high concentration (2 �g/ml) completely abolished expressionof PEG10 in B-ALL and B-CLL CD23�CD5� B cells at mRNAlevels, whereas, low concentration shRNAPEG10 (0.02 �g/ml),DNAPEG10, and vector showed no such effects (data not shown).

FIGURE 5. PEG10 mRNA expression in CD23�CD5� B cells. Thereal-time quantitative detection of RT-PCR (upper panels) and Northernblot (lower panels) for PEG10 mRNA in the CD23�CD5� B cells. Thecells were isolated from normal PBMC (only CD19�) (A), CB of uncom-plicated births (B), B-ALL (C and E) or B-CLL (D) patients as describedin Materials and Methods, which were pretreated at absence or presence ofchemokine as indicated (all at 100 ng/ml) for 24 h at 37°C, followingstimulation with TNF-� (100 ng/ml) for 24 h at 37°C. The procedure forquantitative RT-PCR amplification was described in Materials and Meth-ods. A linear relationship between the cycle threshold (CT) and log startingquantity of standard DNA template or target cDNA (PEG10) was detected(data not shown). The correlation coefficients are �0.97–0.99. The show-ing bars were mean values SD of six similar experiments conducted(�, p � 0.001, untreated vs CXCL13�CXCL19-treated CD23�CD5� Bcells from B-ALL or B-CLL). Total RNA from different cells as indicatedwere isolated, electrophoresed and blotted as described in Materials andMethods. The hybridization signals for PEG10 mRNA in different cellswere shown in upper parts of the panels. The 28S rRNAs in lower parts ofthe panels confirmed the comparable amounts of loaded total RNA. Thedata were from a single experiment, which was representative of six ex-periments performed. In E, the real-time quantitative detection of RT-PCRfor PEG10 mRNA in the CD23�CD5� B cells from B-ALL patients asdescribed in Materials and Methods, which were pretreated at absence orpresence of CXCL13 and CCL19 (each 100 ng/ml) for the time intervals asindicated at 37°C. The data were mean values SD of six experimentsperformed. Statistically significant differences as compared with untreatedcells (0 h) were indicated (�, p � 0.01, untreated vs CXCL13�CXCL19-treated B-ALL CD23�CD5� B cells).

Table II. Analysis of total dead cells in B-CLL CD23�CD5� B cells indifferent treatments

Treatmenta CXCL13 CCL19 CXCL13/CCL19b

Nontreated 65 14c 56 18 14 5Vector (2 �g) 66 18 63 17 12 4DNAPEG10 (2 �g) 62 14 61 18 15 5shRNAPEG10 (0.02 �g) 59 20 68 10 21 11shRNAPEG10 (2 �g) 63 8 67 11 68 13d

a The purified CD23�CD5� B cells from B-ALL patients were cultured for 6 daysin the presence or absence of shRNAPEG10 at low concentration (0.02 �g) and highconcentration (2 �g) as described in Materials and Methods.

b They were then pretreated at presence of CXCL13 or/and CCL19 (all at 100ng/ml) described in Materials and Methods, following stimulation in the presence ofTNF-� (100 ng/ml) for 24 h at 37°C.

c The cells were analyzed by flow cytometry for PI (y-axis) and FITC-conjugatedannexin V (x-axis) as described in Materials and Methods. The data for total deadcells (PI� annexin V� plus PI� annexin V�) were mean values SD of six exper-iments performed.

d Value of p � 0.005 vs nontreated.


The shRNAPEG10 at high concentration significantly blocked theeffects of CXCL13 and CCL19 costimulation on induction of re-sistance to TNF-�-mediated apoptosis in B-ALL CD23�CD5� Bcells, no low concentration shRNAPEG10, DNAPEG10 and vec-tor had such effect (Table II). All treatments did not alter the pat-terns of effects of CXCL13 or CCL19 alone in resistance to TNF-�-mediated apoptosis in B-ALL CD23�CD5� B cells (Table II).Similar results were obtained in B-CLL CD23�CD5� B cells (datanot shown).

Expression levels of activated caspase-3 (Fig. 6A) and caspase-8(Fig. 6B) were measured by intracellular staining in B-ALLCD23�CD5� B cells in cultures with the distinct treatments (40).The shRNAPEG10 at high concentration also significantly blockedthe effects of CXCL13, and CCL19 costimulation promoted sta-bilization of caspase-3 and caspase-8 in B-ALL CD23�CD5� Bcells. In contrast, low concentration shRNAPEG10, DNAPEG10,and vector showed no such effect (data not shown). All treatmentsdid not alter effects of CXCL13 or CCL19 alone on stabilization ofcaspase-3 and caspase-8 in B-ALL CD23�CD5� B cells (Fig. 6).As a control, we also measured caspase-3 and caspase-8 expres-sion in untreated B-ALL CD23�CD5� B cells during TNF-�-mediated apoptosis. The shRNAPEG10 treatment itself did notalter the patterns of caspase-3 and caspase-8 expression duringTNF-�-mediated apoptosis (Fig. 6). We observed similar results ofshRNAPEG10 to block stabilization of caspase-3 and caspase-8expression by CXCL13 and CCL19 costimulation during TNF-�-mediated apoptosis in B-CLL CD23�CD5� B cells (data not shown).

The Raji cells were used for transfection studies. The cells weretransfected with either PEG10 alone (Fig. 7A), or cotransfectedwith CCR7 and varying concentrations of PEG10 (Fig. 7, B–D).The transfection with PEG10 alone conferred neither significantprotection against apoptosis nor stabilization of caspase-3 (Fig.7A) and caspase-8 (data not shown) in Raji cells, indicating thatPEG10 should be activated by two chemokines to fully function.Therefore, we cotransfected Raji cells with PEG10 and CCR7. Thedata showing in Fig. 7B verified that successful transfection ofPEG10, CXCR5 (existed), and CCR7 took place by Western blot.Flow cytometric analysis (Fig. 7C) revealed that only the cellstransfected with high doses of PEG10 (400 or 800 ng) could berescued by CXCL13 and CCL19 costimulation of the cells fromTNF-�-induced apoptosis (Fig. 7C, d and e), whereas, neither lowdose PEG10 (100 ng), vector, nor untransfected control showedany effect (Fig. 7C, a–c). The transfection itself did not causeapoptosis either (Fig. 7Cf ). Expression levels of activatedcaspase-3 were measured by intracellular staining in transfectedcells. Only in the cells transfected with high doses of PEG10 (400or 800 ng) could caspase-3 be stabilized by CXCL13 and CCL19costimulation during TNF-�-induced apoptosis (Fig. 7D, d and e),whereas, in the cells transfected with either low dose PEG10 (100ng), vector, or untransfected cells, caspase-3 could not be stabi-lized (Fig. 7D, a–c). The transfection itself did not cause destabi-lization (Fig. 7Df ). We also obtained similar results of caspase-8in the transfected cells (data not shown). The results strongly sug-gested a critical role of PEG10 in rescuing cells from TNF-�-mediated apoptosis by CXCL13 and CCL19 costimulation.

DiscussionChemokine receptors were reported to express on neoplastic cells ofhemopoietic and nonhematopoietic origin, and overexpression ofsome of these receptors was closely related to tumor progression andmetastasis (41–43). As for B cell-derived lymphoproliferative disor-ders, CXCR5 has been detected in neoplastic B cells from B-ALL(44) and B-CLL (45). CCR7 has been detected in B-CLL (46) and in

FIGURE 6. Activation of caspases in CD23�CD5� B cells. Flow cyto-metric analysis of active caspase-3 (A) and caspase-8 (B) in CD23�CD5� Bcells. The purified CD23�CD5� B cells from B-ALL patients were culturedfor 6 days in the presence or absence of shRNAPEG10 at low concentration(0.02 �g) and high concentration (2 �g) as described in Materials and Meth-ods. They were then pretreated at presence of CXCL13 or/and CCL19 (all at100 ng/ml) described in Materials and Methods, following stimulation at pres-ence of TNF-� (100 ng/ml) for 24 h at 37°C. They were then permeabilizedand fixed as indicated in Materials and Methods and subsequently stained forintracellular activated (cleaved) caspase-3 or caspase-8. Activated caspase-3-or caspase-8-specific fluorescence intensity was measured by flow cytometry.The indicating percentages of cells with activated caspase-3 or caspase-8 werequantitated from relative-frequency histograms. Isotype Ab controls were ex-pressed as dished curves. The data were from a single experiment, which wasrepresentative of six experiments performed.


tumor cells from classical Hodgkin’s disease with lymphocyte pre-dominance (47). In this study, we showed that CD23�CD5� B cellscomprised the largest group �70% of the malignant cells in B-CLL,and 40% in B-ALL. CD5� B cells have been reported to be the majorpopulation in fetal life, but decreased with age, and indicated as aself-replenishing subpopulation, showing an increased propensity tomalignant transformation (1). In the present study, we compared fourdifferent types of cells (CD23�CD5� and CD23�CD5� B cells fromnormal CB, B-ALL, and B-CLL patients) in terms of expression andfunctions of CXCR5 and CCR7. We have found that CXCR5 andCCR7 are selectively, frequently, and functionally expressed on B-ALL and B-CLL CD23�CD5� B cells, whereas, their expression inB-ALL and B-CLL CD23�CD5� B cells was at a low level. Withoutimpressive chemotactic responsiveness, CXCR5 and CCR7 costimu-lation (but not alone) displayed a novel function to induce resistanceto TNF-�-mediated apoptosis in B-ALL and B-CLL CD23�CD5� Bcells.

Chemokine receptor signaling is reported to provide antiapop-totic activity to hemopoietic cells in a natural context (48). CCR9/CCL25 interaction provides a cell survival signal to the receptor-expressing cells (29). CXCL1 and CXCL4 are able to support thesurvival of endothelial cells and monocytes, respectively (49, 50).However, there are some controversial, even contradictory, re-ports. For instance, CXCR4 induces programmed cell death ofhuman peripheral CD4� T cells, malignant T cells, and CD4/CXCR4 transfectants (51). The interaction between HIV R5 Envand CCR5 activates the Fas pathway and caspase-8 and triggersFasL production, ultimately causing CD4� T cell death (52). Wehave also reported that CCR3 expression induced by IL-2 and IL-4functions as a death receptor for B cells (30). The results in thisstudy, together with other observations, suggest that normal B andT cells use CXCR5/CXCL13 and CCR7/CCL19 for migration,homing, development, maturation, selection, and cell homeostasisas well as secondary lymphoid tissue organogenesis. Mean-while, some malignant cells, particularly B-ALL and B-CLLCD23�CD5� B cells, take advantages of CXCR5/CXCL13 andCCR7/CCL19 for infiltration, resistance to apoptosis, and inappro-priate proliferation. To our knowledge, this study is the first reportof differential functions of CXCR5/CXCL13 and CCR7/CCL19 indistinct types of cells in terms of induction of apoptotic resistance,and is direct evidence of the pathophysiological activity of B-ALLand B-CLL CD23�CD5� B cells induced by CXCL13 and CCL19costimulation.

PEG10 is identified on human chromosome 7q21 (21, 53).Mouse homolog PEG10 has recently been located in a large im-printed gene cluster on mouse proximal chromosome 6 and hasbeen confirmed to be imprinted (54). Because the protein productsfrom the predicted open reading frames 1 and 2 of PEG10 showhomology to the gag and pol proteins of vertebrate retrotransposonTy3/Gypsy, PEG10 is speculated to be a retrotransposon-derivedgene. Distinct expression of PEG10 is found in the brain, kidney,lung, testis, and placenta but not in the liver and a number of othertissues (20). In contrast to this, expression of PEG10 is only de-tected in the placenta among the 14 adult mouse tissues examined(54). Some experimental data suggest a role for preferential ex-pression of the imprinted genes in regulating growth control ofliver and pancreatic carcinoma cells (23). Exogenous expression ofPEG10 promotes growth of certain HCC cell lines that do notmanifest endogenous expression of this gene. The interaction ofPEG10 protein with SIAH proteins plays important roles in resis-tance to apoptosis (22). Even PEG10 is suggested to serve as anovel molecular target for treatment of HCCs (22). In the presentstudy, we have found that freshly isolated B-ALL and B-CLL

FIGURE 7. CXCL13 and CCL19 require PEG10 for protection againstapoptosis. Raji cells were transfected with vectors encoding PEG10 (100 or800 ng alone) (A), or cotransfected with vectors encoding CCR7 (400 ngeach) in the absence or presence of increasing concentrations of PEG10(100, 400, or 800 ng) (B–D). The amount of transfected cDNA was keptconstant in each sample by adding control pcDNA3 vector. In A, PEG10-transfected cells were treated with TNF-� (100 ng/ml) for 24 h at 37°C.The cells were then analyzed by flow cytometry as described in Materialsand Methods for apoptotic cells (right panels) and active caspase-3 (leftpanels). In B, Western blotting were showing equal expression levels ofCXCR5 and CCR7 as well as increasing expression levels of PEG10. In Cand D, cotransfected cells were pretreated at absence or presence ofCXCL13 and CCL19 (all 100 ng/ml) described in Materials and Methods,some of cells were followed stimulation with TNF-� (100 ng/ml) for24 h at 37°C before other assay as indicated (�, no stimulation withTNF-�). C, analysis of total dead (necrotic and apoptotic) cells. Thecells were analyzed by flow cytometry as described in the legend forFig. 3. The data were from a single experiment, which was represen-tative of six experiments performed. D, Flow cytometric analysis ofactive caspase-3 in transfected cells as indicated. Cells were pretreatedas mentioned above. They were then permeabilized and fixed as indi-cated in Materials and Methods and subsequently stained for intracel-lular activated (cleaved) caspase-3. The indicating percentages of cellswith activated caspase-3 were quantitated from relative-frequency his-tograms. Isotype Ab controls were expressed as dished curves. The datawere from a single experiment, which was representative of six exper-iments performed.


CD23�CD5� B cells express an elevated level of PEG10, com-pared with that in normal peripheral CD19� B cells and CBCD23�CD5� B cells. After 24 h of culture with CXCL13 andCCL19 costimulation, PEG10 expression levels in the cells hadbeen significantly up-regulated (Fig. 5). By using shRNAs, wehave found that abrogation of PEG10 expression significantlyblocks the protective effects of CXCL13 and CCL19 costimu-lation on TNF-�-mediated apoptosis in B-ALL and B-CLLCD23�CD5� B cells (Table II). We suggest that CXCL13 andCCL19 cooperate via frequently expressed and activated CXCR5and CCR7, which up-regulates PEG10 expression and function,and subsequently stabilizes caspase-3 and caspase-8 in B-ALL andB-CLL CD23�CD5� B cells as an important mechanism to rescuethe cells from TNF-�-mediated apoptosis (Figs. 6 and 7). This isthe first report of this imprinted gene expressed in both humanB-ALL and B-CLL CD23�CD5� B cells. We have also proven amechanism by which overexpression of CXCR5 and CCR7 onB-ALL and B-CLL CD23�CD5� B cells renders direct resistanceto apoptosis in these malignant cells. Understanding the molecularbasis of abnormal imprinting of PEG10 in both human B-ALL andB-CLL will shed new light on the process and mechanism thatleads to malignant lymphoproliferative disorders.

DisclosuresThe authors have no financial conflict of interest.

References1. Dono, M., V. L. Burgio, C. Tacchetti, A. Favre, A. Augliera, S. Zupo,

G. Taborelli, N. Chiorazzi, C. E. Grossi, and M. Ferrarini. 1996. Subepithelial Bcells in the human palatine tonsil. I. Morphologic, cytochemical and phenotypiccharacterization. Eur. J. Immunol. 26: 2035–2042.

2. Muller, G., U. E. Hopken, and M. Lipp. 2003. The impact of CCR7 and CXCR5on lymphoid organ development and systemic immunity. Immunol. Rev. 195:117–135.

3. Wu, M. T., and S. T. Hwang. 2002. CXCR5-transduced bone marrow-deriveddendritic cells traffic to B cell zones of lymph nodes and modify antigen-specificimmune responses. J. Immunol. 168: 5096–5102.

4. Forster, R., T. Emrich, E. Kremmer, and M. Lipp. 1994. Expression of the G-protein-coupled receptor BLR1 defines mature, recirculating B cells and a subsetof T-helper memory cells. Blood 84: 830–840.

5. Saeki, H., M. T. Wu, E. Olasz, and S. T. Hwang. 2000. A migratory populationof skin-derived dendritic cells expresses CXCR5, responds to B lymphocyte che-moattractant in vitro, and co-localizes to B cell zones in lymph nodes in vivo.Eur. J. Immunol. 30: 2808–2814.

6. Yu, P., Y. Wang, R. K. Chin, L. Martinez-Pomares, S. Gordon, M. H. Kosco-Vibois,J. Cyster, and Y. X. Fu. 2002. B cells control the migration of a subset of den-dritic cells into B cell follicles via CXC chemokine ligand 13 in a lymphotoxin-dependent fashion. J. Immunol. 168: 5117–5123.

7. Ansel, K. M., V. N. Ngo, P. L. Hyman, S. A. Luther, R. Forster, J. D. Sedgwick,J. L. Browning, M. Lipp, and J. G. Cyster. 2000. A chemokine-driven positivefeedback loop organizes lymphoid follicles. Nature 406: 309–314.

8. Gunn, M. D., V. N. Ngo, K. M. Ansel, E. H. Ekland, J. G. Cyster, andL. T. Williams. 1998. A B-cell-homing chemokine made in lymphoid folliclesactivates Burkitt’s lymphoma receptor-1. Nature 391: 799–803.

9. Forster, R., A. E. Mattis, E. Kremmer, E. Wolf, G. Brem, and M. Lipp. 1996. Aputative chemokine receptor, BLR1, directs B cell migration to defined lymphoidorgans and specific anatomic compartments of the spleen. Cell 87: 1037–1047.

10. Okada, T., V. N. Ngo, E. H. Ekland, R. Forster, M. Lipp, D. R. Littman, andJ. G. Cyster. 2002. Chemokine requirements for B cell entry to lymph nodes andPeyer’s patches. J. Exp. Med. 196: 65–75.

11. Reif, K., E. H. Ekland, L. Ohl, H. Nakano, M. Lipp, R. Forster, and J. G. Cyster.2002. Balanced responsiveness to chemoattractants from adjacent zones deter-mines B-cell position. Nature 416: 94–99.

12. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Twosubsets of memory T lymphocytes with distinct homing potentials and effectorfunctions. Nature 401: 708–712.

13. Yanagihara, S., E. Komura, J. Nagafune, H. Watarai, and Y. Yamaguchi. 1998.EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation. J. Immunol. 161: 3096–3102.

14. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, andL. T. Williams. 1998. A chemokine expressed in lymphoid high endothelialvenules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc.Natl. Acad. Sci. USA 95: 258–263.

15. Ohl, L., G. Henning, S. Krautwald, M. Lipp, S. Hardtke, G. Bernhardt, O. Pabst,and R. Forster. 2003. Cooperating mechanisms of CXCR5 and CCR7 in de-velopment and organization of secondary lymphoid organs. J. Exp. Med. 197:1199 –1204.

16. Luther, S. A., T. Lopez, W. Bai, D. Hanahan, and J. G. Cyster. 2000. BLCexpression in pancreatic islets causes B cell recruitment and lymphotoxin-depen-dent lymphoid neogenesis. Immunity 12: 471–481.

17. Finke, D., H. Acha-Orbea, A. Mattis, M. Lipp, and J. Kraehenbuhl. 2002.CD4�CD3� cells induce Peyer’s patch development: role of �4�1 integrin ac-tivation by CXCR5. Immunity 17: 363–373.

18. Smith, J. R., R. M. Braziel, S. Paoletti, M. Lipp, M. Uguccioni, andJ. T. Rosenbaum. 2003. Expression of B-cell-attracting chemokine 1 (CXCL13)by malignant lymphocytes and vascular endothelium in primary central nervoussystem lymphoma. Blood 101: 815–821.

19. Mazzucchelli, L., A. Blaser, A. Kappeler, P. Scharli, J. A. Laissue, M. Baggiolini,and M. Uguccioni. 1999. BCA-1 is highly expressed in Helicobacter pylori-induced mucosa-associated lymphoid tissue and gastric lymphoma. J. Clin. In-vest. 104: R49–R54.

20. Ono, R., S. Kobayashi, H. Wagatsuma, K. Aisaka, T. Kohda, T. Kaneko-Ishino,and F. Ishino. 2001. A retrotransposon-derived gene, PEG10, is a novel imprintedgene located on human chromosome 7q21. Genomics 73: 232–237.

21. Ono, R., K. Nakamura, K. Inoue, M. Naruse, T. Usami, N. Wakisaka-Saito,T. Hino, R. Suzuki-Migishima, N. Ogonuki, H. Miki, et al. 2006. Deletion ofPeg10, an imprinted gene acquired from a retrotransposon, causes early embry-onic lethality. Nat. Genet. 38: 101–106.

22. Okabe, H., S. Satoh, Y. Furukawa, T. Kato, S. Hasegawa, Y. Nakajima, Y.Yamaoka, and Y. Nakamura. 2003. Involvement of PEG10 in human hepa-tocellular carcinogenesis through interaction with SIAH1. Cancer Res. 63:3043–3048.

23. Li, C. M., A. A. Margolin, M. Salas, L. Memeo, M. Mansukhani, H. Hibshoosh,M. Szabolcs, A. Klinakis, and B. Tycko. 2006. PEG10 is a c-Myc target gene incancer cells. Cancer Res. 66: 665–672.

24. Bennett, J. M., D. Catovsky, M. T. Daniel, G. Flandrin, D. A. Galton,H. R. Gralnick, and C. Sultan. 1981. The morphological classification of acutelymphoblastic leukaemia: concordance among observers and clinical correla-tions. Br. J. Haematol. 47: 553–561.

25. Cheson, B. D., J. M. Bennett, M. Grever, N. Kay, M. J. Keating, S. O’Brien, andK. R. Rai. 1996. National Cancer Institute-sponsored Working Group guidelinesfor chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment.Blood 87: 4990–4997.

26. Bennett, J. M., D. Catovsky, M. T. Daniel, G. Flandrin, D. A. Galton,H. R. Gralnick, and C. Sultan. 1989. Proposals for the classification of chronic(mature) B and T lymphoid leukaemias. French-American-British (FAB) Coop-erative Group. J. Clin. Pathol. 42: 567–584.

27. Qiuping, Z., L. Qun, H. Chunsong, Z. Xiaolian, H. Baojun, Y. Mingzhen,L. Chengming, H. Jinshen, G. Qingping, Z. Kejian, et al. 2003. Selectively in-creased expression and functions of chemokine receptor CCR9 on CD4� T cellsfrom patients with T-cell lineage acute lymphocytic leukemia. Cancer Res. 63:6469–6477.

28. Jinquan, T., S. Quan, H. H. Jacobi, C. Jing, A. Millner, B. Jensen, H. O. Madsen,L. P. Ryder, A. Svejgaard, H. J. Malling, et al. 2000. CXC chemokine receptor3 expression on CD34� hematopoietic progenitors from human cord blood in-duced by granulocyte-macrophage colony-stimulating factor: chemotaxis and ad-hesion induced by its ligands, interferon �-inducible protein 10 and monokineinduced by interferon �. Blood 96: 1230–1238.

29. Jinquan, T., H. H. Jacobi, C. Jing, A. Millner, E. Sten, L. Hviid, L. Anting,L. P. Ryder, C. Glue, P. S. Skov, et al. 2003. CCR3 expression induced by IL-2and IL-4 functioning as a death receptor for B cells. J. Immunol. 171: 1722–1731.

30. Kruse, N., M. Pette, K. Toyka, and P. Rieckmann. 1997. Quantification of cy-tokine mRNA expression by RT PCR in samples of previously frozen blood.J. Immunol. Methods 210: 195–203.

31. Sica, A., A. Saccani, A. Borsatti, C. A. Power, T. N. Wells, W. Luini,N. Polentarutti, S. Sozzani, and A. Mantovani. 1997. Bacterial lipopolysacchariderapidly inhibits expression of C-C chemokine receptors in human monocytes.J. Exp. Med. 185: 969–974.

32. Massari, P., Y. Ho, and L. M. Wetzler. 2000. Neisseria meningitidis porin PorBinteracts with mitochondria and protects cells from apoptosis. Proc. Natl. Acad.Sci. USA 97: 9070–9075.

33. Youn, B. S., Y. J. Kim, C. Mantel, K. Y. Yu, and H. E. Broxmeyer. 2001.Blocking of c-FLIPL-independent cycloheximide-induced apoptosis or Fas-me-diated apoptosis by the CC chemokine receptor 9/TECK interaction. Blood 98:925–933.

34. Kawasaki, H., and K. Taira. 2003. Short hairpin type of dsRNAs that are con-trolled by tRNA(Val) promoter significantly induce RNAi-mediated gene silenc-ing in the cytoplasm of human cells. Nucleic Acids Res. 31: 700–707.

35. Kanbe, K., N. Shimizu, Y. Soda, K. Takagishi, and H. Hoshino. 1999. A CXCchemokine receptor, CXCR5/BLR1, is a novel and specific coreceptor for humanimmunodeficiency virus type 2. Virology 265: 264–273.

36. Youn, B. S., C. Mantel, and H. E. Broxmeyer. 2000. Chemokines, chemokinereceptors and hematopoiesis. Immunol. Rev. 177: 150–174.

37. Kampen, G. T., S. Stafford, T. Adachi, T. Jinquan, S. Quan, J. A. Grant,P. S. Skov, L. K. Poulsen, and R. Alam. 2000. Eotaxin induces degranulation andchemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases. Blood 95: 1911–1917.

38. Boehme, S. A., S. K. Sullivan, P. D. Crowe, M. Santos, P. J. Conlon,P. Sriramarao, and K. B. Bacon. 1999. Activation of mitogen-activated pro-tein kinase regulates eotaxin-induced eosinophil migration. J. Immunol. 163:1611–1618.


39. Wang, J. F., I. W. Park, and J. E. Groopman. 2000. Stromal cell-derived factor-1�stimulates tyrosine phosphorylation of multiple focal adhesion proteins and in-duces migration of hematopoietic progenitor cells: roles of phosphoinositide-3kinase and protein kinase C. Blood 95: 2505–2513.

40. Thornberry, N. A., and Y. Lazebnik. 1998. Caspases: enemies within. Science281: 1312–1316.

41. Muller, A., B. Homey, H. Soto, N. Ge, D. Catron, M. E. Buchanan,T. McClanahan, E. Murphy, W. Yuan, S. N. Wagner, et al. 2001. Involvement ofchemokine receptors in breast cancer metastasis. Nature 410: 50–56.

42. Geminder, H., O. Sagi-Assif, L. Goldberg, T. Meshel, G. Rechavi, I. P. Witz, andA. Ben-Baruch. 2001. A possible role for CXCR4 and its ligand, the CXC che-mokine stromal cell-derived factor-1, in the development of bone marrow me-tastases in neuroblastoma. J. Immunol. 167: 4747–4757.

43. Scotton, C. J., J. L. Wilson, D. Milliken, G. Stamp, and F. R. Balkwill. 2001.Epithelial cancer cell migration: a role for chemokine receptors? Cancer Res. 61:4961–4965.

44. Durig, J., U. Schmucker, and U. Duhrsen. 2001. Differential expression of che-mokine receptors in B cell malignancies. Leukemia 15: 752–756.

45. Burger, J. A., M. Burger, and T. J. Kipps. 1999. Chronic lymphocytic leukemiaB cells express functional CXCR4 chemokine receptors that mediate spontaneousmigration beneath bone marrow stromal cells. Blood 94: 3658–3667.

46. Till, K. J., K. Lin, M. Zuzel, and J. C. Cawley. 2002. The chemokine receptorCCR7 and �4 integrin are important for migration of chronic lymphocytic leu-kemia cells into lymph nodes. Blood 99: 2977–2984.

47. Hopken, U. E., H. D. Foss, D. Meyer, M. Hinz, K. Leder, H. Stein, and M. Lipp.2002. Up-regulation of the chemokine receptor CCR7 in classical but not in

lymphocyte-predominant Hodgkin disease correlates with distinct disseminationof neoplastic cells in lymphoid organs. Blood 99: 1109–1116.

48. Youn, B. S., K. Y. Yu, J. Oh, J. Lee, T. H. Lee, and H. E. Broxmeyer. 2002. Roleof the CC chemokine receptor 9/TECK interaction in apoptosis. Apoptosis 7:271–276.

49. Han, Z. C., M. Lu, J. Li, M. Defard, B. Boval, N. Schlegel, and J. P. Caen. 1997.Platelet factor 4 and other CXC chemokines support the survival of normal he-matopoietic cells and reduce the chemosensitivity of cells to cytotoxic agents.Blood 89: 2328–2335.

50. Scheuerer, B., M. Ernst, I. Durrbaum-Landmann, J. Fleischer, E. Grage- Griebenow,E. Brandt, H. D. Flad, and F. Petersen. 2000. The CXC-chemokine platelet factor 4promotes monocyte survival and induces monocyte differentiation into macrophages.Blood 95: 1158–1166.

51. Berndt, C., B. Mopps, S. Angermuller, P. Gierschik, and P. H. Krammer. 1998.CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4� T cells.Proc. Natl. Acad. Sci. USA 95: 12556–12561.

52. Algeciras-Schimnich, A., S. R. Vlahakis, A. Villasis-Keever, T. Gomez,C. J. Heppelmann, G. Bou, and C. V. Paya. 2002. CCR5 mediates Fas- andcaspase-8 dependent apoptosis of both uninfected and HIV infected primary hu-man CD4 T cells. AIDS 16: 1467–1478.

53. Nakabayashi, K., L. Bentley, M. P. Hitchins, K. Mitsuya, M. Meguro,S. Minagawa, J. S. Bamforth, P. Stanier, M. Preece, R. Weksberg, et al. 2002.Identification and characterization of an imprinted antisense RNA (MESTIT1) inthe human MEST locus on chromosome 7q32. Hum. Mol. Genet. 11: 1743–1756.

54. Ono, R., H. Shiura, H. Aburatani, T. Kohda, T. Kaneko-Ishino, and F. Ishino.2003. Identification of a large novel imprinted gene cluster on mouse proximalchromosome 6. Genome Res. 13: 1696–1705.


CXC Chemokine Ligand 13 and CC Chemokine Ligand 19 Cooperatively Render Resistance to Apoptosis in B...

Documents

Transcript of CXC Chemokine Ligand 13 and CC Chemokine Ligand 19 Cooperatively Render Resistance to Apoptosis in B...