Aiko-Konno Shirakawa

CCL9 Is Secreted by the Follicle-Associated Epithelium and Recruits Dome Region Peyer’s Patch CD11b+ Dendritic Cells

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3α). CCL20 is chemotactic to the CD11b+ dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer’s patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b+ DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer’s patches, CCL9 (MIP-1γ, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer’s patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer’s patch CD11b+ DCs and in a chemotaxis assay, CD11b+ DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b+ DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b+ DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b+ DC number in the subepithelial dome regions of Peyer’s patches of both wild type and CCR6 −/− mice. Taken together, these results demonstrate an important role of CCL9 in CD11b+ DC recruitment to the dome regions of mouse Peyer’s patches.

1,25-Dihydroxyvitamin D3 Induces CCR10 Expression in Terminally Differentiating Human B Cells

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19+ B cells into IgD−CD38+ plasma cells in vitro. A minor proportion of the resulting CD19+IgD−CD38+ cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamine D3, dramatically increased the proportion of CD19+IgD−CD38+ cells expressing high levels of CCR10. The 1,25-(OH)2D3 also increased the number of CCR10+ cells expressing surface IgA, although the majority of CCR10+ cells remained negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3. We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3.

Tax-Inducible Production of CC Chemokine Ligand 22 by Human T Cell Leukemia Virus Type 1 (HTLV-1)-Infected T Cells Promotes Preferential Transmission of HTLV-1 to CCR4-Expressing CD4+ T Cells

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.

Aberrant expression of Fra-2 promotes CCR4 expression and cell proliferation in adult T-cell leukemia

Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Primary ATL cells frequently express CCR4 at high levels. Since HTLV-1 Tax does not induce CCR4 expression, transcription factor(s) constitutively active in ATL may be responsible for its strong expression. We identified an activator protein-1 (AP-1) site in the CCR4 promoter as the major positive regulatory element in ATL cells. Among the AP-1 family members, Fra-2, JunB and JunD are highly expressed in fresh primary ATL cells. Consistently, the Fra-2/JunB and Fra-2/JunD heterodimers strongly activated the CCR4 promoter in Jurkat cells. Furthermore, Fra-2 small interfering RNA (siRNA) or JunD siRNA, but not JunB siRNA, effectively reduced CCR4 expression and cell growth in ATL cells. Conversely, Fra-2 or JunD overexpression promoted cell growth in Jurkat cells. We identified 49 genes, including c-Myb, BCL-6 and MDM2, which were downregulated by Fra-2 siRNA in ATL cells. c-Myb, BCL-6 and MDM2 were also downregulated by JunD siRNA. As Fra-2, these proto-oncogenes were highly expressed in primary ATL cells but not in normal CD4+ T cells. Collectively, aberrantly expressed Fra-2 in association with JunD may play a major role in CCR4 expression and oncogenesis in ATL.

Expression of CCL17 and CCL22 by latent membrane protein 1-positive tumor cells in age-related Epstein-Barr virus-associated B-cell lymphoproliferative disorder

Age‐related Epstein–Barr virus‐positive (EBV+) B‐cell lymphoproliferative disorder (ALPD) is a disease entity identified from a large‐scale re‐survey of cases diagnosed as diffuse large B‐cell lymphoma. ALPD is a group of EBV+ polymorphic B‐cell lymphoma typically seen in elderly patients. An age‐associated decline in host immunity against EBV might be partly responsible for the pathogenesis of ALPD. Histologically, ALPD is often characterized by a minor proportion of EBV‐encoded RNA‐positive tumor cells in a background of extensive cellular infiltration, similar to that of classical Hodgkins lymphoma. In contrast to Hodgkin and Reed–Sternberg cells, ALPD tumor cells are clearly positive for B cell markers CD20 and/or CD79a. Hodgkin and Reed–Sternberg cells produce various chemokines, including CCL17 and CCL22, that attract chemokine receptor CCR4‐expressing Th2 cells and regulatory T cells. Previously, we have shown that EBV‐immortalized B cells also produce CCL17 and CCL22 through latent membrane protein 1 (LMP1)‐mediated activation of nuclear factor κB. Here we examined expression of CCL17 and CCL22 in ALPD. ALPD tumor cells were often heterogeneous in size in accordance with the differential expression of EBV latent genes at the single cell level. LMP1‐expressing tumor cells were typically large in size and selectively positive for CCL17 and CCL22. CCR4+ cells and forkhead box protein 3+ regulatory T cells were abundantly present, and the majority of forkhead box protein 3+ cells were CCR4+. Collectively, our data show production of CCL17 and CCL22 by LMP1+ large‐sized tumor cells and accumulation of CCR4‐expressing cells including regulatory T cells in ALPD. (Cancer Sci 2008; 99: 296–302)

Expression of CCR9 in HTLV‐1+ T cells and ATL cells expressing Tax

Adult T‐cell leukemia (ATL) is a highly aggressive mature CD4+ T‐cell malignancy that is etiologically associated with human T‐lymphotropic virus Type 1 (HTLV‐1). ATL is characterized by frequent infiltration of lymph nodes, spleen, liver, skin and gut. Previously, we and others have shown that the majority of ATL cases are strongly positive for CCR4, which may explain the frequent skin invasion of ATL. Here, we examined whether ATL cells express CCR9, which is involved in T‐cell homing to the gastrointestinal tract. Human T cell lines carrying HTLV‐1 consistently expressed CCR9 together with the HTLV‐1‐encoded transcriptional activator Tax. Although ATL cells freshly isolated from peripheral blood hardly expressed CCR9, ATL cells cultured for 1 day consistently expressed CCR9 in parallel with the upregulation of Tax. Induction of Tax by Cd2+ in JPX‐9, a subline of Jurkat human T cell line carrying Tax under the control of metallothionein promoter, led to upregulation of CCR9. A luciferase reporter gene under the control of the CCR9 promoter was expressed by cotransfection of an expression vector for Tax or in Cd2+‐treated JPX‐9 cells. Furthermore, immunohistochemical staining demonstrated that ATL cells infiltrating gastrointestinal tract were frequently positive for CCR9. Collectively, CCR9 is inducible in ATL cells expressing Tax and may play a role in the gastrointestinal involvement of ATL.

CXCR7 is inducible by HTLV‐1 Tax and promotes growth and survival of HTLV‐1‐infected T cells

Human T‐lymphotropic virus type 1 (HTLV‐1), the etiological agent of adult T‐cell leukemia (ATL), encodes the potent transcriptional activator Tax, which is required for HTLV‐1‐induced immortalization of T cells. CXCR7 is an atypical chemokine receptor frequently expressed by tumor cells and known to promote cell growth and survival. We found that HTLV‐1‐immortalized T cells expressing Tax consistently expressed CXCR7. Induction of Tax in JPX‐9 upregulated CXCR7. Wild‐type Tax efficiently activated the CXCR7 promoter via a proximal NF‐κB site, while a mutant Tax selectively defective in NF‐κB activation did not. CCX754, a synthetic CXCR7 antagonist, inhibited cell growth and increased apoptosis of HTLV‐1‐immortalized T cells. Knockdown of CXCR7 by small interfering RNA also reduced cell growth. Stable expression of CXCR7 in a CXCR7‐negative ATL cell line promoted cell growth and survival. Taken together, CXCR7 is inducible by Tax and may play an important role in HTLV‐1‐induced immortalization of T cells by promoting growth and survival of HTLV‐1‐infected T cells.

Pathway-selective suppression of chemokine receptor signaling in B cells by LPS through downregulation of PLC-β2

Lymphocyte activation leads to changes in chemokine receptor expression. There are limited data, however, on how lymphocyte activators can alter chemokine signaling by affecting downstream pathways. We hypothesized that B cell-activating agents might alter chemokine responses by affecting downstream signal transducers, and that such effects might differ depending on the activator. We found that activating mouse B cells using either anti-IgM or lipopolysaccharide (LPS) increased the surface expression of CCR6 and CCR7 with large increases in chemotaxis to their cognate ligands. By contrast, while anti-IgM also led to enhanced calcium responses, LPS-treated cells showed only small changes in calcium signaling as compared with cells that were freshly isolated. Of particular interest, we found that LPS caused a reduction in the level of B-cell phospholipase C (PLC)-β2 mRNA and protein. Data obtained using PLC-β2−/− mice showed that the β2 isoform mediates close to one-half the chemokine-induced calcium signal in resting and anti-IgM-activated B cells, and we found that calcium signals in the LPS-treated cells were boosted by increasing the level of PLC-β2 using transfection, consistent with a functional effect of downregulating PLC-β2. Together, our results show activator-specific effects on responses through B-cell chemokine receptors that are mediated by quantitative changes in a downstream signal-transducing protein, revealing an activity for LPS as a downregulator of PLC-β2, and a novel mechanism for controlling chemokine-induced signals in lymphocytes.