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CXC Chemokine Ligand 13 and CC Chemokine Ligand 19 Cooperatively Render Resistance to Apoptosis in B Cell Lineage Acute 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 Jinquan^3,*,

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

	Abstract

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CXCL13/CXCR5 and CCL19/CCR7 play a quite important role in normal physiological conditions, but the functions of both chemokine/receptor pairs in pathophysiological events are not well-investigated. We have investigated expression and functions of CXCL13/CXCR5 and CCL19/CCR7 in CD23⁺CD5⁺ and CD23⁺CD5^– B cells from cord blood (CB) and patients with B cell lineage 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 chemotactic responsiveness was observed, CXCL13 and CCL19 cooperatively induce significant resistance to TNF--mediated apoptosis in B-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 elevated levels of paternally expressed gene 10 (PEG10). CXCL13 and CCL19 together significantly up-regulate PEG10 expression in the same cells. We have found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulate PEG10 expression and function, subsequently stabilize caspase-3 and caspase-8 in B-ALL and B-CLL CD23⁺CD5⁺ B cells, and further rescue 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 lymphoid tissues organogenesis. In addition, certain malignant cells take advantages of CXCL13/CXCR5 and CCL19/CCR7 for infiltration, resistance to apoptosis, and inappropriate proliferation.

	Introduction

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Although they decrease with age, CD5⁺ B cells are the major population in fetal life Most human IgM⁺ cord blood (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 subpopulation, showing an increased propensity to malignant transformation (1). CD23⁺CD5⁺ B cells comprise the largest group of the malignant cells in B cell chronic lymphocytic leukemia (B-CLL) and an important component in B cell lineage acute (B-ALL).

CCL19 (EBV-induced gene-1 ligand chemokine (ELC)) and CCL21 (secondary lymphoid tissue chemokine/6Ckine) are ligands for the chemokine receptor CCR7 (Burkitt lymphoma receptor-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, and CCL19, as well as their corresponding receptors, CXCR5 and CCR7, have been shown to closely cooperate in the development of lymphoid organs and the maintenance of lymphoid tissue microarchitecture (2). Expression of CXCR5 can be detected on mature recirculating B cells, small subsets of normal CD4⁺ and CD8⁺ T cells, and skin-derived migratory dendritic cells (3, 4, 5, 6). CXCR5 is essentially responsible for guiding B cells into the B cell zones of secondary lymphoid organs (7, 8, 9). However, the expression of CXCR5 on a subset of T cells strongly suggests an additional role for this receptor in T cell migration (9, 10). CCR7 is highly expressed on naive T cells, but expressed at lower levels on peripheral B cells. T cells and B cells show a transient increase in receptor expression following their activation (11), whereas T cell differentiation toward effector cells is accompanied by a down-regulation of CCR7 on the cell surface (12, 13, 14).

The synergy of CXCL13/CXCR5 and CCL19/CCR7 has been shown in both physiological and pathological situations. CXCR5 cooperated with CCR7 control T cells and dendritic cell homing to secondary lymphoid organs (12, 13, 14). The balanced expression of CCR7 and CXCR5 determines the positioning and proper function of follicular Th cells (11). CXCR5 and CCR7 double-deficient mice lack lymphoid follicles due to an impaired migration of B cells (15). Overexpression of CXCR5 and CCR7 on tumor lymphocytes closely related to cells apoptosis as well as their migration and infiltration (16, 17, 18, 19). The coexpression of CXCR5 and CCR7 were also found in B cell leukemia. However, the functional importance of CXCR5-CXCL13/BCA-1 and CCR7-CCL19/ELC receptor-ligand pairs in the pathophysiological events of malignant B cells trafficking, homing, and survival is not fully understood.

A novel paternally expressed imprinted gene, paternally expressed gene 10 (PEG10), is identified as a paternally expressed gene from a newly defined imprinted region at human chromosome 7q21 (20). PEG10 shows parent-of-origin-specific expression in monochromosomal hybrids (21). PEG10 knockout mice show 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 majority of the human hepatocellular carcinoma (HCC) cells (22, 23). Exogenous expression of PEG10 confers oncogenic activity and transfection of hepatoma cells with PEG10 antisense suppressing its expression results in cancer cell growth inhibition (22). In addition, PEG10 protein associates with human homolog of Drosophila seven in absenia (SIAH1), a mediator of apoptosis. Overexpression of PEG10 reduces the cell from death mediated by SIAH1 (22). Knockdown of PEG10 inhibits the proliferation of pancreatic carcinoma and HepG2 hepatocellular carcinoma cells (23).

In this study, we have found that CXCR5 and CCR7 are selectively expressed on B-ALL, B-CLL, and CB CD23⁺CD5⁺ B cells at high frequency. CXCL13 and CCL19 cooperate selectively to induce resistance to apoptosis in B-ALL and B-CLL CD23⁺CD5⁺ B cells by means of activation of PEG10.

	Materials and Methods

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Patients 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 National Cancer Institute Working Group (25, 26). All patients gave informed consent according to institutional guidelines. CD19⁺ (from normal periphery), CD23⁺, or CD23⁺CD5⁺ cells were purified from PBMCs from peripheral blood of normal subjects, CB of uncomplicated births (IgM undetectable), or patients with B-ALL or B-CLL using a FACStar^Plus sorting (27, 28). The viability of all cultured cells >95% was tested by trypan blue exclusion. The malignancy of purified B-ALL or B-CLL CD23⁺CD5⁺ cells was confirmed by expression of CD20 and FMC-7. The cell line was Raji cell (B cell Burkitt lymphoma cell line) obtained from the American Type Culture Collection. 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-labeled CD23, FITC-labeled CD5 (DakoCytomation) and PerCP-labeled chemokine receptor Ab (R&D Systems), or matched isotype Ab (DakoCytomation) at 5 µg/ml in PBS containing 2% BSA and 0.1% sodium azide for 20 min, followed washing twice with staining buffer (28). The analyses were performed with flow cytometer (Coulter XL; Coulter). For detection of apoptosis, cells were stained in staining medium (RPMI 1640, 2% FBS, and 0.1% sodium azide) with 1 µg/ml propidium iodide (PI) for 30 min at 4°C, then stained with FITC-conjugated annexin V with binding buffer (BD Pharmingen) as previously described (29, 30). Coulter XL was used for analyses. For detection of intracellular active caspases, Cytofix/Cytoperm buffer (BD Pharmingen) was used according to the manufacturer’s instructions to permeabilize cells, and cells were subsequently stained with anti-active-capsase-3 or anti-active-caspase-8 mAb (BD Pharmingen). After washing, active caspase-3 or caspase-8 fluorescence intensity was measured by flow cytometry. Data were analyzed by means of the WinList program (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 special optical tubes in a 96-well microtiter plate (Applied Biosystems) with an ABI PRISM 7700 Sequence Detector Systems (Applied Biosystems). By using the SYBR Green PCR Core Reagents kit, fluorescence signals were generated during each PCR cycle via the 5' to 3' endonuclease activity of AmpliTaq Gold to provide real-time quantitative PCR information. The sequences 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 -actin cDNA. PCR retain conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles with 15 s at 95°C, 60 s at 60°C for amplifications. Potential PCR product contamination was digested by uracil-N-glycosylase because dTTP is substituted by dUTP.

Northern and Western blot assays

For mRNA detection (Northern blot), as previously described (27, 32), each 5 µg of total RNA was electrophoresed under denaturing conditions, followed by blotting onto Nytran membranes, and cross-linked by UV irradiation. CXCR5 and CCR7 cDNA probes, labeled by [-³²P]dCTP, were obtained by PCR amplification of the sequence mentioned above from total RNA from PBMC from normal adults (for CXCR5) or thymocytes from the specimen of thymusectomy (for CCR7), or human hepatoma cell line HepG2 (for PEG10). The membranes were hybridized overnight with 1 x 10⁶ cpm/ml ³²P-labeled probe, followed by intensively washing with 0.2x SSC and 0.1% SDS before being autoradiographed. For protein detection (Western blot), the cells were lysed in lysis buffer. Cell lysis was performed for 30 min at 4°C with lysis buffer. Expression of inhibitor of apoptosis protein (IAP) family proteins (or other proteins indicated) was semiquantified after Western blot analysis (33). Lysates were centrifuged at 10,000 rpm for 5 min at 4°C. Protein concentration was measured by Bio-Rad protein assay. Protein (around 40 µg) was loaded onto 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes after electrophoresis, and incubated with the appropriate Abs at 0.5 µg/ml. Analyses were conducted using ECL detection (Amersham Biosciences). All Abs (Bcl-2, Bcl-x, c-FLIP_L, c-IAP1, c-IAP2, X-linked mammalian, and survivin) were obtained from Santa Cruz Biotechnology, except anti-livin which was obtained 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 (Neuro Probe) technique (27, 29). Briefly, chemokines in RPMI 1640 with 0.5% BSA were placed in the lower wells (25 µl). Twenty-five microliters of cell suspension (2 x 10⁶ cells/ml) was added to the upper well of the chamber, which was separated from the lower well by a 5-µm pore size, polycarbonate, polyvinylpyrolidone-free membrane (Nucleopore). The chamber was incubated for 60 min at 37°C and 5% CO₂. The membrane was then carefully removed, fixed in 70% methanol, and stained for 5 min in 1% Coomassie brilliant blue. The migrating cells were counted using a light microscopy. Approximately 6% of the cells will migrate spontaneously (known as migrating cells on negative control (MCNC)). The chemotactic index (CI) = migrating cell number at tested well of chemoattractant sample/MCNC_. The results were expressed as CI with SD.

Gene silencing assay

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

Plasmids and cell transfection

Plasmids encoding PEG10 and CCR7 used in this study have been previously described (23, 35). The cells were transiently transfected with vectors encoding target genes as described elsewhere (23, 35). Briefly, the cells were cultured with DMEM containing 10% FCS, penicillin, and streptomycin. Cells were grown to 70% confluence in 60-mm dishes for 24 h before transfection. The DNA constructs of expression vectors (0.4 µg unless indicated) or vector only were mixed with 12 µl of LipofectAMINE (Invitrogen Life Technologies) in 2 ml of opti-DMEM serum-free medium and added to cells, and incubated for 6 h. The cells were further cultured in 2.5 ml of DMEM containing 10% FCS in 5% CO₂.

Statistical analysis

Statistical significance was assessed by the paired or unpaired Student t test. Values of p < 0.05 were considered statistically significant.

	Results

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CXCR5 and CCR7 are selectively expressed on B-ALL CD23⁺CD5⁺ B cells at high frequency

We screened some CXC and CC chemokine receptors on different CD23⁺CD5⁺ and CD23⁺CD5^– B cells (as well as normal peripheral CD19⁺CD5⁺ and CD19⁺CD5^– cells). Flow cytometric analysis revealed that the data of CXCR3, CXCR6, CCR4, and CCR9 were either in agreement with previous reports or that there were no differences among four types of cell sources in a total of 41 cases of typical B-ALL and B-CLL patients (Table I). Interestingly, CXCR5 and CCR7 were selectively expressed on B-ALL and B-CLL CD23⁺CD5⁺ B cells at high frequency (86, 81, 42, and 51%, respectively) (Fig. 1, A and B), whereas, they were expressed 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 were expressed at similar levels on CD23⁺CD5⁺ and CD23⁺CD5^– B cells from CB.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. CXCR5 and CCR7 selectively express on CD23+CD5+ B cells. Triple-color flow cytometric analysis of the distribution of CXCR5 and CCR7 on CD23+CD5+ and CD23+CD5– B cells from B-ALL (A) and B-CLL (B) patients. The CD23+ B cells were freshly isolated and stained in triple colors of CD23 (PE), CD5 (FITC), and CXCR5 or CCR7 (PerCP) as described in Materials and Methods. The indicated numbers in the graphs were percentages of CD23+CD5+ and CD23+CD5– B cells. The indicated percentages in the graphs were numbers of CXCR5+ or CCR7+ B cells. The data were from a single experiment, which was representative of 23 (B-ALL) and 18 (B-CLL) similar experiments performed. Isotype Ab controls were expressed as dished curves. CXCR5 expression on CD23+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 quantitative RT-PCR amplification was described in Materials and Methods. The showing 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 of CXCR5 and CCR7 were by Northern blot for freshly isolated CD23+CD5+ B cells from normal PBMC (only CD19+), CB of uncomplicated births, B-ALL, or B-CLL patients. Total RNA from different cells was treated as described in Materials and Methods. The hybridization signals for CXCR5 or CCR7 mRNA from different cells were shown in each upper picture. The 28S rRNAs in lower pictures confirmed that comparable amounts of total RNA were used. In C and D (lower panels), the CXCR5 or CCR7 protein was examined using Western blot analyses. Actins in each lower picture indicated the quantity of total cellular protein from the tested samples loaded in each lane. Arrows indicate markers used to verify equivalent molecular weights of appropriate proteins in each lane.

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 CB CD23⁺CD5⁺ B cells (Fig. 2).

View larger version (22K): [in this window] [in a new window]   FIGURE 2. The chemotaxis of CD23+CD5+ B cells. The chemotaxis of freshly isolated in CD23+CD5+ T cells from CB of uncomplicated births and B-ALL patients toward CXCL13/BCA-1 (A) (), CCL19/ELC (B) (), CCL3/MIP-1 () (100 ng/ml), or PBS control (). All results were expressed as CI or percentage of adhesive cells with SD (±SD), and based on triplicate determination of chemotaxis and adhesion on each concentration of chemokine indicated as nanograms per milliliter. The showing bars were mean values ± SD of eight experiments conducted. Statistical significant differences as compared with controls are indicated 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.

We examined the protective effects of CXCL13 and CCL19 on different types of cells on TNF--mediated apoptosis. Flow cytometric analysis (Fig. 3) revealed that the number of apoptotic and necrotic cells was significantly decreased in cultured B-ALL and B-CLL CD23⁺CD5⁺ B cells in presence of both CXCL13 and CCL19 (Fig. 3A, o and p), in comparison with those in the absence of CXCL13 and CCL19 (Fig. 3A, c and d). Interestingly, CXCL13 or CCL19 alone had no such effect in B-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 in normal peripheral CD19⁺ B cells and CB CD23⁺CD5⁺ B cells (Fig. 3A, e, f, i, j, m, and n), in comparison with those in the absence of CXCL13 and CCL19 (Fig. 3A, a and b). Neither CCL13 nor CXCL19 had such an effect as to rescue different types of B cells (normal peripheral CD19⁺ B cells, and CB, B-ALL B-CLL CD23⁺CD5⁺ B cells) from TNF--mediated apoptotic response (data not shown). Abs against CXCR5 and CCR7 could completely block the combined protective effect of CXCL13 and CCL19, indicating that the rescuing effect was indeed induced by means of interaction of CXCL13/CCL19 and CXCR5/CCR7 (data shown in Fig. 4). The total numbers of dead cells (including apoptotic and necrotic) in different types of the cells exhibited a similar pattern as shown in Fig. 3. Thus, CXCL13 and CCL19 cooperatively rescued B-ALL and B-CLL CD23⁺CD5⁺ B cells from TNF--mediated apoptosis, but not either normal peripheral CD19⁺ B cells or CB CD23⁺CD5⁺ B cells, confirming antiapoptotic effects mediated by CXCR5 and CCR7 in B-ALL and B-CLL CD23⁺CD5⁺ B cells.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Analysis of apoptotic and total dead (necrotic and apoptotic) cells for inhibition of TNF--mediated apoptosis of CD23+CD5+ B cells. Flow cytometric analysis of apoptotic (A) and total dead cells (B), the CD23+CD5+ B cells were freshly isolated from normal PBMC (only CD19+), CB of uncomplicated births, B-ALL or B-CLL patients and were pretreated at absence or presence of chemokine as indicated (all at 100 ng/ml) for 24 h at 37°C, following stimulation with TNF- (100 ng/ml) for 24 h at 37°C. The cells were analyzed by flow cytometry for PI (y-axis) and FITC-conjugated annexin V (x-axis) as described in Materials and Methods. The gating in the forward scatter and side scatter histograms were adhered to the lymphocyte region. The percentages of PI– annexin V+ cells and PI+ annexin V+ cells were indicated in the figure. The data (A) were from a single experiment, which was representative of six experiments 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 untreated cells were indicated (*, p < 0.001, untreated vs CXCL13+CXCL19-treated CD23+CD5+ B cells from B-ALL or B-CLL).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Analysis of the inhibition of TNF--mediated apoptosis in CD23+CD5+ B cells. Flow cytometric analysis of apoptotic (A) and total dead cells (B), B-ALL CD23+CD5+ B cells were freshly isolated from B-ALL patients as described in Materials and Methods, then serum starved overnight and treated with anti-CXCR5 plus anti-CCR7 (each 5 µg/ml, Abs), isotype Abs (each 5 µg/ml, Isotypes), pertussis toxin (PT) (1 µg/ml), wortmannin (WT, 50 nM), PD98059 (50 nM), or vehicle DMSO for 1 h, followed culture in the presence of CXCL13 and CCL19/ELC (, each 100 ng/ml) for 6 h before apoptotic assay. As a control, Z-VAD-FMK (20 nM), a caspase inhibitor, was used for blocking apoptosis. The cells were analyzed by flow cytometry as described in the legend for Fig. 3. The data were from a single experiment, which was representative of six experiments 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 cells were indicated (*, p < 0.001, Ab- or PT-treated vs PD98059-, WT- or Z-VAD-FMK-treated CD23+CD5+ B cells from B-ALL).

PEG10 expression is selectively increased in B-ALL CD23⁺CD5⁺ B cells by CXCL13 and CCL19 costimulation

Western blot showed that the protein levels of one group of antiapoptotic members (Bcl-2, Bcl-x, and c-FLIP_L) in different types of freshly isolated CD23⁺CD5⁺ B cells (CB, B-CLL, and B-ALL) were identical (data not shown). Interestingly, after stimulation with CXCL13 or/and CCL19 and further apoptotic induction with TNF-, their expression levels were still not significantly altered in different types of CD23⁺CD5⁺ B cells (data not shown). The expression levels of another group of antiapoptotic proteins in the IAP family (XIAP, c-IAP1, c-IAP2, survivin, and livin) in different types of freshly isolated CD23⁺CD5⁺ B cells (CB, B-CLL and B-ALL) were identical as well (data not shown). After stimulation with CXCL13 or/and CCL19 and apoptotic induction with TNF-, their expression levels were still not significantly changed in different types of B cell CD23⁺CD5⁺ B cells (data not shown).

We further examined the expression levels of PEG10 in distinct CD23⁺CD5⁺ B cells during stimulation with CXCL13/CCL19 and TNF-. Data obtained from real-time quantitative RT-PCR and Northern blot analyses (Fig. 5) revealed that freshly isolated normal peripheral CD19⁺ B cells and CB CD23⁺CD5⁺ B cells expressed almost no PEG10 (Fig. 5A) or at very low levels (Fig. 5B). PEG10 expression levels in the cells were not significantly altered after 24 h culture with CXCL13 and/or CCL19. Costimulation with TNF- did not change PEG10 expression levels (data not shown). In contrast, freshly isolated B-ALL and B-CLL CD23⁺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 CCL19 costimulation, CXCL13 or CCL19 alone did not show such an effect (Fig. 5, C and D). Costimulation with TNF- did not change PEG10 expression levels (data not shown). The time-course study (Fig. 5E) showed a significant up-regulation of PEG10 mRNA level by CXCL13 and CCL19 costimulation was already observed within 8 h and peaked at 24 h. The data suggested that PEG10 expression and functionality might contribute to might involved in the mechanism of resistance to TNF--mediated apoptosis in B-ALL and B-CLL CD23⁺CD5⁺ B cells as afforded by CXCL13 and CCL19 costimulation.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. PEG10 mRNA expression in CD23+CD5+ B cells. The real-time quantitative detection of RT-PCR (upper panels) and Northern blot (lower panels) for PEG10 mRNA in the CD23+CD5+ B cells. The cells were isolated from normal PBMC (only CD19+) (A), CB of uncomplicated births (B), B-ALL (C and E) or B-CLL (D) patients as described in Materials and Methods, which were pretreated at absence or presence of chemokine as indicated (all at 100 ng/ml) for 24 h at 37°C, following stimulation with TNF- (100 ng/ml) for 24 h at 37°C. The procedure for quantitative RT-PCR amplification was described in Materials and Methods. A linear relationship between the cycle threshold (CT) and log starting quantity of standard DNA template or target cDNA (PEG10) was detected (data not shown). The correlation coefficients are 0.97–0.99. The showing bars were mean values ± SD of six similar experiments conducted (*, p < 0.001, untreated vs CXCL13+CXCL19-treated CD23+CD5+ B cells from B-ALL or B-CLL). Total RNA from different cells as indicated were isolated, electrophoresed and blotted as described in Materials and Methods. The hybridization signals for PEG10 mRNA in different cells were shown in upper parts of the panels. The 28S rRNAs in lower parts of the panels confirmed the comparable amounts of loaded total RNA. The data were from a single experiment, which was representative of six experiments performed. In E, the real-time quantitative detection of RT-PCR for PEG10 mRNA in the CD23+CD5+ B cells from B-ALL patients as described in Materials and Methods, which were pretreated at absence or presence of CXCL13 and CCL19 (each 100 ng/ml) for the time intervals as indicated at 37°C. The data were mean values ± SD of six experiments performed. Statistically significant differences as compared with untreated cells (0 h) were indicated (*, p < 0.01, untreated vs CXCL13+CXCL19-treated B-ALL CD23+CD5+ B cells).

We applied shRNA of PEG10 (shRNAPEG10) to knockdown the endogenous PEG10 expression to verify whether it contributes to CXCL13/CCL19 afforded resistance to TNF--mediated apoptosis. Northern blot results showed that culture with shRNAPEG10 at high concentration (2 µg/ml) completely abolished expression of PEG10 in B-ALL and B-CLL CD23⁺CD5⁺ B cells at mRNA levels, whereas, low concentration shRNAPEG10 (0.02 µg/ml), DNAPEG10, and vector showed no such effects (data not shown). The shRNAPEG10 at high concentration significantly blocked the effects of CXCL13 and CCL19 costimulation on induction of resistance to TNF--mediated apoptosis in B-ALL CD23⁺CD5⁺ B cells, no low concentration shRNAPEG10, DNAPEG10 and vector had such effect (Table II). All treatments did not alter the patterns 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 (data not shown).

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Activation of caspases in CD23+CD5+ B cells. Flow cytometric analysis of active caspase-3 (A) and caspase-8 (B) in CD23+CD5+ B cells. The purified CD23+CD5+ B cells from B-ALL patients were cultured for 6 days in the presence or absence of shRNAPEG10 at low concentration (0.02 µg) and high concentration (2 µg) as described in Materials and Methods. They were then pretreated at presence of CXCL13 or/and CCL19 (all at 100 ng/ml) described in Materials and Methods, following stimulation at presence of TNF- (100 ng/ml) for 24 h at 37°C. They were then permeabilized and fixed as indicated in Materials and Methods and subsequently stained for intracellular 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 were quantitated from relative-frequency histograms. Isotype Ab controls were expressed as dished curves. The data were from a single experiment, which was representative of six experiments performed.

View larger version (42K): [in this window] [in a new window]   FIGURE 7. CXCL13 and CCL19 require PEG10 for protection against apoptosis. Raji cells were transfected with vectors encoding PEG10 (100 or 800 ng alone) (A), or cotransfected with vectors encoding CCR7 (400 ng each) in the absence or presence of increasing concentrations of PEG10 (100, 400, or 800 ng) (B–D). The amount of transfected cDNA was kept constant 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 Materials and Methods for apoptotic cells (right panels) and active caspase-3 (left panels). In B, Western blotting were showing equal expression levels of CXCR5 and CCR7 as well as increasing expression levels of PEG10. In C and D, cotransfected cells were pretreated at absence or presence of CXCL13 and CCL19 (all 100 ng/ml) described in Materials and Methods, some of cells were followed stimulation with TNF- (100 ng/ml) for 24 h at 37°C before other assay as indicated (, no stimulation with TNF-). C, analysis of total dead (necrotic and apoptotic) cells. The cells were analyzed by flow cytometry as described in the legend for Fig. 3. The data were from a single experiment, which was representative of six experiments performed. D, Flow cytometric analysis of active caspase-3 in transfected cells as indicated. Cells were pretreated as mentioned above. They were then permeabilized and fixed as indicated in Materials and Methods and subsequently stained for intracellular activated (cleaved) caspase-3. The indicating percentages of cells with activated caspase-3 were quantitated from relative-frequency histograms. Isotype Ab controls were expressed as dished curves. The data were from a single experiment, which was representative of six experiments performed.

	Discussion

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Chemokine receptor signaling is reported to provide antiapoptotic 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 the survival of endothelial cells and monocytes, respectively (49, 50). However, there are some controversial, even contradictory, reports. For instance, CXCR4 induces programmed cell death of human peripheral CD4⁺ T cells, malignant T cells, and CD4/CXCR4 transfectants (51). The interaction between HIV R5 Env and CCR5 activates the Fas pathway and caspase-8 and triggers FasL production, ultimately causing CD4⁺ T cell death (52). We have also reported that CCR3 expression induced by IL-2 and IL-4 functions as a death receptor for B cells (30). The results in this study, together with other observations, suggest that normal B and T cells use CXCR5/CXCL13 and CCR7/CCL19 for migration, homing, development, maturation, selection, and cell homeostasis as well as secondary lymphoid tissue organogenesis. Meanwhile, some malignant cells, particularly B-ALL and B-CLL CD23⁺CD5⁺ B cells, take advantages of CXCR5/CXCL13 and CCR7/CCL19 for infiltration, resistance to apoptosis, and inappropriate proliferation. To our knowledge, this study is the first report of differential functions of CXCR5/CXCL13 and CCR7/CCL19 in distinct types of cells in terms of induction of apoptotic resistance, and is direct evidence of the pathophysiological activity of B-ALL and B-CLL CD23⁺CD5⁺ B cells induced by CXCL13 and CCL19 costimulation.

PEG10 is identified on human chromosome 7q21 (21, 53). Mouse homolog PEG10 has recently been located in a large imprinted gene cluster on mouse proximal chromosome 6 and has been confirmed to be imprinted (54). Because the protein products from the predicted open reading frames 1 and 2 of PEG10 show homology to the gag and pol proteins of vertebrate retrotransposon Ty3/Gypsy, PEG10 is speculated to be a retrotransposon-derived gene. Distinct expression of PEG10 is found in the brain, kidney, lung, testis, and placenta but not in the liver and a number of other tissues (20). In contrast to this, expression of PEG10 is only detected in the placenta among the 14 adult mouse tissues examined (54). Some experimental data suggest a role for preferential expression of the imprinted genes in regulating growth control of liver and pancreatic carcinoma cells (23). Exogenous expression of PEG10 promotes growth of certain HCC cell lines that do not manifest endogenous expression of this gene. The interaction of PEG10 protein with SIAH proteins plays important roles in resistance to apoptosis (22). Even PEG10 is suggested to serve as a novel molecular target for treatment of HCCs (22). In the present study, we have found that freshly isolated B-ALL and B-CLL CD23⁺CD5⁺ B cells express an elevated level of PEG10, compared with that in normal peripheral CD19⁺ B cells and CB CD23⁺CD5⁺ B cells. After 24 h of culture with CXCL13 and CCL19 costimulation, PEG10 expression levels in the cells had been significantly up-regulated (Fig. 5). By using shRNAs, we have found that abrogation of PEG10 expression significantly blocks the protective effects of CXCL13 and CCL19 costimulation on TNF--mediated apoptosis in B-ALL and B-CLL CD23⁺CD5⁺ B cells (Table II). We suggest that CXCL13 and CCL19 cooperate via frequently expressed and activated CXCR5 and CCR7, which up-regulates PEG10 expression and function, and subsequently stabilizes caspase-3 and caspase-8 in B-ALL and B-CLL CD23⁺CD5⁺ B cells as an important mechanism to rescue the cells from TNF--mediated apoptosis (Figs. 6 and 7). This is the first report of this imprinted gene expressed in both human B-ALL and B-CLL CD23⁺CD5⁺ B cells. We have also proven a mechanism by which overexpression of CXCR5 and CCR7 on B-ALL and B-CLL CD23⁺CD5⁺ B cells renders direct resistance to apoptosis in these malignant cells. Understanding the molecular basis of abnormal imprinting of PEG10 in both human B-ALL and B-CLL will shed new light on the process and mechanism that leads to malignant lymphoproliferative disorders.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the National Key Basic Research Program of China from the Ministry of Science and Technology of People’s Republic of China (Nos. 2001CB510004 and 2001CB510008), and by the National Natural Science Foundation of China (Nos. 39870674, 30572119, 30030130, and 30471509), Science Foundation of Anhui Province, China (No. 98436630), and Education and Research Foundation of Anhui Province, China (No. 98JL063) and Research Foundation from Health Department of Hubei Provincial Government, China (No. 301140344), and a special grant from the Personnel Department of Wuhan University, China. T.J. is a Chang Jiang Scholar supported by Chang Jiang Scholars Program from Ministry of Education, 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 of Immunology, School of Basic Medical Sciences, Anhui Medical University, 230032 Hefei, China or Department of Immunology, Wuhan University School of Medicine, Wuhan University, Dong Hu Road 115, 430071 Wuchang, Wuhan, People’s Republic of China. E-mail address: jinquan_tan{at}hotmail.com

⁴ Abbreviations used in this paper: CB, cord blood; B-CLL, B cell chronic lymphocytic leukemia; B-ALL, B cell lineage acute; ELC, EBV-induced gene-1 ligand chemokine; 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 Drosophila seven in absenia.

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

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