Tan Jinquan

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 cells

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-α-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-α-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.

CC chemokine ligand 25 enhances resistance to apoptosis in CD4+ T cells from patients with T-cell lineage acute and chronic lymphocytic leukemia by means of livin activation.

We investigated CD4 and CD8 double-positive thymocytes, CD4+ T cells from typical patients with T-cell lineage acute lymphocytic leukemia (T-ALL) and T cell lineage chronic lymphocytic leukemia (T-CLL), and MOLT4 T cells in terms of CC chemokine ligand 25 (CCL25) functions of induction of resistance to tumor necrosis factor α (TNF-α)–mediated apoptosis. We found that CCL25 selectively enhanced resistance to TNF-α–mediated apoptosis in T-ALL and T-CLL CD4+ T cells as well as in MOLT4 T cells, but CD4 and CD8 double-positive thymocytes did not. One member protein of the inhibitor of apoptosis protein (IAP) family, Livin, was selectively expressed in the malignant cells at higher levels, particularly in T-ALL CD4+ T cells, in comparison with the expression in CD4 and CD8 double-positive thymocytes. After stimulation with CCL25 and apoptotic induction with TNF-α, the expression levels of Livin in these malignant cells were significantly increased. CCL25/thymus-expressed chemokine (TECK), by means of CC chemokine receptor 9 (CCR9) ligation, selectively activated Livin to enhance resistance to TNF-α–mediated apoptosis in c-jun-NH2-kinase 1 (JNK1) kinase-dependent manner. These findings suggested differential functions of CCR9/CCL25 in distinct types of cells. CD4 and CD8 double-positive thymocytes used CCR9/CCL25 for migration, homing, development, maturation, selection, cell homeostasis, whereas malignant cells, particularly T-ALL CD4+ T cells, used CCR9/CCL25 for infiltration, resistance to apoptosis, and inappropriate proliferation.

CXC chemokine receptor 4 expression and stromal cell-derived factor-1α-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10

We report that interleukin (IL)‐4 and IL‐10 can significantly up‐ or down‐regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell‐derived factor‐1α (SDF‐1α)‐induced CD4+ T‐lymphocyte chemotaxis was also correspondingly regulated by IL‐4 and IL‐10. IL‐4 and IL‐10 up‐ or down‐regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real‐time quantitative reverse transcription–polymerase chain reaction (RT–PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd≈ 6·3 n m), and ≈ 70 000 SDF‐1α‐binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1≈ 4·4 n m and Kd2≈ 14·6 n m), and a total of ≈ 130 000 SDF‐1α‐binding sites per cell, among IL‐4‐stimulated CD4+ T lymphocytes. The regulation of CXCR4 expression in CD4+ T lymphocytes by IL‐4 and IL‐10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP‐ and cGMP‐dependent protein kinase (H‐8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both cAMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL‐4 and IL‐10 on CXCR4 expression implied that the capacity of IL‐4 and IL‐10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium‐mobilization stimulation. These results indicate that the effects of IL‐4 and IL‐10 on the CXCR4–SDF‐1 receptor–ligand pair may be of particular importance in the cytokine/chemokine environment concerning the inflammatory processes and in the progression of human immunodeficiency virus (HIV) infection.

Peptide Nucleic Acid Antisense Prolongs Skin Allograft Survival by Means of Blockade of CXCR3 Expression Directing T Cells into Graft

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-γ-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-γ/CXCL9. It was reported that CXC chemokines IFN-γ-inducible protein 10/CXCL10 and monokine induced by IFN-γ/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 ± 2.4 days) compared with physiological saline treatment (7.5 ± 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 ± 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.

Androgen activates PEG10 to promote carcinogenesis in hepatic cancer cells

The molecular mechanism of striking higher prevalence of hepatocellular carcinoma (HCC) in male subjects has not yet been fully elucidated. Here, we report that androgen receptor (AR) is differentially expressed in different HCC cell lines. AR agonist dihydrotestosterone (DHT) enhances HCC cell growth and apoptotic resistance. Antagonist flutamide (FLU) blocks the effects of DHT on the HCC cell lines. Paternally expressed gene 10 (PEG10) is expressed in HCC cell lines at substantial high level. Using small interfering RNAs against AR and PEG10 in AR- and PEG10-expressing BEL-7404 hepatoma cells and HuH7 hepatoma cells (HuH7) cells, and AR-transfection technique in AR-lacking and PEG10-expressing HepG2 cells, we have confirmed that through upregulation and activation of PEG10, DHT enhances HCC cell growth and apoptotic resistance. We have further demonstrated that DHT upregulates expression of human telomerase reverse transcriptase (hTERT) in HCC cell lines in a PEG10-dependent manner. Moreover, AR directly interacts in vivo with androgen-responsive elements in the regions of promoter and exon 2 of PEG10 gene in HCC cell lines. DHT promotes the hepatoma formation in vivo nude mice through PEG10 activation. AR antagonists (FLU and valproate) inhibit the hepatoma formation. These findings suggest that PEG10 plays an essential role in hepatocarcinogenesis. The PEG10 inhibition can be a novel approach for therapy of HCC.

Different Neurotropic Pathogens Elicit Neurotoxic CCR9- or Neurosupportive CXCR3-Expressing Microglia

What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9+Irg1+ (immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-α; priming and rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3+Irg1− microglia, which are sensitive to apoptosis and produce a high level of IL-10 and TGF-β. Administration of CCR9 and/or Irg1 small interfering RNA alters the frequency and functional profiles of neurotoxic CCR9+Irg1+ and neurosupportive CXCR3+Irg1− microglia in vivo. Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of microglia has been confirmed as neurotoxic CCR9+Irg1+ and neurosupportive CXCR3+Irg1− cells in these mouse models. These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer’s disease and AIDS dementia.

CCR3 Expression Induced by IL-2 and IL-4 Functioning as a Death Receptor for B Cells

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (∼98% CCR3+). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19+ (∼40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.

CD19+CD5+ B Cells in Primary IgA Nephropathy

The source of IgA and the mechanism for deposition of IgA in the mesangium remain unknown for primary IgA nephropathy. Because CD19(+)CD5(+) B cells are important producers of IgA and contribute to several autoimmune diseases, they may play an important role in IgA nephropathy. In this study, flow cytometry, quantitative PCR, and confocal microscopy were used to assess the frequency, distribution, Ig production, CD phenotypes, cytokine production, and sensitivity to apoptosis of CD19(+)CD5(+) B cells in the peripheral blood, peritoneal fluid, and kidney biopsies of 36 patients with primary IgA nephropathy. All patients with IgA nephropathy were significantly more likely to have CD19(+)CD5(+) B cells in the peripheral blood, peritoneal fluid, and kidney biopsies than were five control subjects and 10 patients with active systemic lupus erythematosus. The 33 patients who had IgA nephropathy and responded to treatment demonstrated a significant decrease in CD19(+)CD5(+) B cells in the peripheral blood, peritoneal fluid, and kidney (all P < 0.01). In the three patients who had IgA nephropathy and did not respond to treatment, the frequency of CD19(+)CD5(+) B cells did not change. CD19(+)CD5(+) B cells isolated from patients with untreated IgA nephropathy expressed higher levels of IgA, produced more IFN-gamma, and were more resistant to CD95L-induced apoptosis than cells isolated from control subjects and patients with lupus; these properties reversed with effective treatment of IgA nephropathy. In conclusion, these results strongly suggest that CD19(+)CD5(+) B cells play a prominent role in the pathogenesis of primary IgA nephropathy.

Aberration of CCR7+ CD8+ memory T cells from patients with systemic lupus erythematosus: an inducer of T helper type 2 bias of CD4+ T cells

Chemokine receptors are important in the entry of leucocytes into the inflammatory sites of systemic lupus erythematosus (SLE). CCR7+ and CCR7– memory T cells exert different functions in homing, cytokine production and cytotoxicity. To determine whether differential expression and functions of the CCR7 occur in SLE patients, we examined CCR3, CCR4, CCR5, CCR7 and CCR9 on CD4+ and CD8+ T cells from normal and SLE subjects. Flow cytometry, real‐time quantitative reverse transcription polymerase chain reactions and Northern blotting were used to detect the expression of chemokine receptors and cytokines; a chemotaxis assay was used to detect their functions. CD4+ T‐cell stimulation with syngeneic CCR7+ CD8+ CD45RO+ T cells and dendritic cells (including transwell chambers) was used to induce cytokine expression. We demonstrated that CCR7 was selectively, frequently and functionally expressed on CD8+ (94·8%) but not on CD4+ (16·1%) T cells from patients with active SLE, whereas this phenomenon was not seen in normal subjects and in those whose SLE was inactive. CCR7+ CD8+ CD45RO+ memory T cells from patients with active SLE, themselves T helper type 2 (Th2) biased, were inducers of Th2 bias in CD4+ T cells in a cell–cell contact manner in vitro, meanwhile, the cells from both normal subjects and those whose SLE was inactive drove CD4+ T cells into a regulatory T‐cell‐derived cytokine pattern. Our findings might provide new clues to understanding the functions of CCR7+ CD8+ CD45RO+‘central’ memory T cells in autoimmue diseases (such as SLE). We suggest that in the case of active SLE, CCR7+ central memory T cells were able to enter peripheral blood and inflammatory sites from secondary lymphoid organs, were continuously expressing CCR7, and interacted with dendritic cells and functioned as CCR7–‘effector’ memory T cells, which were described in normal humans.

Selectively frequent expression of CXCR5 enhances resistance to apoptosis in CD8 + CD34 + T cells from patients with T-cell-lineage acute lymphocytic leukemia

We investigated CD4+CD34+, CD8+CD34+, CD4+CD34−, and CD8+CD34− T cells from cord blood and from typical patients with T-cell-lineage acute lymphocytic leukemia and T-cell-lineage chronic lymphocytic leukemia in terms of expression and functions of CXCR5/CXCL13. We found that CXCR5 was selectively frequently expressed on T-cell-lineage acute (chronic) lymphocytic leukemia (T-ALL) CD8+CD34+ T cells, but not on T-ALL CD4+CD34+, CD4+CD34−, and CD8+CD34− T cells. CXCR5 was rarely expressed on all types of CD34+ and CD34− CB or T-CLL T cells. CXCL13/B cells attracting chemokine 1 induced significant resistance to TNF-α-mediated apoptosis in T-ALL CD8+CD34+ T cells, instead of induction of chemotactic and adhesive responsiveness. A proliferation-inducing ligand expression in T-ALL CD8+CD34+ T cells was upregulated by CXCL13/BCA-1 (B-cell attracting chemokine 1). The CXCR5/CXCL13 pair by means of activation of APRIL (A proliferation-inducing ligand) induced resistance to apoptosis in T-ALL CD8+CD34+ T cells in livin-dependent manner. In this process, cell–cell contact in culture was necessary. Based on our findings, we suggested that there were differential functions of CXCR5/CXCL13 in distinct types of cells. Normal lymphocytes, especially naïve B and T cells, utilized CXCR5/CXCL13 for migration, homing, maturation, and cell homeostasis, as well as secondary lymphoid tissue organogenesis. Meanwhile, certain malignant cells took advantages of CXCR5/CXCL13 for infiltration, resistance to apoptosis, and inappropriate proliferation.