Conventional type 1 dendritic cells (cDC1s) play a crucial role in antitumor immunity through the induction and activation of tumor-specific CD8+ cytotoxic T cells (CTLs). The chemokine XCL1 is a major chemotactic factor for cDC1s and its receptor XCR1 is selectively expressed on cDC1s. Here, we investigated the effect of intratumoral delivery of a highly active form of murine XCL1 (mXCL1-V21C/A59C) on cDC1-mediated antitumor immunity using a hydrophilic gel patch. The hydrophilic gel patch containing mXCL1-V21C/A59C increased cDC1 accumulation in the tumor masses and promoted their migration to the regional lymph nodes, resulting in enhanced induction of tumor-specific CTLs. Tumor-infiltrating cDC1s not only expressed XCR1 but also produced CXCL9, a ligand for CXCR3 which is highly expressed on CTLs and NK cells. Consequently, CTLs and NK cells were increased in the tumor masses of mice treated with mXCL1-V21C/A59C, while immunosuppressive cells such as monocyte-derived suppressive cells and regulatory T cells were decreased. We also confirmed that anti-CXCL9 treatment decreased the tumor infiltration of CTLs. The intratumoral delivery of mXCL1-V21C/A59C significantly decreased tumor growth and prolonged survival in E.G7-OVA and B16-F10 tumor-bearing mice. Furthermore, the antitumor effect of mXCL1-V21CA59C was enhanced in combination with anti-programmed cell death protein 1 treatment. Finally, using The Cancer Genome Atlas database, we found that XCL1 expression was positively correlated with tumor-infiltrating cDC1s and a better prognosis in melanoma patients. Collectively, our findings provide a novel therapeutic approach to enhance tumor-specific CTL responses through the selective recruitment of CXCL9-expressing cDC1s into the tumor masses.

Cancer tissue generally possesses an immunosuppressive microenvironment. However, some cancers are associated with lymphoid stroma (i.e., a widely developed tertiary lymphoid structure). The T-cell zone (paracortex) of secondary lymphoid organs, particularly lymph nodes, is characterized by an abundance of T-cell zone fibroblastic reticular cells (TCZ-FRCs) that express C-C motif chemokine ligand 21 (CCL21) and smooth muscle actin (SMA). We analyzed the presence of TCZ-FRCs in 30 cases of carcinomas with lymphoid stroma of the breast, stomach, colon, tongue, and skin. Immunohistochemistry corroborated the abundance of CCL21+ SMA+ TCZ-FRCs in the normal lymph nodes. In sharp contrast, all 30 carcinomas with lymphoid stroma displayed no CCL21+ SMA+ TCZ-FRCs despite the affluence of T cells. Real-time reverse transcription polymerase chain reaction confirmed a marked decrease in the messenger ribonucleic acid expression of CCL21 and its receptor C-C motif chemokine receptor 7 in cancer lymphoid stroma compared to that in lymph nodes. Next, we analyzed the T cell phenotypes. The cancer lymphoid stroma demonstrated an abundance of CD3+ CD62L- memory-type T cells, in contrast to the presence of CD3+ CD62L+ naïve- and central memory T cells in the T cell zone of lymphoid tissues. Our data demonstrated the following: 1) Cancer lymphoid stroma lacked TCZ-FRCs with abundance of more activated T cells than in lymph nodes and 2) these were common phenomena in cancer lymphoid stroma irrespective of the histological types and organs involved.

The oral cavity serves as the primary path through which substances from the outside world enter our body. Therefore, it functions as a critical component of host defense. Saliva is essential for maintaining a stable oral environment by catching harmful agents, including pathogens, allergens, and chemicals, in the air or food. CCL28, highly expressed in mucosal tissues, such as the colon and salivary glands, is a chemokine that attracts CCR10/CCR3 expressing cells. However, the role of CCL28 in salivary gland formation remains unclear. In this study, we investigated the salivary gland structure in CCL28-deficient mice. Histological analysis showed decreased staining intensity of Alcian blue, which detects acidic mucous, reduced expression of MUC2, and higher infiltration of gram-positive bacteria in the salivary glands of CCL28-deficient mice. In addition, CCL28-deficient mice contained ectopically MUC2-expressed cells in the ducts and reduced the expression of cytokeratin 18, a marker for ductal cells, within the submandibular glands, resulting in decreased duct numbers. Additionally, the submandibular glands of CCL28-deficient mice showed reduced expression of several stem cell markers. These results suggest that CCL28 regulates saliva production via proper differentiation of salivary gland stem cells and could be a valuable biomarker of salivary gland function.

Increasing evidence indicates that immune abnormalities are associated with the pathogenesis of depression. CCR4 is a chemokine receptor that regulates regulatory T cell (Treg) and Th17 cell migration. Here, using a lipopolysaccharide (LPS)-induced depression mouse model, we demonstrated that CCR4 deficiency exacerbated depressive-like behavior. Tregs and M2 macrophages, but not Th17 cells, were decreased in the brain of CCR4-deficient mice. Consistently, treatment with a CCR4 inhibitor reduced Tregs and M2 macrophages in the brain and exacerbated depressive-like behavior. Thus, CCR4 may contribute to the reduction of depressive symptoms by promoting Treg recruitment to the brain and subsequent M2 macrophage polarization.

Four major mucosal-associated chemokines, CCL25, CCL28, CXCL14, and CXCL17, play an important role in protecting mucosal surfaces from infectious pathogens. However, their role in protection against genital herpes remains to be fully explored. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). In this study, we investigated the role of the CCL28/CCR10 chemokine axis in mobilizing protective antiviral B and T cell subsets into the VM site of herpes infection. We report a significant increase in the frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10, in herpes-infected asymptomatic (ASYMP) women compared with symptomatic women. Similarly, a significant increase in the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP C57BL/6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+CD62L-CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected ASYMP mice. Inversely, compared with wild-type C57BL/6 mice, the CCL28 knockout (CCL28-/-) mice (1) appeared to be more susceptible to intravaginal infection and reinfection with HSV type 2, and (2) exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+CD62L-CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. These findings suggest a critical role of the CCL28/CCR10 chemokine axis in the mobilization of antiviral memory B and T cells within the VM to protect against genital herpes infection and disease.

Abstract CCR4 is a major trafficking receptor for Th2 cells and Th17 cells and is considered as a potential therapeutic target for atopic dermatitis (AD). The CCR4 ligands CCL17 and CCL22 have been reported to be upregulated in the skin lesions of AD patients. Of note, Thymic stromal lymphopoietin (TSLP), a master regulator of the Th2 immune response, promotes the expression of CCL17 and CCL22 in AD skin lesions. Here, we investigated the role of CCR4 in an AD mouse model induced by MC903, a TSLP inducer. Topical application of MC903 to ear skin increased the expression of not only TSLP but also CCL17, CCL22, the Th2 cytokine IL-4, and the Th17 cytokine IL-17A. Consistently, MC903 induced AD-like skin lesions as shown by increased epidermal thickness; increased infiltration of eosinophils, mast cells, type 2 innate lymphoid cells, Th2 cells, and Th17 cells; and elevated serum levels of total IgE. We also found increased expansion of Th2 cells and Th17 cells in the regional lymph nodes (LNs) of AD mice. Compound 22, a CCR4 inhibitor, ameliorated AD-like skin lesions with reduction of Th2 cells and Th17 cells in the skin lesions and regional LNs. We further confirmed that compound 22 diminished the expansion of Th2 cells and Th17 cells in the coculture of CD11c + dendritic cells and CD4 + T cells derived from the regional LNs of AD mice. Collectively, CCR4 antagonists may exhibit anti-allergic effects by inhibiting both the recruitment and expansion of Th2 cells and Th17 cells in AD.

Vaginal mucosa-resident anti-viral effector memory B- and T cells appeared to play a crucial role in protection against genital herpes. However, how to mobilize such protective immune cells into the vaginal tissue close to infected epithelial cells remains to be determined. In the present study, we investigate whether and how, CCL28, a major mucosal-associated chemokine, mobilizes effector memory B- and T cells in leading to protecting mucosal surfaces from herpes infection and disease. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). We found the presence of significant frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10 receptor, in herpes-infected asymptomatic (ASYMP) women compared to symptomatic (SYMP) women. A significant amount of the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP B6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+ CD62L- CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected asymptomatic mice. In contrast, compared to wild-type (WT) B6 mice, the CCL28 knockout (CCL28(-/-)) mice: (i) Appeared more susceptible to intravaginal infection and re-infection with HSV-2; (ii) Exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+ CD62L- CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. The results imply a critical role of the CCL28/CCR10 chemokine axis in the mobilization of anti-viral memory B and T cells within the VM to protect against genital herpes infection and disease.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma associated with chronic inflammation (DLBCL-CI) develops in the setting of long-standing inflammation. This type of lymphoma may have specific expression profiles of chemokines involved in the pathogenesis of DLBCL-CI. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and represents a valuable model for the study of this disease category. Using a panel of PAL cell lines, we found that PAL cells expressed and secreted C-X-C motif chemokine ligands 9 and 10 (CXCL9 and CXCL10), the ligands of CXCR3, in contrast to EBV-negative DLBCL cell lines, which did not. Culture supernatants from PAL cell lines attracted CXCR3-expressing CD4+ T cells, CD8+ T cells, and CD56+ natural killer cells from human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CXCR3-positive cytotoxic lymphocytes that expressed interferon-γ. The expression of CXCL9 and CXCL10 was detected in PAL tumor biopsy samples from patients, and CXCR3-positive lymphocytes were abundant in the tissue samples. Collectively, these findings suggest that CXCL9 and CXCL10 are produced by PAL cells and can elicit cytotoxic responses via CXCR3. This chemokine system is also likely to contribute to tissue necrosis, which is a signature histological feature of DLBCL-CI. Further studies are warranted to determine whether the CXCL9-CXCL10/CXCR3 axis exerts antitumor effects in DLBCL-CI.

Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.

Th17 cells express CCR4 and secrete cytokines such as IL-17A and GM-CSF, while dendritic cells (DCs) produce CCL22, a CCR4 ligand, upon stimulation with GM-CSF. Th17 cells are known to play a critical role in the pathogenesis of rheumatoid arthritis (RA). CCL22 has also been shown to be upregulated in the synovial tissues of RA patients. Here, we investigated the role of CCR4 in collagen-induced arthritis (CIA), a mouse model of RA. DBA/1J mice efficiently developed CIA as shown by erythema, paw swelling, joint rigidity, and joint destruction. Th17 cells were increased in the arthritic joints and regional lymph nodes (LNs) of CIA mice. A fraction of Th17 cells were also shown to produce GM-CSF. On the other hand, we observed no significant increases of Th2 cells or Treg cells, the T cell subsets also known to express CCR4, in these tissues. We further observed clusters of CCR4-expressing memory Th17 cells and CCL22-producing DCs in the regional LNs of CIA mice, supporting the role of the CCR4-CCL22 axis in the expansion of Th17 cells in the regional LNs. Compound 22, a CCR4 inhibitor, ameliorated the disease severity with reduction of Th17 cells in the arthritic joints and regional LNs and Th17-DC clusters in the regional LNs. We further confirmed that CCR4-deficient mice in the C57BL/6J background were highly resistant to CIA induction compared with wild-type mice. Collectively, CCR4 contributes to the pathogenesis of CIA and may thus represent a new therapeutic target for RA.

Atopic dermatitis (AD) is the most common inflammatory skin disease, which is characterized by excessive Th2 immune responses. In AD patients, the expression of the chemokines CCL17 and CCL22 is increased in skin lesions, leading to the infiltration of Th2 cells. In addition, typical pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, have also been shown to be associated with the pathogenesis of AD. Recently, DDH-1, an ascorbic acid derivative, has been synthesized and demonstrated to have a more stabilized structure and better skin penetrability. Furthermore, DDH-1 has been shown to suppress pro-inflammatory cytokine expression in vitro and in vivo. Therefore, using an AD mouse model, we evaluated the effect of DDH-1 to reduce allergic skin inflammation. We found that cutaneous administration of DDH-1 significantly reduced the expression levels of TNF-α, IL-1β and IL-6 in the skin lesions of AD-like mice. Additionally, DDH-1 administration also significantly reduced the expression levels of CCL17 and CCL22, resulting in decreased skin infiltration of Th2 cells. Consequently, DDH-1 reduced ear and epidermal thickness, the serum IgE levels and the number of infiltrating inflammatory cells and mast cells into the AD-like skin lesions. Combination treatment with DDH-1 and corticosteroid more efficiently improved the skin lesions compared with corticosteroid alone. Collectively, our results suggest that DDH-1 has an anti-allergic effect in an AD mouse model by reducing not only the pro-inflammatory cytokine expression but also the Th2-associated chemokine expression. Thus, DDH-1 may be beneficial for AD treatment and prevention as a monotherapy or in combination with corticosteroids.

Lung function deterioration is significantly associated with poor prognosis in patients with chronic obstructive pulmonary disease (COPD). We previously reported that CC chemokine ligand 17/thymus and activation-regulated chemokine (CCL17/TARC) could be a predictive factor of lung function decline in patients with COPD. However, the role of CCL17 in the pathogenesis of COPD is unclear. Here we examined the role of CCL17 in lung inflammation using mouse COPD models. Exposure to cigarette smoking induced CCL17 production in bronchial epithelial cells and accumulation of alveolar macrophages in the lungs. Intranasal administration of recombinant CCL17 further enhanced cigarette smoke-induced macrophage accumulation and also aggravated elastase-induced pulmonary emphysema. We confirmed that cigarette smoke (CS) extract as well as hydrogen peroxide upregulated CCL17 in BAES-2B cells. Of note, macrophages of both M1 and M2 surface markers were accumulated by cigarette smoke. Both alveolar macrophage accumulation via exposure to cigarette smoking and emphysematous changes induced by elastase administration were significantly reduced in CCL17-deficient mice. We further demonstrated that CCL17 strongly induced the expression of CC chemokine ligand 2 (CCL2), a chemoattractant for macrophages, in RAW264.7 cells, and its production was inhibited by knockdown of CCR4, the receptor of CCL17. Collectively, the present results demonstrate that CCL17 is produced by lung epithelial cells upon CS exposure. Furthermore, CCL17 is involved in CS-induced accumulation of alveolar macrophages and development of elastase-induced pulmonary emphysema, possibly through CCL17-induced production of CCL2 by macrophages. Our findings may provide a new insight into the pathogenesis of COPD.

Various immune cells are involved in host tumor immune responses. In particular, there are many T cell subsets with different roles in tumor immunity. T-helper (Th) 1 cells are involved in cellular immunity and thus play the major role in host anti-tumor immunity by inducing and activating cytotoxic T lymphocytes (CTLs). On the other hand, Th2 cells are involved in humoral immunity and suppressive to Th1 responses. Regulatory T (Treg) cells negatively regulate immune responses and contribute to immune evasion of tumor cells. Th17 cells are involved in inflammatory responses and may play a role in tumor progression. However, recent studies have also shown that Th17 cells are capable of directly inducting CTLs and thus may promote anti-tumor immunity. Besides these T cell subsets, there are many other innate immune cells such as dendritic cells (DCs), natural killer (NK) cells, and myeloid-derived suppressor cells (MDSCs) that are involved in host immune responses to cancer. The migratory properties of various immune cells are critical for their functions and largely regulated by the chemokine superfamily. Thus, chemokines and chemokine receptors play vital roles in the orchestration of host immune responses to cancer. In this review, we overview the various immune cells involved in host responses to cancer and their migratory properties regulated by the chemokine superfamily. Understanding the roles of chemokines and chemokine receptors in host immune responses to cancer may provide new therapeutic opportunities for cancer immunotherapy.

Psoriasis is a chronic skin disease associated with T helper (Th)17-mediated inflammation. Because CCR4 is a major chemokine receptor expressed on Th17 cells, we investigated the role of CCR4 in a modified imiquimod-induced psoriasis model that showed enhanced skin infiltration of Th17 cells. CCR4-deficient mice had less severe skin disease than wild-type mice. Th17 cells were decreased in the skin lesions and regional lymph nodes of CCR4-deficient mice. In the regional lymph nodes of wild-type mice, CD44+ memory Th17 cells expressing CCR4 were found to be clustered with dendritic cells expressing CCL22, a ligand for CCR4. Such dendritic cell‒Th17 cell clusters were significantly decreased in CCR4-deficient mice. Similar results were obtained using the IL-23‒induced psoriasis model. In vitro, compound 22, a CCR4 antagonist, significantly reduced the expansion of Th17 cells in the coculture of CD11c+ dendritic cells and CD4+ T cells separately prepared from the regional lymph nodes of wild-type mice with psoriasis. In vivo, compound 22 ameliorated the psoriasis-like skin disease in wild-type mice with significant decreases of Th17 cells in the regional lymph nodes and skin lesions. Collectively, CCR4 is likely to play a role in the pathogenesis of psoriasis through the expansion of Th17 cells.

Cancer immunotherapy aims to treat cancer by enhancing cancer-specific host immune responses. Recently, cancer immunotherapy has been attracting much attention because of the successful clinical application of immune checkpoint inhibitors targeting the CTLA-4 and PD-1/PD-L1 pathways. However, although highly effective in some patients, immune checkpoint inhibitors are beneficial only in a limited fraction of patients, possibly because of the lack of enough cancer-specific immune cells, especially CD8+ cytotoxic T-lymphocytes (CTLs), in the host. On the other hand, studies on cancer vaccines, especially DC-based ones, have made significant progress in recent years. In particular, the identification and characterization of cross-presenting DCs have greatly advanced the strategy for the development of effective DC-based vaccines. In this review, we first summarize the surface markers and functional properties of the five major DC subsets. We then describe new approaches to induce antigen-specific CTLs by targeted delivery of antigens to cross-presenting DCs. In this context, the chemokine receptor XCR1 and its ligand XCL1, being selectively expressed by cross-presenting DCs and mainly produced by activated CD8+ T cells, respectively, provide highly promising molecular tools for this purpose. In the near future, CTL-inducing DC-based cancer vaccines may provide a new breakthrough in cancer immunotherapy alone or in combination with immune checkpoint inhibitors.

FoxP3+ regulatory T cells (Tregs) play crucial roles in peripheral immune tolerance. In addition, Tregs that reside or accumulate in nonlymphoid tissues, called tissue Tregs, exhibit tissue-specific functions and contribute to the maintenance of tissue homeostasis and repair. In an experimental mouse model of crescentic glomerulonephritis induced by an anti-glomerular basement membrane antibody, Tregs started to accumulate in the kidney on day 10 of disease onset and remained at high levels (~30-35% of CD4+ T cells) during the late stage (days 21-90), which correlated with stable disease control. Treg depletion on day 21 resulted in the relapse of renal dysfunction and an increase in Th1 cells, suggesting that Tregs are essential for disease control during the convalescence stage. The Tregs that accumulated in the kidney showed tissue Treg phenotypes, including high expression of GATA3, ST2 (the IL33 receptor subunit), amphiregulin (Areg), and PPARγ. Although T-bet+ Tregs and RORγt+ Tregs were observed in the kidney, GATA3+ Tregs were predominant during the convalescence stage, and a PPARγ agonist enhanced the accumulation of GATA3+ Tregs in the kidney. To understand the function of specific genes in kidney Tregs, we developed a novel T cell transfer system to T cell-deficient mice. This experiment demonstrates that ST2, Areg, and CCR4 in Tregs play important roles in the accumulation of GATA3+ Tregs in the kidney and in the amelioration of renal injury. Our data suggest that GATA3 is important for the recruitment of Tregs into the kidney, which is necessary for convalescence after renal tissue destruction.

Exposure to moderate doses of ionizing radiation (IR), which is sufficient for causing skin injury, can occur during radiation therapy as well as in radiation accidents. Radiation-induced skin injury occasionally recovers, although its underlying mechanism remains unclear. Moderate-dose IR is frequently utilized for bone marrow transplantation in mice; therefore, this mouse model can help understand the mechanism. We had previously reported that bone marrow-derived cells (BMDCs) migrate to the epidermis-dermis junction in response to IR, although their role remains unknown. Here, we investigated the role of BMDCs in radiation-induced skin injury in BMT mice and observed that BMDCs contributed to skin recovery after IR-induced barrier dysfunction. One of the important mechanisms involved the action of CCL17 secreted by BMDCs on irradiated basal cells, leading to accelerated proliferation and recovery of apoptosis caused by IR. Our findings suggest that BMDCs are key players in IR-induced skin injury recovery.

Extracellular ATP is known to promote Th17 cell differentiation in the intestinal lamina propria by stimulating CD70+CD11clow dendritic cells (DCs) via P2X receptors (P2XRs). Recent studies have also shown that Th17 cells enhance antitumor immunity by directly promoting proliferation of cytotoxic T lymphocytes (CTLs). These finding led us to test a P2XR agonist, αβ-methylene ATP (αβ-ATP), as a mucosal vaccine adjuvant to promote CTL responses through Th17 induction. We demonstrated that (i) CD70+CD11clow DCs were present in the nasal lamina propria and expressed P2X1R, P2X2R and P2X4R; (ii) CD70+CD11clow DCs isolated from the nasal lamina propria enhanced Th17 cell differentiation of cocultured splenic CD4+ T cells upon stimulation with αβ-ATP; (iii) mice intranasally immunized with ovalbumin (OVA) and αβ-ATP had increased OVA-specific Th17 cells and CTLs in the nasal lamina propria and regional lymph nodes; (iv) mice intranasally immunized with OVA and αβ-ATP also had elevated resistance to E.G7-OVA tumor growth compared with those intranasally immunized with OVA alone; (v) suramin, a broad-range inhibitor of P2 receptors, suppressed the increases of OVA-specific Th17 cells and CTLs in mice intranasally immunized with OVA and αβ-ATP; and (vi) suramin also abrogated the enhanced antitumor immunity of mice intranasally immunized with OVA and αβ-ATP against E.G7-OVA. Collectively, αβ-ATP may be a promising mucosal adjuvant that promotes antigen-specific CTL responses via CD70+CD11clow DC-mediated Th17 induction.

Memory CD8+ cytotoxic T-lymphocytes (CTLs) play a key role in protective immunity against infection and cancer. However, the induction of memory CTLs with currently available vaccines remains difficult. The chemokine receptor XCR1 is predominantly expressed on CD103+ cross-presenting dendritic cells (DCs). Recently, we have demonstrated that a high activity form of murine lymphotactin/XCL1 (mXCL1-V21C/A59C), a ligand of XCR1, can induce antigen-specific memory CTLs by increasing the accumulation of CD103+ DCs in the vaccination site and the regional lymph nodes. Here, we combined a hydrophilic gel patch as a transcutaneous delivery device and mXCL1-V21C/A59C as an adjuvant to further enhance memory CTL responses. The transcutaneous delivery of ovalbumin (OVA) and mXCL1-V21C/A59C by the hydrophilic gel patch increased CD103+ DCs in the vaccination site and the regional lymph nodes for a prolonged period of time compared with the intradermal injection of OVA and mXCL1-V21C/A59C. Furthermore, the hydrophilic gel patch containing OVA and mXCL1-V21C/A59C strongly induced OVA-specific memory CTLs and efficiently inhibited the growth of OVA-expressing tumors more than the intradermal injection of OVA and mXCL1-V21C/A59C. Collectively, this type of hydrophilic gel patch and a high activity form of XCL1 may provide a useful tool for the induction of memory CTL responses.

免疫細胞は，生体内を循環することで異物侵入に対して全身をパトロールしているが，いざ異物が侵入してくると，その侵入部位へ即座に動員される．このような免疫細胞の細胞遊走には，方向性を持たないランダムな運動(chemokinesis)と細胞遊走因子の濃度勾配にしたがった方向性を持った運動(chemotaxis)に分けられる．Chemotaxis反応では，標的部位におけるケモカインの発現と免疫細胞におけるケモカイン受容体の選択的発現によって，免疫細胞の体内動態は緻密に制御されている．また細胞が，細胞外マトリックスに結合した細胞遊走因子と接触することで引き起こされる運動(haptotaxis)もある．

免疫系の主役となるリンパ球は，血管系とリンパ系を絶えず循環し，外界からの異物侵入に対してパトロール役として機能する．骨髄や胸腺などの一次リンパ組織で産生されたリンパ球は，二次リンパ組織に流入し，さらにその場を離れて全身を巡り，再び特定の二次リンパ組織に戻ってくる．この現象をリンパ球ホーミングという．この特定の組織への流入を制御する分子メカニズムには，リンパ球上に発現する接着分子やケモカイン受容体と標的組織の血管に発現するケモカインの組み合わせが重要である．例えば，リンパ節ホーミングには，接着分子L-セレクチンおよびケモカイン受容体CCR7が重要な役割を果たすことが明らかにされている．

Populations of CD8+ lung-resident memory T (TRM) cells persist in the interstitium and epithelium (airways) following recovery from respiratory virus infections. While it is clear that CD8+ TRM cells in the airways are dynamically maintained via the continuous recruitment of new cells, there is a vigorous debate about whether tissue-circulating effector memory T (TEM) cells are the source of these newly recruited cells. Here we definitively demonstrate that CD8+ TRM cells in the lung airways are not derived from TEM cells in the circulation, but are seeded continuously by TRM cells from the lung interstitium. This process is driven by CXCR6 that is expressed uniquely on TRM cells but not TEM cells. We further demonstrate that the lung interstitium CD8+ TRM cell population is also maintained independently of TEM cells via a homeostatic proliferation mechanism. Taken together, these data show that lung memory CD8+ TRM cells in the lung interstitium and airways are compartmentally separated from TEM cells and clarify the mechanisms underlying their maintenance.

Chemokines interact with their G protein-coupled receptors (GPCRs) through a two-step, two-site mechanism and, through this interaction, mediate various homeostatic and immune response mechanisms. Upon initial recognition of the chemokine by the receptor, the amino terminus of the chemokine inserts into the orthosteric pocket of the GPCR, causing conformational changes that trigger intracellular signaling. There is considerable structural and functional evidence to suggest that the amino acid composition and length of the chemokine amino terminus is critical for GPCR activation, complementing the size and amino acid composition of the orthosteric pocket. However, very few structures of a native chemokine-receptor complex have been solved. Here, we used a hybrid approach that combines structure-function data with Rosetta modeling to describe key contacts within a chemokine-GPCR interface. We found that the extreme amino-terminal residues of the chemokine XCL1 (Val1, Gly2, Ser3, and Glu4) contribute a large fraction of the binding energy to its receptor XCR1, whereas residues near the disulfide bond-forming residue Cys11 modulate XCR1 activation. Alterations in the XCL1 amino terminus changed XCR1 activation, as determined by assessing inositol triphosphate accumulation, intracellular calcium release, and directed cell migration. Computational analysis of XCL1-XCR1 interactions revealed functional contacts involving Glu4 of XCL1 and Tyr117 and Arg273 of XCR1. Subsequent mutation of Tyr117 and Arg273 led to diminished binding and activation of XCR1 by XCL1. These findings demonstrate the utility of a hybrid approach, using biological data and homology modeling, to study chemokine-GPCR interactions.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas associated with chronic inflammation (DLBCL-CI) develop in patients with chronic inflammation but without any predisposing immunodeficiency. Given the expression of the EBV latent genes, DLBCL-CI should have mechanisms for evasion of host antitumor immunity. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and may provide a valuable model for the study of immune evasion by DLBCL-CI. This study demonstrates that PAL cell lines express and secrete CCL17 and/or CCL22 chemokines, the ligands of C-C motif chemokine receptor 4 (CCR4), in contrast to EBV-negative DLBCL cell lines. Accordingly, culture supernatants of PAL cell lines efficiently attracted CCR4-positive regulatory T (Treg) cells in human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CCR4-expressing Treg cells. Furthermore, this study confirmed that CCR4-expressing Treg cells were abundantly present in primary PAL tissues. Collectively, these findings provide new insight into the mechanisms of immune evasion by PAL, and further studies are warranted on whether such mechanisms eventually lead to the development of DLBCL-CI.

CCR4 is a chemokine receptor highly expressed by Th2 cells, and regarded as a potential therapeutic target for atopic dermatitis (AD). CCL17 and CCL22 are the CCR4 ligands, and thymic stromal lymphopoietin (TSLP) is shown to promote the expression of CCL17 and CCL22 by dendritic cells. Here, by using dibutyl phthalate (DBP), a TSLP inducer, and a hydrogel patch as a transcutaneous delivery device for ovalbumin, we developed a novel murine AD model and investigated the effect of Compound 22, a CCR4 antagonist. We first found that the mRNA expression of TSLP together with CCL17 and CCL22 was increased in the skins treated with DBP. Furthermore, the topical application of ovalbumin and DBP efficiently and rapidly induced AD-like skin lesions in BALB/c mice, which were characterized by ear swelling accompanied by infiltration of eosinophils, mast cells, and CCR4-expressing Th2 cells in the skin lesions, and elevated total IgE levels in the sera. Using this AD model, we demonstrated that cutaneous administration of Compound 22 inhibited Th2 cell infiltration and ameliorated the AD-like skin lesions. These results suggest that our AD model could be useful for studying new therapeutic strategies. Collectively, CCR4 antagonists may be a promising approach for treating AD.

In addition to maintaining immune tolerance, FOXP3+ regulatory T (Treg) cells perform specialized functions in tissue homeostasis and remodelling1,2. However, the characteristics and functions of brain Treg cells are not well understood because there is a low number of Treg cells in the brain under normal conditions. Here we show that there is massive accumulation of Treg cells in the mouse brain after ischaemic stroke, and this potentiates neurological recovery during the chronic phase of ischaemic brain injury. Although brain Treg cells are similar to Treg cells in other tissues such as visceral adipose tissue and muscle3-5, they are apparently distinct and express unique genes related to the nervous system including Htr7, which encodes the serotonin receptor 5-HT7. The amplification of brain Treg cells is dependent on interleukin (IL)-2, IL-33, serotonin and T cell receptor recognition, and infiltration into the brain is driven by the chemokines CCL1 and CCL20. Brain Treg cells suppress neurotoxic astrogliosis by producing amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand. Stroke is a leading cause of neurological disability, and there are currently few effective recovery methods other than rehabilitation during the chronic phase. Our findings suggest that Treg cells and their products may provide therapeutic opportunities for neuronal protection against stroke and neuroinflammatory diseases.

Psoriasis is a chronic inflammatory skin disease in which inflammatory cytokines play a major role in its pathogenesis. Because DDH-1, a novel amphipathic ascorbic acid derivative, has been recently shown to reduce inflammatory cytokine expression in human keratinocytes in vitro, we investigated its effect on imiquimod-induced psoriasis-like skin lesions in C57BL/6 mice. We first found that IL-1β and TNF-α mRNA expression was significantly decreased in the skin lesions treated with DDH-1. Furthermore, cutaneous administration of DDH-1 ameliorated psoriasis-like skin lesions. These results suggest that DDH-1 may be effective in the prevention and supplemental treatment of psoriasis.

Atopic dermatitis is a chronic inflammatory skin disease involving T-helper (Th) 2 cells, eosinophils, and mast cells. Although CCR4 is a major chemokine receptor expressed on Th2 cells and regarded as a potential therapeutic target for allergic diseases, its role in atopic dermatitis remains unclear. Here, by using a hydrogel patch as a transcutaneous delivery device for ovalbumin (an antigen) and Staphylococcus aureus δ-toxin (a mast cell activator), we efficiently induced acute atopic dermatitis-like skin lesions in BALB/c mice, a strain prone to Th2 responses, which were characterized by increased numbers of eosinophils, mast cells, and CCR4-expressing Th2 cells in the skin lesions; elevated levels of total and ovalbumin-specific IgE in the sera; and increased expression of IL-4, IL-17A, IL-22, CCL17, CCL22, and CCR4 in the skin lesions. Of note, the same model was less efficient in C57BL/6 mice, a strain prone to Th1 responses. Using this atopic dermatitis model in BALB/c mice, we demonstrated that CCR4-deficiency or a CCR4 antagonist ameliorated the allergic responses. Collectively, these results demonstrate that CCR4 plays a pivotal role in skin allergic inflammation of BALB/c mice by recruiting CCR4-expressing Th2 cells and Th17 cells.

CCR4 is a major chemokine receptor expressed by Treg cells that downregulate immune responses. Here, we investigated the role of CCR4-mediated Treg cell recruitment in antigen-specific immune responses. CCR4-deficient mice immunized intramuscularly with ovalbumin (OVA) showed enhanced OVA-specific IgG responses. Furthermore, intramuscular administration of OVA induced the expression of MDC/CCL22, a ligand for CCR4, in macrophages of the muscle tissues, and enhanced the recruitment of CCR4+ Treg cells in wild-type mice, whereas this recruitment of Treg cells was severely impaired in CCR4-deficient mice. Furthermore, OVA-loaded dendritic cells (DCs) derived from the muscle injection site of CCR4-deficient mice had an upregulated expression of the DC activation marker CD40 and 86, and the lymphoid organ homing receptor CCR7 resulting in an increased number of migratory DCs in the regional lymph node. Compound 22, a CCR4 antagonist, also inhibited the recruitment of Treg cells to the muscle tissue, and further enhanced DC activation and homing to the regional lymph node. Consequently, Compound 22 enhanced OVA-specific IgG responses, and the expression levels of IL-4 and IFN-γ in CD4+ T cells and the levels of IFN-γ in CD8+ T cells. Finally, intramuscular administration of OVA and Compound 22 significantly inhibited the growth of OVA-expressing tumors. Collectively, CCR4 plays a pivotal role in Treg cell recruitment to the muscle tissue, and intramuscular administration of CCR4 antagonists may be a promising approach for enhancing vaccine efficacy.

CCL28 induces the migration of IgA Ab-secreting cells (ASCs) via CCR10 and also displays a potent antimicrobial activity in vitro. To explore the role of CCL28 in vivo, we generated CCL28-deficient mice. The mice exhibited a significant reduction and abnormal distribution of IgA ASCs in the lamina propria of the colon. The concentrations of total and Ag-specific IgA in the fecal extracts of CCL28-deficient mice were also drastically reduced. The average amount of IgA secreted by a single IgA ASC derived from the colon was also substantially reduced in CCL28-deficient mice. Furthermore, CCL28 was found to significantly increase the average amount of IgA secreted by a single IgA ASC derived from the colon in vitro. In contrast, the generation of IgA ASCs in Peyer's and cecal patches was not significantly impaired in CCL28-deficient mice. We also found a relative increase in the Class Bacilli in the fecal extracts of CCL28-deficient mice and demonstrated a potent antimicrobial activity of CCL28 against Bacillus cereus and Enterococcus faecalis, both of which belong to Class Bacilli. Thus, CCL28 may also suppress the outgrowth of some bacterial species by its direct antimicrobial activity. Finally, CCL28-deficient mice exhibited a highly aggravated dextran sodium sulfate-induced colitis that was ameliorated by pretreatment with antibiotics. Collectively, CCL28 plays a pivotal role in the homing, distribution, and function of IgA ASCs in the colon and may also affect the intestinal microbiota through its direct antimicrobial activity.

The chemokine receptor XCR1 is known to be selectively expressed by cross-presenting dendritic cells (DCs), while its ligand XCL1/lymphotactin is mainly produced by activated CD8+ T cells and natural killer cells. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8+ T cell responses by preferentially delivering antigens to XCR1+ DCs. However, XCL1 per se was found to be a poor adjuvant for induction of CD8+ T cell responses. XCL1 is unique because of its lack of one of the two disulfide bonds commonly conserved in all other chemokines and thus has an unstable structure with a relatively weak chemokine activity. In the present study, we generated a variant form of murine XCL1 termed mXCL1-V21C/A59C that contained a second disulfide bond to stabilize its chemokine structure. We confirmed that mXCL1-V21C/A59C had much more potent chemotactic and calcium mobilization activities than the wild type XCL1 (mXCL1-WT). Intradermal injection of mXCL1-V21C/A59C, but not that of mXCL1-WT, significantly increased the accumulation of XCR1+CD103+ DCs in the injection site, and most of the accumulated XCR1+CD103+ DCs were found to take up co-injected ovalbumin (OVA). Furthermore, recruited XCR1+CD103+ DCs efficiently migrated to the draining lymph nodes and stayed for a prolonged period of time. Consequently, mXCL1-V21C/A59C strongly induced OVA-specific CD8+ T cells. The combination of OVA and mXCL1-V21C/A59C well protected mice from E.G7-OVA tumor growth in both prophylactic and therapeutic protocols. Finally, memory CTL responses were efficiently induced in mice immunized with OVA and mXCL1-V21C/A59C. Although intradermal injection of OVA and polyinosinic-polycytidylic acid (poly(I:C)) as an adjuvant also induced CD8+ T cell responses to OVA, poly (I:C) poorly recruited XCR1+CD103+ DCs in the injection site and failed to induce significant memory CTL responses to OVA. Collectively, our findings demonstrate that a highly active form of XCL1 is a promising vaccine adjuvant for cross-presenting DCs to induce antigen-specific effector and memory CD8+ T cells.

Knock-in mice lacking PKN1 kinase activity were generated by introducing a T778A point mutation in the catalytic domain. PKN1[T778A] mutant mice developed to adulthood without apparent external abnormalities, but exhibited lower T and B lymphocyte counts in the peripheral blood than those of wild-type (WT) mice. T and B cell development proceeded in an apparently normal fashion in bone marrow and thymus of PKN1[T778A] mice, however, the number of T and B cell counts were significantly higher in the lymph nodes and spleen of mutant mice in those of WT mice. After transfusion into WT recipients, EGFP-labelled PKN1[T778A] donor lymphocytes were significantly less abundant in the peripheral circulation and more abundant in the spleen and lymph nodes of recipient mice compared with EGFP-labelled WT donor lymphocytes, likely reflecting lymphocyte sequestration in the spleen and lymph nodes in a cell-autonomous fashion. PKN1[T778A] lymphocytes showed significantly lower chemotaxis towards chemokines and sphingosine 1-phosphate (S1P) than WT cells in vitro. The biggest migration defect was observed in response to S1P, which is essential for lymphocyte egress from secondary lymphoid organs. These results reveal a novel role of PKN1 in lymphocyte migration and localization.

Resistance to the breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitor (TKI) imatinib poses a major problem when treating chronic myeloid leukemia (CML). Imatinib resistance often results from a secondary mutation in BCR-ABL1. However, in the absence of a mutation in BCR-ABL1, the basis of BCR-ABL1-independent resistance must be elucidated. To gain insight into the mechanisms of BCR-ABL1-independent imatinib resistance, we performed an array-based comparative genomic hybridization. We identified various resistance-related genes, and focused on MET. Treatment with a MET inhibitor resensitized K562/IR cells to BCR-ABL1 TKIs. Combined treatment of K562/IR cells with imatinib and a MET inhibitor suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation, but did not affect AKT activation. Our findings implicate the MET/ERK and MET/JNK pathways in conferring resistance to imatinib, providing new insights into the mechanisms of BCR-ABL1 TKI resistance in CML.

CCR4 is a major chemokine receptor expressed by Treg cells and Th17 cells. While Treg cells are known to suppress antitumor immunity, Th17 cells have recently been shown to enhance the induction of antitumor cytotoxic T lymphocytes. Here, CCR4-deficient mice displayed enhanced tumor growth upon intradermal inoculation of B16-F10 melanoma cells. In CCR4-deficient mice, while IFN-γ+CD8+ effector T cells were decreased in tumor sites, IFN-γ+CD8+ T cells and Th17 cells were decreased in regional lymph nodes. In wild-type mice, CD4+IL-17A+ cells, which were identified as CCR4+CD44+ memory Th17, were found to be clustered around dendritic cells expressing MDC/CCL22, a ligand for CCR4, in regional lymph nodes. Compound 22, a CCR4 antagonist, also enhanced tumor growth and decreased Th17 cells in regional lymph nodes in tumor-bearing mice treated with Dacarbazine. In contrast, CCR6 deficiency did not affect the tumor growth and the numbers of Th17 cells in regional lymph nodes. These findings indicate that CCR4 is critically involved in regional lymph node DC-Th17 cell interactions that are necessary for Th17 cell-mediated induction of antitumor CD8+ effector T cells in mice bearing B16 melanoma.

CC chemokine receptor 3 (CCR3) is expressed selectively in eosinophils, basophils, and some Th2 cells and plays a major role in allergic diseases. A methanol extract from the arils of Myristica fragrans inhibited CC chemokine ligand 11-induced chemotaxis in CCR3-expressing L1.2 cells at 100 mu g/mL. From this extract, eight new neolignans, maceneolignans A-H (1-8), were isolated, and their stereostructures were elucidated from their spectroscopic values and chemical properties. Of those constituents, compounds 1, 4, 6, and 8 and (+)-erythro-(7S,8R)-Delta(8)'-7-hydroxy-3,4-methylenedioxy-3,5'-dimethoxy-8-O-4'-neolignan (11), (-)- (8R)-Delta(8,)-3,4-methylene dioxy-3',5-dimethoxy-8-O-4'-neolignan (17), (+)-licarin A (20), nectandrin B (25), verrucosin (26), and myristicin (27) inhibited CCR3-mediated chemotaxis at, a concentration of 1 mu M. Among them, 1 (EC50 1.6 mu M), 6 (1.5 mu M), and 8 (1.4 mu M) showed relatively strong activities, which were comparable to that of a synthetic CCR3 selective antagonist, SB328437 (0.78 mu M).

Adjuvants are required to enhance antigen-specific immune responses by vaccines. Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X and P2Y receptors and triggers the activation of dendritic cells (DCs). Here we investigated the in vivo adjuvant efficacy of alpha,beta-methylene-ATP (alpha beta-ATP), a non-hydrolysable form of ATP. We found that intradermal injection of ovalbumin (OVA), as a model antigen, combined with alpha beta-ATP, as the adjuvant, enhanced OVA-specific immune responses more than OVA alone. Additionally, DCs in the skin of mice injected with OVA and alpha beta-ATP had increased expression of major histocompatibility complex class II and co-stimulator molecules, CD40, CD80, and CD86, suggesting that alpha beta-ATP activated DC. These findings indicate that alpha beta-ATP functions as a potent vaccine adjuvant.

Chemokines and chemokine receptors play important roles in the immune response. We previously reported the pathogenic role of C-C chemokine receptor type 4 (CCR4) in experimental autoimmune encephalomyelitis (EAE). Here, we examined whether CCR4 antagonism modulates the disease course of EAE. Wild-type and CCR4-knockout mice were induced EAE and were administered Compound 22, an antagonist of CCR4. Compound 22 significantly ameliorated the severity of EAE in wild-type mice, but not in the CCR4-knockout mice. Compound 22 inhibited Th1 and Th17 polarization of antigen-induced T-cell responses. Therefore, CCR4 antagonists might be potential therapeutic agents for multiple sclerosis. (C) 2015 Elsevier B.V. All rights reserved.

Chemokine receptors CCR3 and CCR4 are preferentially expressed by TH2 cells, mast cells, and/or eosinophils, all of which are involved in the pathogenesis of allergic diseases. Therefore, CCR3 and CCR4 have long been highlighted as potent therapeutic targets for allergic diseases. Japanese traditional herbal medicine Kampo consists of multiple crude drugs/herbs, which further consist of numerous chemical substances. Recent studies have demonstrated that such chemical substances appear to promising sources in the development of novel therapeutic agents. Based on these findings, we hypothesize that Kampo-related crude drugs/herbs would contain chemical substances that inhibit the cell migration mediated by CCR3 and/or CCR4. To test this hypothesis, we screened 80 crude drugs/herbs to identify candidate substances using chemotaxis assay. Among those tested, Ephedra Herb inhibited the chemotaxis mediated by both CCR3 and CCR4, Cornus Fruit inhibited that mediated by CCR3, and Rhubarb inhibited that mediated by CCR4. Furthermore, Ephedra Herb specifically inhibited the chemotaxis mediated by not only CCR3 and CCR4 but CCR8, all of which are selectively expressed by TH2 cells. This result led us to speculate that ephedrine, a major component of Ephedra Herb, would play a central role in the inhibitory effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. However, ephedrine exhibited little effects on the chemotaxis. Therefore, we fractionated Ephedra Herb into four subfractions and examined the inhibitory effects of each subfraction. As the results, ethyl acetate-insoluble fraction exhibited the inhibitory effects on chemotaxis and calcium mobilization mediated by CCR3 and CCR4 most significantly. In contrast, chloroform-soluble fraction exhibited a weak inhibitory effect on the chemotaxis mediated by CCR8. Furthermore, maoto, one of the Kampo formulations containing Ephedra Herb, exhibited the inhibitory effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. Taken together, our data suggest that these crude drugs/herbs might be useful sources to develop new drugs targeting TH2-mediated allergic diseases.

Several chemokines/chemokine receptors such as CXCL12, CCL3, CXCR4 and CCR1 attract multiple myelomas to specific microenvironments. In the present study, we investigated whether the CX3CL1/CX3CR1 axis is involved in the interaction of the multiple myeloma cells with their microenvironment. The expression of CX3CR1 (also known as fractalkine) was detected in three of the seven human myeloma cell lines. CX3CL1-induced phosphorylation of Akt and ERK1/2 was detected in the CX3CR1-positive cell lines, but not in the CX3CR1-negative cell lines. In addition, CX3CL1-induced cell adhesion to fibronectin and vascular cell adhesion molecule-1 (VCAM-1) in the human myeloma RPMI-8226 cell line. We also investigated whether a relationship existed between myeloma cells and osteoclasts that may function via the CX3CL1/CX3CR1 axis. Conditioned medium from CX3CL1-stimulated RPMI-8226 cells drastically increased the osteoclast differentiation. Collectively, the results from the present study support the concept of the CX3CL1-mediated activation of the progression of the multiple myeloma via CX3CR1. Thus, CX3CR1 may represent a potential therapeutic target for the treatment of multiple myeloma in a bone microenvironment.

Known for its unusual metamorphic native state structure, XCL1 has been the focus of most efforts to elucidate the structural, functional, and physiological properties of chemokines in the C subfamily. By comparison, its closely related paralog XCL2 remains virtually uncharacterized. Based on the importance of the chemokine N-terminus in receptor activation, it was hypothesized that two amino acid differences in XCL2 would alter its agonist activity relative to XCL1 for their shared receptor XCR1. This present study reveals several properties of XCL2 that were unexamined until now. Structurally, XCL1 and XCL2 are very similar, exchanging between the monomeric chemokine fold and an unrelated dimeric state under physiological NaCl and temperature conditions. Ca2+ flux, chemotaxis, and heparin binding assays showed that the monomer form of XCL2 is responsible for G protein-coupled receptor activation while the dimeric form is important for GAG binding. Despite their high structural similarity, XCL2 displays a slightly higher affinity for heparin than XCL1. Because their in vitro functional profiles are virtually identical, distinct physiological roles for XCL1 and XCL2 are probably encoded at the level of expression. (C) 2014 Elsevier Ltd. All rights reserved.

Background: Inhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients. Methods: We generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages. Results: CXCL16 expression enhanced TNF-alpha-induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-kappa B-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF-alpha-induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF-alpha-induced apoptosis through the induction of M1 macrophages, which released TNF-alpha. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis. Conclusions: Collectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.

Background: Lung cancer cells have been reported to produce cytokines, resulting in systemic reactions. There have been few reports showing that these cytokines induced the formation of an inflammatory mass around lung cancers. Case presentation: We encountered a patient with a pleomorphic carcinoma of the lung. This tumor produced interleukin (IL)-8, granulocyte colony-stimulating factor and IL-6, which in turn recruited inflammatory cells, such as CD8 positive lymphocytes, around the tumor, resulting in a rapidly growing tumor shadow. Conclusion: 18 F-fluoro-deoxy-glucose positron emission tomography, in addition to a conventional radiological approach such as computed tomography, may detect immunological responses around a tumor.

Our previous study indicated that consuming (-)-epigallocatechin gallate (EGCG) before or after traumatic brain injury (TBI) eliminated free radical generation in rats, resulting in inhibition of neuronal degeneration and apoptotic death, and improvement of cognitive impairment. Here we investigated the effects of administering EGCG at various times pre- and post-TBI on cerebral function and morphology. Wistar rats were divided into five groups and were allowed access to (1) normal drinking water, (2) EGCG pre-TBI, (3) EGCG pre- and post-TBI, (4) EGCG post-TBI, and (5) sham-operated group with access to normal drinking water. TBI was induced with a pneumatic controlled injury device at 10 weeks of age. Immunohistochemistry and lipid peroxidation studies revealed that at 1, 3, and 7 days post-TBI, the number of 8-Hydroxy-2'-deoxyguanosine-, 4-Hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells, and levels of malondialdehyde around the damaged area were significantly decreased in all EGCG treatment groups compared with the water group (P < 0.05). Although there was a significant increase in the number of surviving neurons after TBI in each EGCG treatment group compared with the water group (P < 0.05), significant improvement of cognitive impairment after TBI was only observed in the groups with continuous and post-TBI access to EGCG (P < 0.05). These results indicate that EGCG inhibits free radical-induced neuronal degeneration and apoptotic death around the area damaged by TBI. Importantly, continuous and post-TBI access to EGCG improved cerebral function following TBI. In summary, consumption of green tea may be an effective therapy for TBI patients.

Previously, we have shown that an AP-1 family member, FRA-2, is constitutively expressed in adult T-cell leukemia/lymphoma (ATL) and, together with JUND, upregulates CCR4 and promotes ATL cell growth. Among the identified potential target genes of FRA-2/JUND was SOX4. Here, we examine the expression and function of SOX4 in ATL. SOX4 was indeed consistently expressed in primary ATL cells. FRA-2/JUND efficiently activated the SOX4 promoter via an AP-1 site. Knockdown of SOX4 expression by small interfering RNA (siRNA) strongly suppressed cell growth of ATL cell lines. Microarray analyses revealed that SOX4 knockdown reduced the expression of genes such as germinal center kinase related (GCKR), NAK-associated protein 1 (NAP1), and histone deacetylase 8 (HDAC8). We confirmed consistent expression of GCKR, NAP1, and HDAC8 in primary ATL cells. We also showed direct activation of the HDAC8 promoter by SOX4. Furthermore, siRNA knockdown of GCKR, NAP1, and HDAC8 each significantly suppressed cell growth of ATL cell lines. Taken together, we have revealed an important oncogenic cascade involving FRA-2/JUND and SOX4 in ATL, which leads to the expression of genes such as GCKR, NAP1, and HDAC8.

We have previously reported free radical production after traumatic brain injury (TBI), which induces neural stem cell (NSC) degeneration and death. However, the effects of aging on NSC proliferation around the damaged area following TBI have not been investigated. Therefore, in this study, we used 10-week (young group) and 24-month-old (aged group) rat TBI models to investigate the effects of aging on NSC proliferation around damaged tissue using immunohistochemical and ex vivo techniques. Young and aged rats received TBI. At 1, 3 and 7 days after TBI, immunohistochemical and lipid peroxidation studies were performed. Immunohistochemistry revealed that the number of nestin-positive cells around the damaged area after TBI in the aged group decreased significantly when compared with those in the young group (P < 0.01). However, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells and the level of peroxidation around the damaged area after TBI significantly increased in the aged group, compared with those in the young group (P < 0.01). Furthermore, almost all ssDNA-positive cells in young and aged groups co-localized with NeuN and nestin staining. Ex vivo studies revealed that neurospheres, which differentiated into neurons and glia in culture, could only be isolated from injured brain tissue in young and aged groups at 3 days after TBI. These results indicate that, although there were fewer NSCs that have the potential to differentiate into neurons and glia, these NSCs escaped free radical-induced degeneration around the damaged area after TBI in the aged rat brain.

Colorectal cancer (CRC) is a typical lifestyle-related disease, and it metastasizes mostly to the liver. It is important to understand the molecular mechanisms of CRC metastasis in order to design new and effective treatments for CRC patients. Chemokines are known to have antitumor effects as their chemoattractant properties stimulate the accumulation of infiltrating immune cells (TILs) in tumors. Chemokine (C-X-C motif) ligand 16 (CXCL16), also known as SR-PSOX, is a unique membrane-bound chemokine that induces the expression of its specific receptor CXCR6. We previously reported that the expression of CXCL16 by cancer cells enhances the recruitment of TILs, thereby improving the prognosis of CRC. It has since been reported that CXCL16/CXCR6 expression is involved in the metastasis of various types of cancer. However, there is no report of the association between CXCL16 expression and liver metastasis in CRC. In this study, we investigated the role of cancer-derived CXCL16 and the possibility of gene therapy using CXCL16. Therefore, we examined the metastasis of colon 38 SL4 cells to the liver in an experimental model. Following injection of cancer cells into the intraportal vein, CXCL16-expressing CRC cells drastically inhibited liver metastasis. We also found that CD8 T cells and natural killer T (NKT) cells, known as CXCR6-expressing cells, increased in CXCL16-expressing metastatic tissue. Collectively, the inhibitory effect on metastasis to the liver by CXCL16 was observed in NKT cell-depleted mice but not in CD8 T cell-depleted mice. These results demonstrate the inhibitory effect of CXCL16 on liver metastasis via NKT cells in CRC.

Adult T-cell leukemia/lymphoma (ATLL) and cutaneous T-cell lymphomas (CTCLs) are known to frequently express CC chemokine receptor 4 (CCR4). Previously, we investigated the transcriptional control of CCR4 expression in ATLL and have found that an activating protein 1 (AP1) family member, FBJ murine osteosarcoma viral oncogene homolog (FOS)-related antigen 2 (FRA2), is consistently expressed at high levels in ATLL and, together with v-JUN avian sarcoma virus 17 oncogene homolog D (JUND), up-regulates the expression of CCR4 as well as that of several proto-oncogenes such as v-MYB myeloblastosis viral oncogene homolog (MYB), murine double minute 2 homolog (MDM2), and B-cell lymphoma 6 (BCL6). Here, we examined the expression of these genes in clinical samples of CTCLs. We detected the transcripts of FRA2, JUND, CCR4, MYB, MDM2, and BCL6 at high levels in CTCL skin lesions. Except for BCL6, we confirmed protein expression of FRA2, JUND, CCR4, MYB, and MDM2 in CTCL skin lesions. Furthermore, siRNA-mediated knockdown of FRA2 or JUND suppressed cell growth and the expression of CCR4, MYB, MDM2, and BCL6 in CTCL cell lines. Our results, thus, demonstrate the presence of a common oncogenic cascade initiated by FRA2/JUND in CCR4-expressing mature T-cell malignancies such as ATLL and CTCLs.

Lymphocyte-rich gastric cancer (Ly-rich GC) is characterized by lymphoid stroma. To understand its formation, we studied the expression of a chemokine ligand (CCL)20 and its receptor CCR6 in 36 and 37 cases of Ly-rich- and conventional GC, respectively. Lymphoid tissues in the alimentary tract were studied in parallel. By quantitative polymerase chain reaction, Ly-rich GC contained CCL20 and CCR6 mRNAs at higher levels than conventional GC. By immunohistochemistry, CCL20 was expressed by cancer cells more frequently in Ly-rich GC than in conventional GC. This was comparable with its expression in epithelial cells of the alimentary tract lymphoid tissues. CCR6 was mostly expressed by dendritic cells (DC) and B cells in Ly-rich GC, which was also comparable with its expression in the alimentary tract lymphoid tissues. Cancer cells also expressed CCR6. However, its expression did not differ between Ly-rich- and conventional GC, nor was it related to the stage of cancer. Given that the CCL20-CCR6 axis is involved in the formation of alimentary tract lymphoid tissue, the similarity between the lymphoid stroma of Ly-rich GC and the alimentary tract lymphoid tissues supports the notion that it plays a significant role in the formation of lymphoid stroma in Ly-rich GC.

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1. The Journal of Immunology, 2010, 185: 6472-6479.

Objective. The objective of this study was to clarify the prognostic significance of tumor-infiltrating lymphocytes (TILs) in oral squamous cell carcinoma (OSCC); the present study analyzed various TIL-related parameters. Study design. Immunohistochemistry was performed in 87 patients with OSCC for the following TIL-related parameters: nest-CD8(+) T cells, stromal CD8(+) T cells, CD4(+) T cells, total regulatory T cells (Tregs), CCR4(+) Tregs, ratio of nest CD8(+) T cells/CCR4(+) Tregs, and ratio of stromal CD8(+) T cells/CCR4(+) Tregs. Results. In univariate analyses, the following parameters were associated with decreased survival: few nest-and stromal CD8(+) T cells and more stromal CCR4(+) Tregs, but not total Tregs. Low ratios of nest and stromal CD8(+) T cell/CCR4(+) Treg were associated with worse survival. In multivariate analysis, the stromal CD8(+) T cell/CCR4(+) Treg ratio was an independent prognostic factor. Conclusion. Host immune responses in the stroma of OSCC affect the survival of the patients. The in situ balance between effector T cells and regulatory T cells is the most important factor predicting survival. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 744-752)

The recruitment of osteoclast precursors toward osteoblasts and subsequent cell-cell interactions are critical for osteoclast differentiation. Chemokines are known to regulate cell migration and adhesion. CX3CL1 (also called fractalkine) is a unique membrane-bound chemokine, that has dual functions for cells expressing its receptor CX3CR1: a potent chemotactic factor in its soluble form and a type of efficient cell adhesion molecule in its membrane-bound form. In this paper, we demonstrate a novel role of CX3CL1 in osteoblast-induced osteoclast differentiation. We found that osteoclast precursors selectively expressed CX3CR1, whereas CX3CL1 is expressed by osteoblasts. We confirmed that soluble CX3CL1 induced migration of bone marrow cells containing osteoclast precursors, whereas immobilized CX3CL1 mediated firm adhesion of osteoclast precursors. Furthermore, a blocking mAb against CX3CL1 efficiently inhibited osteoclast differentiation in mouse bone marrow cells cocultured with osteoblasts. Anti-CX3CL1 also significantly suppressed bone resorption in neonatal mice by reducing the number of bone-resorbing mature osteoclasts. Collectively, CX3CL1 expressed by osteoblasts plays an important role in osteoclast differentiation, possibly through its dual functions as a chemotactic factor and adhesion molecule for osteoclast precursors expressing CX3CR1. The CX3CL1-CX3CR1 axis may be a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis, osteoporosis, and cancer bone metastasis. The Journal of Immunology, 2009, 183: 7825-7831.

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-kappa B site, while a mutant Tax selectively defective in NF-kappa B 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. (C) 2009 UICC

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. © 2008 Nature Publishing Group All rights reserved.

The product of the human cytomegalovirus (HCMV) gene UL144, expressed at early times postinfection, is located in the UL/b' region of the viral genome and is related to members of the tumor necrosis factor receptor superfamily, but it does not bind tumor necrosis factor superfamily ligands. However, UL144 does activate NF-kappa B, resulting in NF-kappa B-mediated activation of the cellular chemokine CCL22. Consistent with this finding, isolates of HCW lacking the UL/b' region show no such activation of CCL22. Recently, it has been suggested that activation of NF-kappa B is repressed by the product of the viral gene IE86: IE86 appears to block NF-kappa B binding to DNA but not nuclear translocation of NF-kappa B. Intriguingly, IE86 is detectable throughout an infection with the virus, so how UL144 is able to activate NF-kappa B in the presence of continued IE86 expression is unclear. Here we show that although IE86 does repress the UL144-mediated activation of a synthetic NF-kappa B promoter, it is unable to block UL144-mediated activation of the CCL22 promoter, and this lack of responsiveness to IE86 appears to be regulated by binding of the CREB transcription factor.

CCL27 is one of the CC chemokines produced by epidermal keratinocytes and is suggested to be involved in the pathogenesis of inflammatory skin diseases. To clarify the contribution of CCL27 in skin inflammation, we created transgenic C57BL/6 mice that constitutively produce CCL27 in epidermal keratinocytes. These mice had high serum CCL27 levels and did not show any phenotypical change. Thus we stimulated these mice with various reagents by single and repeated application. Interestingly, only contact hypersensitivity to repeated application with fluorescein isothiocyanate was significantly enhanced in transgenic mice compared to non-transgenic mice. Under this condition, the numbers of inflammatory cells, CCR10-positive cells, CCR4-positive cells and cutaneous lymphocyte-associated antigen-positive cells were increased, and IL-4 mRNA expression was higher in the lesional skin of transgenic mice. Increased number of mast cells and higher serum IgE levels, which were similar to atopic dermatitis, were also observed. These results indicated that CCL27 modified inflammation by attracting CCR10-positive and CCR4-positive cells into the lesional skin, and may participate in the pathogenesis of Th2-shifted skin diseases such as atopic dermatitis.

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 D-3 (1,25-(OH)(2)D-3), the active metabolite of vitamine D-3, 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)(2)D-3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)(2)D-3 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)(2)D-3. We confirmed the specific binding of Ets-1 and 1,25-(OH)(2)D-3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)(2)D-3 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)(2)D-3.

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 Hodgkin's 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 kappa B. 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.

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.

The significance of tumor-infiltrating lymphocytes (TIL) has attracted much attention in relation to the prognosis of patients. We herein examined the activation status of the TILs in relation to the tumor microenvironment. By using frozen sections of human early-stage tongue cancers (n = 22), the TILs in the cancer nests and those in the cancer stroma were compared for the expression of PD-1, NKG2A, NKG2D, CD69, and Ki-67. The lymphocytes in oral lichen planus, an active immune response-mediated mucosal disease, were also analyzed for comparison purposes. All of the cancer specimens were abundantly infiltrated by CD8(+) T cells and CD56(+) natural killer (NK) cells in the stroma, as well as in the tumor nest. The tumor nest-infiltrating (intraepith,elial) CD8(+) T cells frequently expressed PD-1, an inhibitory receptor, in sharp contrast to those in the stroma or in the lichen planus. Conversely, the intraepithelial CD8(+) T cells only infrequently expressed NKG2D), an activating receptor, in contrast to those in the stroma or in the lichen planus. No intraepithelial CD8(+) T cells expressed Ki-67, a proliferation-associated marker, whereas those in the stroma frequently expressed it. Furthermore, the intraepithelial NK cells expressed NKG2A, an inhibitory receptor, more frequently than those in the stroma or the lichen planus. Collectively, the intraepithelial CD8(+) T cells and NK cells are phenotypically inactivated, whereas stromal counterparts are phenotypically just as active as those in the lichen planus. These results suggest the first-step occurrence of an immune evasion mechanism in the tumor nest of oral squamous cell carcinoma.

CXCL16 is a new member of the chemokine superfamily, which exists in a transmembrane as well as a soluble form. Its receptor CXCR6 is detected on CD4(+) T cells, CD8(+) T cells, and natural killer T cells. Here, we report a significant correlation of CXCL16 expression by tumor cells with the infiltration of T cells and prognosis in colorectal cancer (CRC). We first found that CXCL16 expression was consistently up-regulated more in tumor tissues than in normal mucosa derived from the same CRC patients. Four human CRC cell lines also expressed CXCL16 mRNA and secreted soluble CXCL16. We next examined the expression of CXCL16 and infiltration of lymphocytes in CRC specimens (n = 58) by immunohistochemistry. CRC patients with high levels of CXCL16 expression (n = 43) had higher levels of CD4(+) and CD8(+) tumor-infiltrating lymphocytes (TIL; P < 0.01) than those with tow levels of CXCL16 expression (n = 15). Furthermore, the high CXCL16 expression group showed significantly better prognosis than the low CXCL16 expression group (P < 0.05). Collectively, our data suggest that the expression of CXCL16 by tumor cells enhances the recruitment of TILs, thereby bringing about a better prognosis in CRC. Thus, CXCL16 is a new prognostic biomarker and may be useful for the development of a more effective therapeutic strategy for CRC.

Adult T-cell leukemia (ATL) is a highly aggressive mature CD4(+) T-cell malignancy that is etiologically associated with human T-lymphotropic virus Type I (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 I 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. (c) 2007 Wiley-Liss, Inc.

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T-cell origin with multi-organ involvement. Because the chemokine receptors play crucial roles in tissue-specific homing of mature lymphocytes, particular chemokine receptors expressed on ATLL cells may be involved in their tissue infiltration. We thus performed a comprehensive survey on the chemokine receptor expression in ATLL. ATLL cells expressed transcripts of CCR1, CCR4, CCR7, CCR8, CCR10 and CXCR4 but hardly expressed those of CCR2, CCR3, CCR5, CCR6, CCR9, CXCR1, CXCR2, CXCR3 and CXCR5. These results were confirmed at the protein level by flow cytometric analysis. Notably, patients who have skin lesions showed significantly higher levels of CCR10 mRNA expression than patients without skin lesions. ATLL cells migrated efficiently to the CCR4 ligand, CCL22, and moderately to the CCR10 ligands, CCL27 and CCL28. Moreover, ATLL skin lesions consistently contained transcripts of CCR10 and its ligands CCL27 and CCL28 besides those of CCR4 and its ligands CCL17 and CCL22 that have been reported previously. Collectively, the frequent co-expression of CCR4 and CCR10, the known pair of skin-homing chemokine receptors, may play an important role in ATLL invasion into the skin.

CC chemokine ligand (CCL)17 is implicated in the pathogenesis of atopic dermatitis (AD). To study the effect of CCL17 produced by keratinocytes (KC) during inflammation, we created transgenic (Tg) mice in which CCL17 is overexpressed in KC. Th2-type contact hypersensitivity (CHS) was enhanced and Th1-type CHS was suppressed in these mice. Increased numbers of CC chemokine receptor (CCR)4(+) cells and mast cells infiltrated in Tg mice. Levels of IL-4 mRNA were higher and those of IFN-gamma mRNA were lower in both acute and chronic CHS. Higher levels of serum IgE were observed after CHS. Numbers of CCR4(+) cells among PBMC were increased in Tg mice challenged acutely on the trunk. Chronic irritation with croton oil induced dermatitis and an elevation of serum IgE levels. Tg mice showed enhanced ear swelling after tape stripping. CCL17 was thought to modify the inflammation caused by sensitizing reagents as well as irritant reagents by attracting CCR4(+) cells into the lesional skin and creating a Th2-dominant condition. AD-like conditions such as increased number of mast cells and elevated levels of serum IgE were observed. Thus, CCL17 may participate in the pathogenesis of skin diseases such as AD by regulating both allergic and irritant inflammation.

Antimicrobial peptides are a diverse family of small, mostly cationic polypeptides that kill bacteria, fungi and even some enveloped viruses, while chemokines are a group of mostly cationic small proteins that induce directed migration of leukocytes through interactions with a group of seven transmembrane G protein-coupled receptors. Recent studies have shown that antimicrobial peptides and chemokines have substantially overlapping functions. Thus, while some antimicrobial peptides are chemotactic for leukocytes, some chemokines can kill a wide range of bacteria and fungi. Here, we examined a possible direct antiviral activity of chemokines against an enveloped virus HSV-1. Among 22 human chemokines examined, chemokines such as MIP-1 alpha/CCL3,MIP-1 beta/CCL4 and RANTES/CCL5 showed a significant direct antiviral activity against HSV-1. It is intriguing that these chemokines are mostly known to be highly expressed by effector CD8(+) T cells. The chemokines with a significant anti-HSV-1 activity commonly bound to HSV-1 virions via envelope glycoprotein gB. Electron microscopy revealed that the chemokines with a significant anti-HSV-1 activity were commonly capable of generating pores in the envelope of HSV-1. Thus, some chemokines have a significant direct antiviral activity against HSV-1 in vitro and may have a potential role in host defense against HSV-1 as a direct antiviral agent. (c) 2006 Elsevier Inc. All rights reserved.

Objective. Mononuclear cell infiltration of the salivary glands is a major feature of Sjogren's syndrome (SS) and its animal model. Local generation of chemokines and the presence of chemokine receptors on the infiltrating cells may be involved in this process. We undertook the present study to investigate the expression of chemokines during the development of autoimmune sialadenitis in MRL/lpr mice and the therapeutic effect of chemokine antagonists on sialadenitis. Methods. NH2-terminal-truncated interferon-inducible protein 10 (IP-10)/CXCL10 analogs were transfected into a nonmetastatic fibroblastoid cell line, MRL/N-1, and injected subcutaneously into MRL/lpr mice, and the effects on sialadenitis were monitored. Results. IP-10 analogs truncated by 5 or more amino acid residues from the N-terminal failed to induce chemotaxis and calcium influx by CXCR3-expressing cells. Of these, the most potent antagonist (AT) (IP-10-AT) was a molecule with methionine added after removal of the 5 N-terminal amino acid residues. Significantly increased expression of the Th1-associated chemokines IP-10, monokine induced by interferon-gamma/ CXCL9, and interferon-inducible T cell chemo-attractant/CXCL11 was induced in the ductal epithelium by interferon-gamma produced in the salivary glands, whereas expression of the Th2-associated chemokines thymus and activation-regulated chemokine (TARC)/ CCL17 and monocyte-derived chemokine/CCL22 was almost undetectable during sialadenitis. Inoculation of IP-10-AT into MRL/lpr mice during the early stage of sialadenitis significantly reduced periductal mononuclear cell infiltration and parenchymal destruction compared with these features in control and TARC-AT-bearing mice. This was due to a significant reduction in infiltration of CXCR3+ T cells, predominantly Th1 cells, resulting in decreased interferon-gamma production. Conclusion. We prepared a novel potent IP-10 antagonist and demonstrated its ability to ameliorate the progression of autoimmune sialadenitis. This agent may provide a new therapeutic approach to SS.

Chemokines are now known to play an important role in cancer growth and metastasis. Here we report that differentiating osteoclasts constitutively produce CCL22 (also called macrophage-derived chemokine) and potentially promote bone metastasis of lung cancer expressing its receptor CCR4. We first examined expression of chemokines by differentiating osteoclasts. CCL22 was selectively upregulated in osteoclast-like cells derived from RAW264.7 cells and mouse bone marrow cells upon stimulation with RANKL (receptor activator of nuclear factor-kappa B ligand). In addition, a human lung cancer cell line SBC-5 that efficiently metastasized to bone when intravenously injected into NK cell-depleted SCID mice was found to express CCR4. Stimulation of SBC-5 cells with CCL22 induced cell migration and also enhanced phosphorylation of protein kinase B/Akt and extracellular signal-regulated kinase (ERK). Furthermore, immunohistochemical analysis of bone metastasis lesions demonstrated close co-localization of tartrate-resistant alkaline phosphatase (TRAP)-positive osteoclasts expressing CCL22 and SBC-5 cells expressing CCR4. Collectively, these results suggest that osteoclasts may promote bone metastasis of cancer cells expressing CCR4 in the bone marrow by producing its ligand CCL22.

Peritoneal carcinomatosis is a frequent cause of death in patients with advanced gastric carcinoma. Because chemokines are now considered to play an important role in the metastasis of various malignancies, we hypothesized that they may be involved in the development of peritoneal carcinomatosis by gastric carcinoma. Human gastric carcinoma cell lines, which were all highly efficient in generating malignant ascites in nude mice upon i.p. inoculation, selectively expressed CXCR4 mRNA and protein. In particular, NUGC4 cells expressed CXCR4 mRNA at high levels and showed vigorous migratory responses to its ligand CXCL12. CXCL12 enhanced proliferation and rapid increases in phosphorylation of protein kinase B/Akt and extracellular signal-regulated kinase of NUGC4 cells. We also showed that AMD3100 (a specific CXCR4 antagonist) effectively reduced tumor growth and ascitic fluid formation in nude mice inoculated with NUGC4 cells. Additionally, we examined human clinical samples. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high concentrations of CXCL12 (4.67 ng/mL). Moreover, immunohistochemical analysis showed that 22 of 33 primary gastric tumors with peritoneal metastasis were positive for CXCR4 expression (67%), whereas only 4 of 16,with other distant metastasis were positive (25%). Notably, 22 of 26 CXCR4-expressing primary tumors developed peritoneal metastases (85%). CXCR4 positivity of primary gastric carcinomas significantly correlated with the development of peritoneal carcinomatosis (P < 0.001). Collectively, our results strongly suggest that the CXCR4/CXC12 axis plays an important role in the development of peritoneal carcinomatosis from gastric carcinoma. Thus, CXCR4 may be a potential therapeutic target for peritoneal carcinomatosis of gastric carcinoma.

The nervous systems affect immune functions by releasing neurohormones and neurotransmitters. A neurotransmitter dopamine signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. The secondary lymphoid tissues are highly innervated by sympathetic nerve fibers that store dopamine at high contents. Lymphocytes also produce dopamine. In this study, we examined expression and function of dopamine receptors in lymphocytes. We found that D3 was the predominant subtype of dopamine receptors in the secondary lymphoid tissues and selectively expressed by naive CD8(+) T cells of both humans and mice. Dopamine induced calcium flux and chemotaxis in mouse L1.2 cells stably expressing human D3. These responses were almost completely inhibited by pertussis toxin, indicating that D3 was coupled with the G alpha i class of G proteins. Consistently, dopamine selectively induced chemotactic responses in naive CD8(+) T cells of both humans and mice in a manner sensitive to pertussis toxin and D3 antagonists. Dopamine was highly synergistic with CCL19, CCL21, and CXCL12 in induction of chemotaxis in naive CD8(+) T cells. Dopamine selectively induced adhesion of naive CD8(+) T cells to fibronectin and ICAM-1 through activation of integrins. Intraperitoneal injection of mice with dopamine selectively attracted naive CD8(+) T cells into the peritoneal cavity. Treatment of mice with a D3 antagonist U-99194A selectively reduced homing of naive CD8(+) T cells into lymph nodes. Collectively, naive CD8(+) T cells selectively express D3 in both humans and mice, and dopamine plays a significant role in migration and homing of naive CD8(+) T cells via D3.

T cell infiltration in colorectal cancer is associated with a favorable prognosis, suggesting an occurrence of a certain degree of anti-tumor immunity. T helper type 1 (Th1) and Th2 cells are now known to selectively express CC-chemokine receptor 5 (CCR5)/CXC-chemokine receptor 3 (CXCR3) and CCR4, respectively. To clarify the mechanism of T cell infiltration, we examined in situ expression of these chemokine receptors and their respective chemokine ligands in 40 cases of human colorectal cancer. Immunohistochemistry showed a predominant accumulation of T cells expressing CCR5 and CXCR3 mainly along the invasive margin, whereas those expressing CCR4 were rare. Flow cytometric analysis showed that more than half of CD8(+) T cells and a fraction of CD4(+) cells isolated from fresh tumor tissues co-expressed CCR5 and CXCR3, and CD8(+) T cells and CD4+ cells predominantly produced interferon-gamma (IFN-gamma) over interleukin-4 (IL-4) after in vitro stimulation. RANTES/CCL5, a ligand of CCR5, was localized within infiltrating CD8+ T cells in a granular pattern, whereas IP-10/CXCL10, a ligand of CXCR3, was localized in cancer cells and macrophages along the invasive margin. These data were consistent with an active recruitment of T cells expressing CCR5 or CXCR3 into the invasive margin of colorectal cancer. With the previous clinicopathological studies showing a favorable prognostic impact of T cell infiltration in colorectal cancer, our study supports the occurrence of a certain level of Th1-shifted cellular immune responses in human colorectal cancer. (C) 2005 Wiley-Liss, Inc.

Considerable attention has recently been paid to the application of chemokines to cancer immunotherapy because of their chemotactic affinity for a variety of immune cells and because several chemokines are strongly angiostatic. In the present study, the recombinant adenovirus vectors encoding chemokine CCL19 or XCL1 in an El cassette (AdRGD-mCCL19 and AdRGD-mXCL1) were developed. The constructed fiber-mutant adenovirus vector, which contained the integrin-targeting Arg-Gly-Asp (RGD) sequence in the fiber knob, notably enhanced the transfection efficiency to OV-HM ovarian carcinoma cells compared to that induced by conventional adenovirus vector. The results of an in vitro chemotaxis assay for chemokine-encoding vector demonstrated that both AdRGD-mCCL19 and AdRGD-mXCL1 could induce the migration of cells expressing specific chemokine receptors. Of the two chemokine-en coding vectors evaluated in vivo, AdRGD-mCCL19 showed significant tumor-suppressive activity in R6C3F1 mice via transduction into OV-HM cells, whereas XCL1 did not exhibit any notable anti-tumor effects, suggesting that CCL19 may be a candidate for cancer immunotherapy.

CCL25 (also known as thymus-expressed chemokine) and CCL28 (also known as mucosae-associated epithelial chemokine) play important roles in mucosal immunity by recruiting IgA Ab-secreting cells (ASCs) into mucosal lamina propria. However, their exact roles in vivo still remain to be defined. In this study, we first demonstrated in mice that IgA ASCs in small intestine expressed CCR9, CCR10, and CXCR4 on the cell surface and migrated to their respective ligands CCL25, CCL28, and CXCL12 (also known as stromal cell-derived factor 1), whereas IgA ASCs in colon mainly expressed CCR10 and CXCR4 and migrated to CCL28 and CXCL12. Reciprocally, the epithelial cells of small intestine were immunologically positive for CCL25 and CCL28, whereas those of colon were positive for CCL28 and CXCL12. Furthermore, the venular endothelial cells in small intestine were positive for CCL25 and CCL28, whereas those in colon were positive for CCL28, suggesting their direct roles in extravasation of IgA ASCs. Consistently, in mice orally immunized with cholera toxin (CT), anti-CCL25 suppressed homing of CT-specific IgA ASCs into small intestine, whereas anti-CCL28 suppressed homing of CT-specific IgA ASCs into both small intestine and colon. Reciprocally, CT-specific ASCs and IgA titers in the blood were increased in mice treated with anti-CCL25 or anti-CCL28. Anti-CXCL12 had no such effects. Finally, both CCL25 and CCL28 were capable of enhancing alpha(4) integrin-dependent adhesion of IgA ASCs to mucosal addressin cell adhesion molecule-1 and VCAM-1. Collectively, CCL25 and CCL28 play essential roles in intestinal homing of IgA ASCs primarily by mediating their extravasation into intestinal lamina propria.

Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine that is constitutively expressed by the liver parenchymal cells and present in the normal plasma at high concentrations. Previous studies have shown that CCL16 is a low-affinity ligand for CCR1, CCR2, CCR5, and CCR8 and attracts monocytes and T cells. Recently, a novel histamine receptor termed type 4 (H4) has been identified and shown to be selectively expressed by eosinophils and mast cells. In this study, we demonstrated that CCL16 induced pertussis toxin-sensitive calcium mobilization and chemotaxis in murine L1.2 cells expressing H4 but not those expressing histamine receptor type 1 (H1) or type 2 (H2). CCL16 bound to H4 with a K-d of 17 nM. By RT-PCR, human and mouse eosinophils express H4 but not H3. Accordingly, CCL16 induced efficient migratory responses in human and mouse eosinophils. Furthermore, the responses of human and mouse eosinophils to CCL16 were effectively suppressed by thioperamide, an antagonist for H3 and H4. Intravenous injection of CCL16 into mice induced a rapid mobilization of eosinophils from bone marrow to peripheral blood, which was also suppressed by thioperamide. Collectively, CCL16 is a novel functional ligand for H4 and may have a role in trafficking of eosinophils.

Chemokines are a family of cytokines that induce directed migration of various types of leukocytes through specific interactions with a group of seven transmembrane receptors. Scavenger receptors are a heterogenous family of transmembrane molecules that commonly bind and uptake oxidized low density lipoprotein and bacteria. Here, we show that not only CXC chemokine 16 (CXCL16)/SR-PSOX, a transmembrane chemokine with scavenger receptor activity, but also 12 out of 15 chemokines examined efficiently bound scavenger receptor ligands in competition with cells expressing their specific chemokine receptors. Furthermore both the chemotactic and scavenger receptor activities of SR-PSOX/CXCL16 were similarly impaired in a series of mutants altered in the chemokine domain, indicating that SR-PSOX/CXCL16 binds scavenger receptor ligands as well as CXCR6 using highly overlapping binding motifs. Taken together, chemokines generally have scavenger receptor-like activity through their receptor-binding domain, suggesting a close evolutionary relationship between chemokines and scavenger receptors.

Purpose. CC chemokine-ligand 20 (CCL20) is known to be selectively expressed by surface-lining mucosal epithelial cells and skin epidermal keratinocytes and to attract cells such as immature dendritic cells and effector T cells via CCR6. This study evaluated the ability of corneal epithelial cells and stromal keratocytes to produce CCL20 in vitro and in vivo. Methods. Human corneal epithelial cells (HCE) and corneal keratocytes (HCK) were treated without or with various cytokines and expression of CCL20 mRNA and secreion of its protein were evaluated by RT-PCR and ELISA. Induction of CCL20 mRNA in HCE and HCK was also examined upon in vitro infection with HSV-1. Using a mouse model of herpetic stromal keratitis (HSK), induction of CCL20 expression and accumulation of cells expressing CCR6 were evaluated by RT-PCR and immunohistochemistry. Results. Not only corneal epithelial cells but also stromal keratocytes efficiently expressed CCL20 mRNA and protein upon stimulation with IL-1beta and TNF-alpha. In vitro infection with HSV-1 also induced CCL20 mRNA in both types of cells. In a mouse herpetic stromal keratitis model, prominent accumulation of CCL20 and CCR6 mRNA was revealed in HSV-1-infected corneas. Furthermore, immunohistochemistry demonstrated production of CCL20 by corneal epithelial cells as well as, stromal keratocytes and stromal infiltration of DEC205(+) dendritic cells, CD4(+) T cells and CD8(+) T cells. Double staining revealed that CCR6-expressing cells were mostly MHC class II+ dendritic cells. Conclusions. Not only epithelial cells but also stromal keratocytes are efficient producers of CCL20 in the cornea and recruit CCR6-expressing cells such as dendritic cells into inflamed cornea.

In this study, we screened the anti-tumor activity of murine chemokines including CCL17. CCL19, CCL20, CCL21, CCL22, CCL27, XCL1, and CX3CL1 by inoculating murine B16BL6, CT26, or OV-HM tumor cells, all of which were transfected with chemokine-expressing fiber-mutant adenovirus vector, into immunocompetent mice. A tumor-suppressive effect was observed in mice inoculated with CCL19/B16BL6 and XCK1/B16BL6, and CCL22/OV-HM showed considerable retardation in tumor growth. In the cured mice inoculated with CCL22/OV-HM, a long-term specific immune protection against parental tumor was developed. However, we were unable to identify the chemokine that had a suppressive activity common to all three tumor models. Furthermore, an experiment using chemokine-transfected B16BL6 cells was also performed on mice sensitized with melanoma-associated antigen. A drastic enhancement of the frequency of complete rejection was observed in mice inoculated with CCL17-, CCL19-, CCL22-, and CCL27-transfected B16BL6. Altogether, our results suggest that the tumor-suppressive activity of chemokine-gene immunotherapy is greatly influenced by the kind of tumor and the activation state of the host's immune system. (C) 2004 Elsevier Inc. All rights reserved.

Chronic gastritis is frequently associated with infection of Helicobacter pylori and characterized by tissue infiltration of neutrophils, lymphocytes, and plasma cells. To address the mechanism of lymphocyte infiltration in chronic gastritis, we examined the expression of chemokines and their receptors using frozen sections of chronic gastritis, obtained from 23 patients who underwent gastrectomy for gastric cancer. By immuno histochemistry, lymphocytes in inflamed gastric mucosa expressed CCR5 abundantly, CXCR3 less frequently, and CCR4 sparsely. The numbers of CCR5(+) cells, which were composed of mainly CD8(+) and partly CD4(+) T cells, were positively correlated with the degree of neutrophil infiltration, and decreased in areas with intestinal metaplasia or mucosal atrophy. RANTES/CCL5, one of the ligands of CCR5, was localized mainly in CD8(+) and partly CD4(+) T cells with a characteristic dotted pattern, and such lymphocytes were most densely distributed around the neck region of gastric glands. In situ hybridization confirmed the expression of CCLS mRNA in these cells, and immunoelectron microscopy revealed localization of CCL5 in the membrane-bound granules, which most probably corresponded to the cytolytic granules of cytotoxic T cells. The numbers of CCL5(+) lymphocytes showed a close correlation with the degree of neutrophil infiltration and markedly decreased in intestinal metaplasia. In conclusion, our data suggest that, together with neutrophils, CCL5+ T cells, presumably activated cytotoxic T cells, would play important roles in the active inflammatory process of chronic gastritis. Our data also suggest a self-recruiting mechanism involving CCR5 and CCL5 for tissue accumulation of such T cells.

Classical Hodgkin's disease (HD) is characterized by rare neoplastic Hodgkin and Reed-Sternberg (H-RS) cells within abundant reactive cellular backgrounds. In most cases, H-RS cells originate from the B-cell lineage, but their immunophenotypes are unusual. Here we newly found frequent expression of chemokine receptors CXCR6 and CCR10 and their respective ligands CXCL16 and CCL28 in RD-derived cell lines. CCR10 is known to be selectively expressed by plasma cells, whereas CCL28 attracts eosinophils via CCR3 and plasma cells via CCR10 and CCR3. Therefore, we examined their expression in HD tissues by immunohistochemistry. We found that H-RS cells in 15 of 19 cases were positive for CCL28. Among them, seven cases were also positive for CCR10, suggesting a potential autocrine effect. In situ hybridization confirmed the expression of CCL28 mRNA in H-RS cells. The CCL28 positivity in H-RS cells did not significantly correlate with that of LMP-1, CCL17, CCL22, or CCL11. However, it significantly correlated with the background accumulation of eosinophils, plasma cells, and CCR10(+) cells. Thus, the production of CCL28 by H-RS cells may play a major role in tissue accumulation of eosinophils and/or plasma cells in classical HD. The frequent expression of CCR10 in H-RS cells themselves also supports their close relationship to plasma cells.

Direct contacts between dendritic cells (DCs) and T cells or natural killer T (NKT) cells play important roles in primary and secondary immune responses. SR-PSOX/CXC chemokine ligand 16 (CXCL16), which is selectively expressed on DCs and macrophages, is a scavenger receptor for oxidized low-density lipoprotein and also the chemokine ligand for a G protein-coupled receptor CXC chemokine receptor 6 (CXCR6), expressed on activated T cells and NKT cells. SR-PSOX/CXCL16 is the second transmembrane-type chemokine with a chemokine domain fused to a mucin-like stalk, a structure very similar to that of fractalkine (FNK). Here, we demonstrate that SR-PSOX/CXCL16 functions as a cell adhesion molecule for cells expressing CXCR6 in the same manner that FNK functions as a cell adhesion molecule for cells expressing CX3C chemokine receptor 1 (CX(3)CR1) without requiring CX(3)CR1-mediated signal transduction or integrin activation. The chemokine domain of SR-PSOX/CXCL16 mediated the adhesion of CXCR6-expressing cells, which was not impaired by treatment with pertussis toxin, a Galphai protein blocker, which inhibited chemotaxis of CXCR6-expressing cells induced by SR-PSOX/CXCL16. Furthermore, the adhesion activity was up-regulated by treatment of SR-PSOX/CXCL16-expressing cells with a metalloprotease inhibitor, which increased surface expression levels of SRPSOX/CXCL16. Thus, SR-PSOX/CXCL16 is a unique molecule that not only attracts T cells and NKT cells toward DCs but also supports their firm adhesion to DCs.

Chemokines are likely to play important roles in the pathophysiology of diseases associated with Epstein-Barr virus (EBV). Here, we have analyzed the repertoire of chemokines expressed by EBV-infected B cells. EBV infection of B cells induced expression of TARC/CCL17 and MDC/CCL22, which are known to attract Th2 cells and regulatory T cells via CCR4, and also upregulated constitutive expression of MIP-1alpha/CCL3, MIP-1beta/CCL4, and RANTES/CCL5, which are known to attract Th1 cells and cytotoxic T cells via CCR5. Accordingly, EBV-immortalized B cells secreted these chemokines, especially CCL3, CCL4, and CCL22, in large quantities. EBV infection or stable expression of LMP1 also induced CCL17 and CCL22 in a B-cell line, BJAB. The inhibitors of the TRAF/NF-kappaB pathway (BAY11-7082) and the p38/ATF2 pathway (SB202190) selectively suppressed the expression of CCL17 and CCL22 in EBV-immortalized B cells and BJAB-LMP1. Consistently, transient-transfection assays using CCL22 promoter-reporter constructs demonstrated that two NF-kappaB sites and a single AP-1 site were involved in the activation of the CCL22 promoter by LMP1. Finally, serum CCL22 levels were significantly elevated in infectious mononucleosis. Collectively, LMP1 induces CCL17 and CCL22 in EBV-infected B cells via activation of NF-kappaB and probably ATF2. Production of CCL17 and CCL22, which attract Th2 and regulatory T cells, may help EBV-infected B cells evade immune surveillance by Th1 cells. However, the concomitant production of CCL3, CCL4, and CCL5 by EBV-infected B cells may eventually attract Th1 cells and cytotoxic T cells, leading to elimination of EBV-infected B cells at latency III and to selection of those with limited expression of latent genes.

SR-PSOX and CXC chemokine ligand (CXCL) 16, which were originally identified as a scavenger receptor and a transmembrane-type chemokine, respectively, are indicated to be identical, In this study, we demonstrate that membrane-bound SR-PSOX/CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria. Importantly, our prepared anti-SR-PSOX mAb, which suppressed chemotactic activity of SR-PSOX, signficantly inhibited bacterial phagocytosis by human APCs including dendritic cells. Various scavenger receptor ligands inhibited the bacterial phagocytosis of SR-PSOX. In addition, the recognition specificity for bacteria was determined by only the chemokine domain of SR-PSOX/CXCL16. Thus, SR-PSOXICXCL16 may play an important role in facilitating uptake of various pathogens and chemotaxis of T and NKT cells by APCs through its chemokine domain.

In this study, we examined. antitumor activity of a mouse CC chemokine ILC/CCL27 and a mouse CX3C chemokine fractalkine/CX(3)CL1 in vivo. We generated recombinant adenovirus vectors with a fiber mutation, encoding mILC (Ad-RGD-mILC) and mFKN (Ad-RGD-mFKN). We confirmed tumor cells infected with Ad-RGD-mILC and Ad-RGD-mFKN to express and release these chemokines. Tumor rejection experiments in vivo were carried out by inoculating OV-HM cells infected with Ad-RGD-mILC or Ad-RGD-mFKN into immunocompetent mice. mILC significantly suppressed the tumor growth, whereas no such significant effect was observed by mFKN. The antitumor activity induced by mILC was T cell dependent, involving both CD4(+) and CD8(+) T cells. Immunohistochemical analysis revealed accumulation of both CD3(+) lymphocytes and NK cells in the tumor tissue transduced with mILC and mFKN. However, there was a significant difference in the distribution of infiltrating cells. Furthermore, mFKN appeared to hake an angiogenic activity, which might have masked its tumor suppressive activity. Collectively, ILC/CCL27 may be a good candidate molecule for cancer gene therapy.

Lichen planus is a chronic inflammatory disease of the skin and oral mucosa in which the cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. To understand its pathophysiology further, the present study examined the in situ expression of chemokines and chemokine receptors in oral lichen planus. Immunohistochemical. analysis of 15 cases has consistently revealed that infiltrating CD4(+) and CD8(+) T cells in the submucosa predominantly expressed CCR5 and CXCR3. Furthermore, infiltrating T cells, particularly CD8(+) T cells, were positive for RANTES/CCL5 and IP-10/CXCL10, the ligands of CCR5 and CXCR3, respectively. By immunoelectron microscopy, these chemokines were localized in the cytolytic granules of CD8(+) T cells. Lesional keratinocytes also overexpressed the ligands of CXCR3, namely, MIG/CXCL9, CXCL10, and I-TAC/CXCL11. Our data suggest that the chemokines signaling via CCR5 and CXCR3, which are known to be selectively expressed by type 1 T cells, are predominantly involved in T-cell infiltration of oral lichen planus. Furthermore, the presence of CCL5 and CXCL10 in the cytolytic granules of tissue-infiltrating CD8(+) T cells expressing CCR5 and CXCR3 reveals a potential self-recruiting mechanism involving activated effector cytotoxic T cells.

We systematically examined the repertoire of chemokine receptors expressed by human plasma cells. Fresh bone marrow plasma cells and myeloma cells consistently expressed CXCR4, CXCR6, CCR10, and CCR3. Accordingly, plasma cells responded to. their respective ligands in chemotaxis and very late Ag-4-dependent cell adhesion to fibronectin. Immobilized CXC chemokine ligand (CXCL)16, a novel transmembrane-type chemokine and CXCR6 ligand, also directly induced adhesion of plasma cells without requiring G(alphai) signaling or divalent cations. Furthermore, we revealed consistent expression of CXCL12 (CXCR4 ligand), CXCL16 (CXCR6 ligand), and CC chemokine ligand 28 (CCR10 and CCR3 ligand) in tissues enriched with plasma cells including bone marrow, and constitutive expression. of CXCL12, CXCL16, and CC chemokine ligand 28 by cultured human bone marrow stromal cells. Collectively, plasma cells are likely to be recruited to bone marrow and other target tissues via CXCR4, CXCR6, CCR10, and CCR3. CXCR6 may also contribute to tissue localization of plasma cells through its direct binding to membrane-anchored CXCL16.

CCL28 is a CC chemokine signaling via CCR10 and CCR3 that is selectively expressed in certain mucosal tissues such as exocrine glands, trachea, and colon. Notably, these tissues commonly secrete low-salt fluids. RT-PCR analysis demonstrated that salivary glands expressed CCL28 mRNA at the highest levels among various mouse tissues. Single cells prepared from mouse parotid glands indeed contained a major fraction of CD3(-)B220(low) cells that expressed CCR10 at high levels and CCR3 at low levels and responded to CCL28 in chemotaxis assays. Morphologically, these cells are typical plasma cells. By immunohistochemistry, acinar epithelial cells in human and mouse salivary glands were strongly positive for CCL28. Furthermore, human saliva and milk were found to contain CCL28 at high concentrations. Moreover, the C terminus of human CCL28 has a significant sequence similarity to histatin-5, a histidine-rich candidacidal peptide in human saliva. Subsequently, we demonstrated that human and mouse CCL28 had a potent antimicrobial activity against Candida albicans, Gram-negative bacteria, and Gram-positive bacteria. The C-terminal 28-aa peptide of human CCL28 also displayed a selective candidacidal activity. In contrast, CCL27, which is most similar to CCL28 and shares CCR10, showed no such potent antimicrobial activity. Like most other antimicrobial peptides, CCL28 exerted its antimicrobial activity in low-salt conditions and rapidly induced membrane permeability in target microbes. Collectively, CCL28 may play dual roles in mucosal immunity as a chemoattractant for cells expressing CCR10 and/or CCR3 such as plasma cells and also as a broad-spectrum antimicrobial protein secreted into low-salt body fluids.

De novo formation of lymphoid tissue is one of the characteristic features of chronic inflammation. The formation of T cell-mature dendritic cell (DC) clusters has been previously demonstrated in chronically inflamed skin infected with Candida albicans. A functional similarity was also found between chronic inflammation and the T-cell zone of lymph nodes (LNs), since a substantial fraction of phenotypically mature DCs in both tissues expressed CCL22 (macrophage-derived chemokine; MDC) and were closely surrounded by memory-type T cells expressing its receptor, CCR4. To analyse the nature of T cell-mature DC interactions further in chronically inflamed skin and LNs, the present study focuses on another chemokine system, namely CCL19 (EBI1 ligand chemokine; ELC), CCL21 (secondary lymphoid tissue chemokine; SLC) and their shared receptor, CCR7. RT-PCR analysis revealed expression of CCL19, CCL21, and CCR7 at high levels in LNs and at low levels in inflamed skin. Using immunohistochemistry, the majority of DC-Lamp(+) mature DCs in the T-cell area of LNs expressed CCL19 and were surrounded by CCR7(+) naive-type lymphocytes, while CCL21 was expressed in reticular stromal cells and vascular endothelial cells. Very few mature DCs in LNs were found to express CCR7. In contrast, the majority of DC-Lamp+ mature DCs in inflamed skin were totally negative for CCL19 and were surrounded by CCR7(-) memory-type T cells. Furthermore, CCL21 expression in the inflamed skin was detected in dermal lymphatic endothelial cells and rare CCR7(+) mature DCs were mostly seen within the lymphatic vessels. In normal skin, on the other hand, no cells immunoreactive for CCL19, CCL21, or CCR7 were found. The present study thus reveals a striking difference in the function of mature DCs between LNs and chronically inflamed skin. Copyright (C) 2002 John Wiley Sons, Ltd.

Thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22 are a pair of CC chemokines known to selectively attract T(h)2 type memory T cells via CCR4. Here we examined circulating levels of TARC and MDC in patients with atopic dermatitis (AD) and control subjects by using plasma samples, which reflect blood contents of chemokines more accurately than serum samples. The plasma levels of TARC and MDC were significantly elevated in AD patients. These values also strongly correlated with disease severity and serum lactate dehydrogenase levels, and weakly correlated with serum total IgE levels and blood eosinophilia. Previous studies demonstrated TARC immunoreactivity in the epidermal layer of AD lesional skin and production of TARC by a human keratinocytic cell line HaCaT upon stimulation with IFN-gamma. Here we demonstrated MDC immunoreactivity in the epidermal layer of AD skin at levels stronger than that of TARC. Furthermore, primary epidermal keratinocytes expressed both TARC and MDC mRNA upon stimulation with IFN-gamma, but efficiently secreted only MDC. These results suggest a post-transcriptional regulation in TARC production. IFN-gamma also induced TARC and MDC mRNA in mouse skin. Collectively, both TARC and MDC play important roles in the local accumulation of T(h)2 cells in AD lesional skin. Production of T(h)2-attracting chemokines by epidermal keratinocytes upon treatment with IFN-gamma, which is also the potent inducer of T(h)1-attracting chemokines, may underline the pivotal role of IFN-gamma in the chronic phase of AD where both T(h)1 and T(h)2 responses are mixed.

Background: T(H)2 cells and eosinophils selectively express CC chemokine receptor 4 and CCR3, respectively, and their chemokine ligands are likely to play important roles in the pathogenesis of atopic dermatitis (AD). Objective: The purpose of this study was to demonstrate the presence of thymus and activation-regulated chemokine (TARC) in platelets and its release during clotting and to evaluate the circulating levels of TARC, macrophage-derived chemokine (MDC), and eotaxin in control subjects and patients with AD. Methods: We compared plasma and serum contents of TARC, MDC, and eotaxin. We measured TARC contents in platelet lysates. We analyzed the correlation of plasma levels of TARC, MDC, and eotaxin with various clinicolaboratory parameters in patients with AD. Results: Serum contents of TARC rapidly, increased during clotting, whereas those or MDC and eotaxin increased only slightly. We demonstrated that platelets contained TARC, and its levels were dramatically elevated in patients with AD. Platelets also released TARC on stimulation with thrombin. We therefore evaluated circulating levels of these chemokines in control subjects and patients with AD by using plasma samples. Plasma TARC levels were significantly increased in patients with AD (P <.0001) and showed significant correlations with severity scoring of atopic dermatitis (SCORAD) index (r = 0.665, P <.00001), serum lactate dehydrogenese levels (r = 0.696, P= .00001), cosinophil counts (r = 0.381, P = .007), and platelet counts (r = 0.562, P <.0001). Similarly, plasma MDC levels were significantly increased in patients with AD (P <.0001) and showed significant correlations with SCORAD index (r = 0.727, P <.0001), serum lactate dehydrogenese levels (r = 0.861, P <.0001), eosinophil counts (r = 0.505, P = .005), and platelet counts (r = 0.370, P = .01). On treatment, plasma TARC and MDC levels were dramatically decreased in accordance with improved SCORAD scores (P = .0012 and P = .0007, respectively). On the other hand, plasma eotaxin levels did not show any significant increase or correlation with any of the clinical parameters in patients with AD. Conclusion: Platelets from patients with AD contain high levels of TARC. Thus platelets might play an important role in AD pathogenesis by releasing T(H)2-attracting TARC on activation. Furthermore, circulating levels of TARC and MDC, but not those of eotaxin, correlate well with the disease activity of AD.

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, gammadelta T cells, and terminally differentiated CD8(+) T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1beta/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.

Chemokines and chemokine receptors play important roles In migration and tissue localization of various lymphocyte subsets. Here, we report the highly frequent expression of CCR4 In adult T-cell leukemia (ATL) and human T-cell leukemia virus type 1 (HTLV-1)-immortalized T cells. Flow cytometric analysis revealed that ATL and HTLV-1-immortalized T-cell lines consistently expressed CCR4. Inducible expression of HTLV-1 transcriptional activator tax in a human T-cell line Jurkat did not, however, up-regulate CCR4 mRNA. In vitro Immortalization of peripheral blood T cells led to preferential outgrowth of CD4(+) T cells expressing CCR4. We further demonstrated highly frequent expression of CCR4 in fresh ATL cells by (1) reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CCR4 expression in peripheral blood mononuclear cells (PBMCs) from patients with ATL and healthy controls; (2) flow cytometric analysis of CCR4-expressing cells in PBMCs from patients with ATL and healthy controls; (3) CCR4 staining of routine blood smears from patients with ATL; and (4) an efficient migration of fresh ATL cells to the CCR4 ligands, TARC/CCL17 and MDC/CCL22, in chemotaxis assays. Furthermore, we detected strong signals for CCR4, TARC, and MDC in ATL skin lesions by RT-PCR. Collectively, most ATL cases have apparently derived from CD4+ T cells expressing CCR4. It is now known that circulating CCR4(+) T cells are mostly polarized to Th2 and also contain essentially all skin-seeking memory T cells. Thus, HTLV-1-infected CCR4+ T cells may have growth advantages by deviating host immune responses to Th2. CCR4 expression may also account for frequent infiltration of ATL into tissues such as skin and lymph nodes. (C) 2002 by The American Society of Hematology.

Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.

Liver and activation-regulated chemokine (LARC)/CCL20 is expressed by surface-lining epithelial and epidermal cells, and is likely to link innate and acquired immunity by attracting immature dendritic cells, effector memory T cells and B cells via CCR6. Here we examined the mechanism of LARC expression in epithelial-type cells. Either IL-1 beta or tumor necrosis factor (TNF)-alpha strongly induced LARC mRNA in intestinal cell lines Caco-2 and T84, while both were effective on HEK 293T cells. Induction of LARC was also demonstrated in the intestinal epithelium of BALB/c mice upon treatment with IL-1 alpha or TNF-alpha. Transient transfection assays using murine LARC promoter-reporter constructs identified a region essential for IL-1 beta, or TNF-alpha -induced promoter activation in Caco-2 and 293T cells. Using site-directed mutagenesis, we demonstrated that an NF-kappaB site located between -96 and -87 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1 beta- or TNF-alpha -induced promoter activation in Caco-2 and 293T cells. Electrophoretic mobility shift assays demonstrated that p50/p65 heterodimer and p65 homodimer of NF-kappaB bound to this site in 293T cells upon treatment with IL-1 beta and TNF-alpha, and p50/p65 heterodimer bound to this site in Caco-2 cells upon treatment with IL-1 beta. Co-expression of constitutively active p65 strongly activated the promoter construct carrying the intact NF-kappaB site in 293T and Caco-2 cells. Collectively, LARC expression in intestinal epithelial-type cells is induced by proinflammatory cytokines such as IL-1 and TNF-alpha primarily through activation of NF-kappaB.

Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine selectively expressed in the liver. Here, we investigated its receptor usage by calcium mobilization and chemotactic assays using mouse L1.2 pre-B cell lines stably expressing a panel of 12 human chemokine receptors. At relatively high concentrations, LEG induced calcium mobilization and chemotaxis via CCR1 and CCR2. LEG also induced calcium mobilization, but marginal chemotaxis via CCR5. Consistently, LEG was found to bind to CCR1, CCR2 and CCR5 with relatively low affinities. The binding of LEG to CCR8 was much less significant. In spite of its binding to CCR5, LEG was unable to inhibit infection of an R5-type HIV-1 to activated human peripheral blood mononuclear cells even at high concentrations. In human liver sections, hepatocytes were strongly stained by anti-LEC antibody. HepG2, a human hepatocarcinoma cell line, was found to constitutively express LEC. LEG was also present in the plasma samples from healthy adult donors at relatively high concentrations (0.3-4 nM). Taken together, LEG is a new low-affinity functional ligand for CCR1, CCR2 and CCR5, and is constitutively expressed by liver parenchymal cells. The presence of LEG in normal plasma at relatively high concentrations may modulate inflammatory responses.

Our previous study demonstrated formation of T cell-dendritic cell (DC) clusters in inflamed dermis of intraorally autotransplanted skin flaps. Such T cell-DC clusters are supposed to be important for close interactions between T cells and DCs including the specific antigen presentation. Here we show the involvement of the macrophage-derived chemokine (MDC/CCL22) and its specific receptor CC chemokine receptor 4 (CCR4) in the formation of T cell-DC clusters. Reverse transcriptase-polymerase chain reaction analysis revealed high levels of mRNA expression for MDC and CCR4 in inflamed skin and neck lymph nodes (LNs), but not In normal skin. Immunohistochemically, MDC+ cells and CCR4(+) cells were mainly located within the T cell-DC clusters both in the dermis of inflamed skin and the T cell area of LNs. MDC+ cells were identified to be DCs both in inflamed skin and LNs. The majority of CCR4+ cells were CD4(+) T cells, accounting for approximately one-third of total CD4(+) T cells in the inflamed skin. Our data suggest that the MDC-CCR4 system plays an important role in the formation of T cell-DC clusters both in inflamed skin and LNs.

Thymus- and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically chemoattracts CC chemokine receptor 4-positive (CCR4(+)) Th2 cells. To establish the pathophysiological roles of TARC in vivo, we investigated here whether an mAb against TARC could inhibit the induction of asthmatic reaction in mice elicited by OVA, TARC was constitutively expressed in the lung and was up-regulated in allergic inflammation. The specific Ab against TARC attenuated OVA-induced airway eosinophilia and diminished the degree of airway hyperresponsiveness with a concomitant decrease in Th2 cytokine levels. Our results for the first time indicate that TARC is a pivotal chemokine for the development of Th2-dominated experimental allergen-induced asthma with eosinophilia and AHR. This study also represents the first success in controlling Th2 cytokine production in vivo by targeting a chemokine.

Liver- and activation-regulated chemokine (LARC)/macrophage inflammatory protein (MIP)3 alpha/ CCL20 is a CC chemokine which is constitutively expressed by follicle-associated epithelial cells in the mucosa, and attracts cells expressing CCR6 such as immature dendritic cells and alpha (4)beta (high)(7) intestine-seeking memory T cells. Here, we examine LARC/CCL20 expression in the skin. LARC/CCL20 mRNA and protein were induced in primary human keratinocytes upon stimulation with proinflammatory cytokines such as IL-1 alpha and tumor necrosis factor (TNF)-alpha, In mice, intradermal injection of IL-l a and TNF-a rapidly induced a local accumulation of transcripts for LARC/CCL20 and its receptor CCR6 with a lag of several hours in the latter. In humans, immunostaining of LARC/CCL20 was weak if any in normal skin tissues but strongly augmented in lesional skin tissues with atopic dermatitis. Furthermore, massive infiltration of cells with markers such as CD1a, CD3 or HLA-DR was present in atopic skin lesions. Many infiltrating cells were also found to be CCR6(+) by a newly generated monoclonal anti-CCR6, However, Langerhans cells residing within the epidermis were hardly stained by anti-CCR6 in normal and atopic skin tissues. Furthermore, plasma levels of LARC/CCL20 were found to be elevated in patients with atopic dermatitis. Collectively, our results suggest that epidermal keratinocytes produce LARC/CCL20 upon stimulation with proinflammatory cytokines such as IL-1 alpha and TNF-alpha, and attract CCR6-expressing immature dendritic cells and memory/effector T cells into the dermis of inflamed skin such as atopic dermatitis. LARC/CCL20 may not, however, play a major role in homeostatic migration of Langerhans cells into the skin.

Molluscum contagiosum virus (MCV) encodes a CC chemokine MC148R which is likely to have been acquired from the host. By a homology search employing MC148R as a probe, we hale identified a novel CC chemokine whose gene exists next to the IL-11 receptor alpha (IL-11R alpha) gene in both humans and mice. Thus, this chemokine maps to chromosome 9p13 in humans where IL-11R alpha has been assigned, We term this novel chemokine IL-11R alpha-locus chemokine (ILC), ILC has the highest homology to MC148R among the known human CC chemokines. Furthermore, ILC is strongly and selectively expressed in the skin where infection of MCV also takes place, Thus, ILC is likely to be the original chemokine of MC148R. (C) 1999 Federation of European Biochemical Societies.

Single C motif-1 (SCM-1)/lymphotactin is a C-type member of the chemokine superfamily, Previously, we identified its specific receptor XCR1. Here we isolated the murine homologue of XCR1 (mXCR1). To demonstrate its biological activity, we produced recombinant mouse SCM-1 by the baculovirus expression system, B300-19 murine pre-B cells expressing mXCR1 responded to mSCM-1 in chemotactic and calcium-mobilization assays. mXCR1 mRNA was weakly expressed in spleen and lung of normal C57BL/6 mice. In spleen, CD8(+) cells and NK1.1(+) cells were found to express mXCR1. Identification of mXCR1 will now allow us to study the role of this unique cytokine system in the mouse models of inflammation and immunity, (C) 1999 Federation of European Biochemical Societies.

N-copine is a novel two C2 domain protein that shows Ca2+-dependent phospholipid binding and membrane association. By using yeast two-hybrid assays, we identified OS-9 as a protein capable of interacting with N-copine, We further revealed that the second C2 domain of N-copine bound with the carboxy-terminal region of OS-9, Their interaction in vivo nas also confirmed by co-immunoprecipitation from 293E cells co-expressing transfected N-copine and OS-9, In vitro binding assays showed that this interaction was Ca2+-dependent. By Northern blot analysis, N-copine and OS-9 were co-expressed in the same regions of human brain. These results reveal that OS-9 is a potential target of N-copine. (C) 1999 Federation of European Biochemical Societies.

N-Copine is a novel protein with two C2 domains. Its expression is brain specific and up-regulated by neuronal activity such as kainate stimulation and tetanus stimulation evoking hippocampal CA1 long-term potentiation. We examined the localization and subcellular distribution of N-copine in mouse brain. In situ hybridization analysis showed that N-copine mRNA was expressed exclusively in neurons of the hippocampus and in the main and accessory olfactory bulb, where various forms of synaptic plasticity and memory formation are known to occur. In immunohistochemical analyses, N-copine was detected mainly in the cell bodies and dendrites in the neurons, whereas presynaptic proteins such as synaptotagmin I and rab3A were detected in the regions where axons pass through. In fractionation experiments of brain homogenate, N-copine was associated with the membrane fraction in the presence of Ca2+ but not in its absence. As a GST-fusion protein with the second C2 domain of N-copine showed Ca2+-dependent binding to phosphatidylserine, this domain was considered to be responsible for the Ca2+-dependent association of N-copine with the membrane. Thus, N-copine may have a role as a Ca2+ sensor in postsynaptic events, in contrast to the known roles of "double C2 domain-containing proteins," including synaptotagmin I, in presynaptic events.

Neuronal activity is often associated with changes in gene expression. By a two-dimensional cDNA-display system, restriction landmark cDNA scanning, we identified a novel gene whose expression in the hippocampus was up-regulated by kainate stimulation. The mRNA expression was detected only in brain and up-regulated by the stimulation making CA3-CA1 long-term potentiation. The encoded protein contains two copies of C2-domain, known as the Ca2+-binding domain of PKC-gamma and shows 49% identity with human copine I. We designated this protein N-copine (neuronal-copine), N-copine may have a role in synaptic plasticity. (C) 1998 Federation of European Biochemical Societies.

我々は最近、Taxを発現しているHTLV-1感染細胞株においてTax siRNAにより発現が変動する遺伝子をマイクロアレイにより解析した結果、Tax依存性発現の可能性のある多数の遺伝子を同定した。その中に最近注目を浴びている翻訳伸長因子のeukaryotic elongation factor 1 alpha 2 (eEF1A2)が含まれていた。eEF1A2はEF-1αサブユニットのイソ型で、eEF1A1がハウスキーピング遺伝子として筋肉以外の多くの組織で発現しているのに対し、eEF1A2は主に筋肉と神経で発現している。両者は異なる染色体上にマップされ、92％のアミノ酸相同性を持つものの互いに排他的な発現調節を受けている。さらに興味深いことに、異所性に発現したeEF1A2はがん化との関連が報告されている。したがって、eEF1A2は発がん遺伝子としての性格も有しているが、その作用メカニズムについてはまだ十分明らかにされていない。 我々がeEF1A2に特に注目する理由は、最近明らかになってきているeEF1A2の翻訳伸長因子以外の作用である。eEF

成人T細胞性白血病において、Fra-2の過剰発現がCCR4と細胞増殖を引き起こすことを報告した。

【目的と意義】HTLV-1が感染してから数十年の潜伏感染の後、一部の感染者に成人T細胞白血病（ATL）が発症する。ATL腫瘍細胞の特徴は、本来は末梢血CD4＋T細胞の20%程度しか発現していないケモカイン受容体CCR4の高頻度発現（＞90%）である。前回我々は、HTLV-1感染細胞がTax依存性にCCL22を産生し、末梢血CCR4＋CD4＋T細胞を選択的に遊走させることを報告した。今回、HTLV-1初期感染におけるCCL22/CCR4の役割をさらに追究し、CD4＋T細胞への感染におけるCCL22/CCR4の役割を検討した｡ 【材料と方法】HTLV-1の感染様式を理解するために、①感染開始となる細胞間接着時､②感染早期(1週間前後)、③感染後期（2～3ヶ月）の各時期に分けて検討した｡HTLV-1感染細胞株と末梢血CCR4＋CD4＋T細胞を共培養し、CCR4特異的阻害剤あるいは百日咳毒素（PTX）処理が細胞間結合に与える影響を検討した｡また、マイトマイシンC処理したHTLV-1感染細胞とPHA処理した末梢血単核球細胞（PBMC）を共培養してHTLV-1感染実験

We examined the effect of Tax on expression of CCL22, a ligand of CCR4. Most Tax-expressing HTLV-1+ T cell lines produced a large amount of CCL22 in their culture supernatants. Transient expression of Tax also induced CCL22. Knockdown of Tax gene by siRNA resulted in a reduction of CCL22 mRNA. These results indicate CCL22 is one of the target genes of Tax. Culture supernatants from the HTLV-1+ T cell lines selectively attracted primary CCR4+CD4+ T cells in chemotaxis assays. The migration was blocked by pretreatment of the supernatants with anti-CCL22 Ab or pretreatment of PBMCs with a CCR4-specific antagonist. Further, HTLV-1+ T cell lines showed significant CCR4-dependent cell-to-cell interactions with sorted primary CCR4+CD4+ T cells, and anti-CCL22 Ab inhibited HTLV-1 primary infection. These results indicate that HTLV-1-infected T cells produce CCL22 upon expression of Tax and selectively recruit CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to these cells.

Most Tax+ HTLV-1+ T cell lines produced large amounts of CCL22 in their culture supernatants. Transient Tax expression also expressed CCL22. siRNA-induced knockdown of Tax resulted in a significant reduction of CCL22 mRNA in Tax+ cells. These results indicated CCL22 is one of the target genes of Tax. By using PBMCs from healthy donors in chemotaxis assays, it was demonstrated that culture supernatants of Tax+ cells strongly mobilized CCR4+CD4+ T cells. The migration was blocked by pretreatment of the supernatants with anti-CCL22 neutralizing antibody or pretreatment of the PBMCs with a CCR4-specific inhibitor. Further, Tax+ cells showed significant CCR4-dependent cell-cell interactions with the sorted CCR4+CD4+ T cells. These results imply that HTLV-1+ T cells, which were vertically transmitted from the breast milk of a carrier mother, produced CCL22 upon expression of Tax and preferentially recruited CCR4+CD4+ T cells in the child.

（目的）ケモカインは白血球の主要な遊走制御因子であるが、近年、一部のケモカインにおいて抗菌活性を有することが報告された。抗菌ペプチドの一部はエンベロープウイルスに対しても抗ウイルス活性を持つことから、今回、ケモカインによる抗ウイルス活性について検討を行った。 （方法）単純ヘルペスウイルス1型（HSV-1）のプラーク解析を指標に、ケモカインによる抗ウイルス活性を探索した。結合解析によりケモカインとHSV-1との直接の結合を確認した。ケモカインのHSV-1粒子構造への影響を電子顕微鏡により検討した。さらに、ヘルペス性角膜実質炎（HSK）モデルマウスを用いて、ケモカインによる治療効果についても検討を行った。 （結果）22のケモカインのうちRANTES、MIP-1aおよびMIP-1bを含む8つのケモカインはHSV-1のVero細胞への感染を優位に抑制した。これらのケモカインは直接HSV-1と結合し、その結合はエンベロープタンパクgBに対する中和抗体で特異的に阻害された

目的と意義】HTLV-1が感染してから数十年の潜伏感染の後、一部の感染者に成人T細胞白血病（ATL）が発症する。我々は以前、ATL腫瘍細胞の特徴として、本来末梢血CD4+T細胞で高々10%程度しか発現していないケモカイン受容体CCR4が高頻度に発現していることを報告した（Blood, 2002）｡今回、ATL細胞がCCR4+CD4＋T細胞に集束されるメカニズムの1つとして、HTLV-1感染初期に発現すると考えられるTax蛋白との関係に注目した。Taxは、細胞遺伝子の発現制御や発がん関連遺伝子の修飾を通して腫瘍化の初期に重要な役割を果たすと考えられている。我々はCCR4発現細胞を選択的に細胞遊走するケモカインMDCとTaxとの関係を検討した｡ 【材料と方法】各種HTLV-1感染細胞株、Tax発現誘導系細胞株JPX-9、Jurkat T細胞株などを用いて、RT-PCR、ELISA、siRNA、プロモーター解析、Noshift転写因子解析などにより検討した｡ 【結果】HTLV-1感染細胞株の多くがTaxを強発現しており、解析したTax発現細胞のほとんどでMDCの遺

角膜上皮細胞と角膜実質細胞は炎症性サイトカインやHSV-1感染により、様々なケモカインの発現を誘導した。マウスヘルペス性角膜実質炎では、その急性期には細胞性免疫を担うTh1、一方治癒に向かう後期には液性免疫を担うTh2が優勢な環境が形成されることが示唆された。

角膜上皮細胞と角膜実質細胞は炎症性サイトカインやHSV-1感染により、それぞれ独自のパターンで、未熟樹状細胞、単球やT細胞などを遊走するケモカインを産生することが明らかとなった。マウスヘルペス性角膜実質炎では、その急性期には細胞性免疫を担うTh1、一方治癒に向かう後期には液性免疫を担うTh2が優勢な環境が形成されることが示唆された。

炎症性サイトカインやHSV-1感染により角膜上皮細胞や角膜実質細胞からCCL20が産生されることが分かった。また、ヘルペス性角膜実質炎において、CCL20はその受容体であるCCR6を介した樹状細胞浸潤を誘導することにより、ヘルペス性角膜実質炎の病態形成に重要な役割を果たすと考えられた。

乾癬の発症には、Th17細胞が重要な役割を果たすことが知られている。ケモカイン受容体CCR4は、Th2細胞に発現し、Th2細胞の遊走を介してアトピー性皮膚炎等のアレルギー性疾患の発症に寄与している。近年、このCCR4がTh17細胞にも発現していることが明らかとなった。しかしながら、乾癬におけるCCR4の関与については不明である。本申請課題では、乾癬におけるCCR4を介したTh17細胞の遊走制御機構の解析を通じて、CCR4の乾癬の発症における役割を追究する。 前年度、イミキモド長期塗布により乾癬様病態を誘発したところ、野生型マウスと比べ、CCR4欠損マウスでは病態の軽減および病変部へのTh17細胞の浸潤の減少が認められた。 本年度は、CCR4の選択的阻害薬の投与による乾癬病態の変化について解析した。CCR4阻害薬compound22の2日に1回の投与により、臨床スコア（皮膚肥厚、紅斑、鱗屑）の軽減、病変皮膚および所属リンパ節でのTh17細胞の減少、乾癬関連遺伝子（IL-17A、IL-22、IL-23、STAT3、Keratin 14）の減少がみられた。以上より、CCR4の阻害が乾癬の治療戦略となる可能性が示された。

本研究の目的は、P2受容体が粘膜特有のCD70陽性樹状細胞に発現すること、また、メモリーCTLの生存維持に関与することに着目し、P2受容体を介したエフェクター/メモリーCTL誘導およびその維持機構を解明し、P2受容体のアゴニストである加水分解性抵抗性APTのワクチンアジュバントとしての有用性を検証することである。 本年度は、加水分解抵抗性ATPによって誘導されたエフェクターあるいはメモリーCTLが抗腫瘍活性を示すかどうかについてin vivo担癌モデルマウスを用いて検討した。モデル抗原OVAと加水分解抵抗性ATPを経鼻投与し、エフェクター期およびメモリー期にOVA発現腫瘍細胞 (E.G7-OVA)を皮内に接種した。経日的に腫瘍体積をモニタリングした結果、加水分解抵抗性ATPを併用した場合において、有意な腫瘍増殖抑制効果を認められた。さらに、この効果はP2受容体の阻害剤であるスラミンの投与によって消失した。これらの結果から、加水分解性抵抗性ATPによって誘導されたCTLによって有意な抗腫瘍効果を示すことが明らかとなり、加水分解抵抗性ATPは機能的なCTLを誘導できるアジュバントとしての活性を有することを示した。

ELC/CCL19 is a functional ligand for CCR7, which is mainly expressed by naive T and B cells. In the present study, we found that ELC/CCL19 is also a functional ligand for GPCR#13, which is broadly expressed by effector cells such as NK cells and monocytes. We revealed that ELC/CCL19 efficiently induced cell migration in murine L1.2 cells stably expressing GPCR#13 (L1.2-GPCR#13) as well as in L1.2-CCR7. Furthermore, In chemotaxis assays using human PBMCs, ELC/CCL19 attracted not only naive T cells but also CD16(+) NK cells and CD14(+) monocytes. In addition, ELC/CCL19-mediated cell migration was suppressed by treatment with a GPCR#13 antagonist. In mouse psoriasis model, intradermal injection of ELC/CCL19 recruited GPCR#13(+)/CCR7(-) effector cells in psoriasis-like skin lesions and exacerbated psoriasis-like inflammation. These results suggest that ELC/CCL19 another agonist for GPCR#13 and may play an important role in psoriasis by recruiting GPCR#13-expressing effector cells.

We identified several regulatory T cell (Treg) subsets in inflamed skin tissue of mouse line expressing photoconvertible protein KikGR, by which we can distinguish skin-coming and -remaining lymphocytes, by multi-parameter single-cell gene and protein expression analysis. Each subset expressed each set of functional and migration-related molecules and showed different capabilities to remain in inflamed skin. We distinguished skin-coming and -remaining Tregs without KikGR expression data by multi-parameter single-cell expression analysis of functional and migration-related molecules. We also tried to identify master molecules of Treg subset differentiation and establish method to collect the Treg subsets. This research suggests that Tregs expressing different functional molecules showed different migration status in inflamed tissues.

CCL28 is expressed in the mucosal tissues and to attract IgA-antibody secreting cells (IgA-ASCs) via CCR10. CCL28 has an antimicrobial activity against microbes in vitro. However, in vivo evidence for the role of CCL28 in the mucosal immunity remains scanty. We generated CCL28-deficient mice and demonstrated that CCL28-deficient mice showed reduced numbers and altered distribution of IgA-ASCs in the colon. The IgA contents in the fecal extracts were low. The average amounts of IgA secreted by a single IgA-ASC isolated from the lamina propria of the colon was reduced. Furthermore, the 16S rRNA sequencing analysis of feces revealed an increase of the Class Bacilli. Consistent with the low IgA production and altered microbiota in the colon, CCL28-deficient mice had aggravated colitis upon treatment with dextran sulfate, which was ameliorated by oral antibiotics. Therefore, CCL28 has an role in the mucosal immunity of the colon as a chemoattractant with a direct antimicrobial activity.

Chemokine receptors CCR3 and CCR4 have been paid attention as potent therapeutic targets for allergic diseases. First, we screened 80 crude drugs/herbs to identify candidate substances using chemotaxis assay. Ephedra Herb inhibited the chemotaxis mediated by both CCR3 and CCR4. Thus, we examined inhibitory effects of ephedrine, a major component of Ephedra Herb. However, ephedrine exhibited little effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. Therefore, we fractionated Ephedra Herb into four subfractions and examined the inhibitory effects of each subfraction. Ethyl acetate-insoluble fraction exhibited the inhibitory effects on chemotaxis and calcium mobilization mediated by CCR3 and CCR4 most significantly. Taken together, our data suggest that these crude drugs/herbs might be useful sources to develop new drugs targeting Th2-mediated allergic diseases.

近年、特発性肺線維症（IPF）の9割にEBウイルス（EBV）を始めとしたヘルペスウイルス感染が報告され、ヘルペスウイルスのIPF発症・増悪化への関与が示唆されている。IPFは肺上皮細胞の障害に伴う慢性炎症を基盤とする難治性疾患であり、筋線維芽細胞が産生する細胞外基質の過剰沈着を主体とする。しかしその病態発症機序については未だ不明な点が多い。 ガレクチン9（Gal-9）は上記肺線維症の発症要因とされる炎症促進作用や細胞外基質産生誘導能を有するβ-ガラクトシド認識結合タンパクである。本研究ではこのGal-9がヘルペスウイルス感染肺上皮細胞に強く発現したことから、EBVに相同性の高いマウスγヘルペスウイルス（MHV68）による肺線維症モデルを構築し、Gal-9の肺線維化における役割を解析してきた。 本年度までに肺線維症モデルマウスを構築し、Gal-9欠損マウスではMHV68感染による炎症反応および肺線維化が著しく軽減されることを明らかにした。さらにMHV68感染後の野生型マウス肺で検出されたIL-27の発現がGal-9欠損マウスで顕著に低下していたため、IL-27に対する中和抗体をMHV68感染後の野生型マウスに連続投与したところ、感染後の肺に認められた一連のケモカイン発現上昇および炎症細胞亜集団の浸潤が劇的に抑制された。また組織学的解析およびハイドロキシプロリン量測定によりMHV68誘導性の肺線維化もIL-27中和抗体投与により有意に抑制された。以上のことから、Gal-9に制御されるIL27が肺線維化を抑制するための治療標的分子となる可能性を示した。

Previously, we have examined the transcriptional regulation of CCR4 expression in adult T-cell leukemia/lymphoma (ATL) and demonstrated that an AP-1 family member FRA-2 is aberrantly expressed in ATL and in association with JUND promotes CCR4 expression and cell growth together with upregulation of various proto-oncogenes such as c-Myb and SOX4 in ATL. In the present study, we have extended our analysis to CCR7, which is highly expressed in ATL and cutaneous T cell lymphomas (CTCLs), and found a novel DNA-binding element GAGGAG in the CCR7 promoter and binding of ZFYVE19 to this element. Our study suggests an existence of another oncogenic cascade in ATL and CTCLs, providing useful information on the diagnosis and treatment of mature T cell malignancies.

Previously, we have shown that Fra-2 is consistently expressed and involved in CCR4 expression as well as cell growth in CCR4+ mature T-cell leukemias/lymphomas, including adult T-cell leukemia/lymphoma (ATL) and cutaneous T-cell lymphomas (CTCLs). Here, we we examine the expression and function of SOX4 in the oncogenesis of ATL and CTCLs. Fra-2 and SOX4 were consistently co-expressed in the clinical samples of ATL and CTCL. SOX4 promoter analysis demonstrated that Fra-2-JunD directly activates the SOX4 promoter via an AP-1 site. Furthermore, SOX4 siRNA significantly suppressed cell growth of ATL and CTCL cell lines. We found that SOX4 knockdown reduced the expression of genes such as GCKR, NAP1 and HDAC8. We also showed direct activation of the HDAC8 promoter by SOX4. Furthermore, HDAC8 knockdown significantly suppressed cell growth of ATL and CTCL cell lines. Taken together, these finding suggest that the Fra-2-SOX4 pathway has an important oncogenic role in ATL and CTCLs.

Previously, we have shown that an AP-1 family member, FRA-2, is consistently expressed at high levels in Adult T-cell leukemia/lymphoma (ATLL) and up-regulates the expression of CCR4 as well as that of several proto-oncogenes such as c-Myb, MDM2, and BCL6. Here, we identified an ABC transporter, ABCG2/BCRP, as a target gene of c-Myb in mature T-cell lymphomas including ATLL and cutaneous T-cell lymphomas (CTCLs). We detected the transcripts and proteins of c-Myb and ABCG2/BCRP in ATLL cells. Furthermore, Ko143, a selective ABCG2/BCRP inhibitor, enhanced the antitumor activity of doxorubicin against ATLL cell lines. Taken together, our findings support the notion that the c-Myb-ABCG2/BCRP pathway plays a role in chemoresistance of mature T-cell lymphomas.

In this study, we showed that Fra-2 and c-Myb were consistently co-expressed in cutaneous T-cell lymphomas (CTCLs), and contributed to cell proliferation. Furthermore, we showed that c-Myb was a direct target gene of Fra-2 in CTCLs. In addition, we identified that RASGRP2 was a c-Myb target gene and contributed to cell proliferation in CTCLs. Taken together, our findings support the notion that the Fra-2-c-Myb oncogenic pathway plays a major role in the oncogenesis of CTCLs.

CCR4はTh2細胞、制御性T細胞、皮膚指向性T細胞などに選択的に発現するケモカイン受容体である。我々は成人T細胞白血病(ATL)でのCCR4の高頻度発現を初めて報告した(Yoshie et al., Blood 2002)。そしてATLでのCCR4発現の転写制御機構を調べることにより、ATLではAP-1ファミリーのFra-2が構成的に異常発現しており、Fra-2はJunDとヘテロダイマーを形成することにより、ATLでのCCR4の発現を誘導し、さらにc-Myb、MDM2、Bcl-6などの原がん遺伝子の発現も誘導していることを明らかにした(Nakayama et al., Oncogene 2008)。ATLでFra-2やこれらの原がん遺伝子の構成的発現が示されたのは初めてである。そこで本年度は、Fra-2/JunDによるc-Mybの発現誘導を転写レベルで証明した。また皮膚T細胞リンパ腫(CTCL)は菌状息肉腫やCD30陽性未分化大細胞型リンパ腫などの多様な成熟型T細胞リンパ腫よりなるが、ATLと同様にCCR4を高頻度で発現する。そこでCTCLでのFra-2の発現と役割を検討した。そ.の結果、CTCLでもCCR4の発現とともにFra-2とJunDの構成的な発現が確認され、ATLと同様にFra-2とJunDはCTCLの増殖に関与すること、さらにFra-2とJunDはCCR4の発現だけでなく、c-Myb、MDM2、Bcl-6の発現も誘導していることを明らかにした。これらの結果は、Fra-2の構成的発現と発がんにおける役割はATLに限られるものではなく、CTCLも含めてCCR4を発現する成熟型T細胞由来の白血病/リンパ腫に普遍的に当てはまるものであることを示す。そしてFra-2で発現が誘導されるCCR4はこれらの悪性腫瘍の有用な腫瘍マーカーであるとともに有望な治療標的でもあると言える。

CCR4 is a chemokine receptor selectively expressed by Th2 cells, regulatory T cells, and skin-homing T cells. We have shown a potent ther apeutic effect of a small molecule CCR4 inhibitor on Th2-induced airway inflammation in mice. We have also shown that CCR4 and its ligand MDC/CCL22 mediate immunosuppressive interactions of dendritic cells and regulatory T cells in mesenteric lymphonodes. Furthermore, human oncogenic viruses such as EBV and HTLV-1 commonly induce expression of MDC/CCL22 in infected host cells to attract Th2 cells and regulatory T cells for immunoevasion and virus propagation.

ケモカインeotaxin-3/CCL26は、好酸球に高発現するCCR3のリガンドであり、炎症局所への好酸球浸潤に重要な役割を果たすことが報告されている.本研究において研究代表者は、CCL26が細胞傷害性リンパ球に高発現するCX3CL1受容体/CX3CR1の新しい機能的リガンドであることを見出した.CCL26は好酸球だけでなく細胞傷害性リンパ球も同時に炎症巣に誘導することにより、アレルギー性疾患の病態形成においてこれまで理解されていた以上の役割を果たすことが示唆された.

CCR4のプロモーター解析によりATLでの転写活性化サイトとしてGATA-3サイトとAP-1サイトを同定した。GATA-3はTh2細胞で選択的に発現し、CCR4のTh2選択的発現に関係すると考えられた。一方、AP-1はFosファミリーとJunファミリーのホモダイマーあるいはヘテロダイマーとして機能する。そこでAP-1ファミリーのATLでの発現を検討した。その結果、ATLの臨床検体ではFra-2、JunD、JunBの構成的発現が見られた。さらにCCR4プロモーターの活性化はFra-2/JunDおよびFra-2/JunBのヘテロダイマーがきわめて特異的に誘導することを見出した。さらにFra-2の発現はHTLV-1の転写因子Taxでは誘導されないことも確認した。発がん遺伝子Fra-2の正常T細胞での発現やATLでの構成的活性化はいまだ報告されていない。そこでATLでのFra-2、JunD、JunBの役割をsiRNAノツクダウンで検討した。その結果、Fra-2とJunDのsiRNAがCCR4発現および細胞増殖を抑制した。またFra-2あるいはJunDをJurkatで強制発現すると細胞増殖を促進した。そこでATLでのFra-2の下流標的遺伝子をFra-2 siRNAによるノックダウンとマイクロアレイ解析で検討した。その結果、Fra-2のsiRNAによりATL細胞株で少なくとも49の遺伝子が3倍以上の低下を示した。この中には発がん遺伝子c-Myb、Bc1-6、MDM2も含まれていた。そしてこれらの発がん遺伝子の発現はJunDのsiRNAでも抑制され、Fra-2/JunDヘテロダイマーの下流遺伝子であることが示された。さらにATLの臨床検体でもこれらの発がん遺伝子の構成的発現が確認された。発がん遺伝子Fra-2、c-Myb、Bc1-6、MDM2のATLでの構成的発現は初めての知見である。

CCR10 has been reported to be expressed by almost all IgA-secreting plasma cells, while its ligand CCL28 is widely expressed in various mucosal tissues. Thus, the CCR10-CCL28 system is likely to contribute a wide distribution of IgA-secreting plasma cells in the common mucosal immune system. Here we examined the mechanism of CCR10 expression in terminally differentiating B cells. As reported previously, activated human CD19^+ B cells treated with IL-21 efficiently differentiated into IgD^-CD38^+ plasma cells. Even though IgD^-CD38^+ cells did not spontaneously expressed CCR10, a substantial fraction turned to express CCR10 if the differentiation was induced in the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)_2D_3), which is known to promote common mucosal immune responses if used as an adjuvant. However, we observed little increases in IgA^+ cells by 1,25-(OH)_2D_3. To determine the transcriptional mechanism regulating 1,25-(OH)_2D_3-inducible expression of CCR10 in terminally differentiating B cells, we next carried out a series of analysis on the CCR10 promoter. The reporter assays involving a series of 5'-deleted promoter fragments and promoter fragments with site-directed mutations revealed that a proximal Ets-1 site and an upstream vitamin D3 response element (VDRE) were critical for CCR10 expression. The NoShift transcription factor binding assays using nuclear extracts from CCR10-expressing myeloma cell lines confirmed specific binding of Ets-1 and 1,25-(OH)_2D_3-activated vitamin D3 receptor (VDR) to the respective elements. Collectively, 1,25-(OH)2D3 efficiently induces CCR10 expression in IL-21-induced human plasma cells in vitro. Furthermore, the CCR10 promoter is cooperatively activated by 1,25-(OH)_2D_3-activated VDR and Ets-1.

上皮細胞でのケモカイン発現に対するEBV感染の影響を明らかにするため、上咽頭癌(NPC)細胞株、EBV陽性と陰性の上皮細胞株を用いてケモカインの網羅的発現解析を行ってきた。これまでに、これらのEBV感染上皮細胞とEBV感染B細胞におけるケモカイン発現プロファイルの比較解析により、EBV感染B細胞ではTh2型ケモカインTARC/CCL17とMDC/CCL22の発現が特異的に誘導され、EBV感染上皮細胞では対照的にTh1型ケモカインMIG/CXCL9とIP-10/CXCL10の発現が特異的に誘導されることを明らかにしている。 今回、EBV関連上皮系腫瘍の病態形成におけるケモカインの役割を臨床検体で明らかにするため、NPC腫瘍組織を用いてケモカインおよびケモカインレセプターの免疫染色を行った。その結果、NPCの腫瘍細胞はMIG/CXCL9とIP-10/CXCL10を強く発現していた。さらに、腫瘍細胞の周囲には、それらのレセプターであるCXCR3陽性の多数のT細胞浸潤が認められた。以上の結果から、MIG/CXCL9とIP-10/CXCL10の産生はCXCR3を介してTh1細胞を誘導することが示唆された。また、腫瘍細胞の周囲には、Foxp3^+IL-10^+CD69^-制御性T細胞の多数の浸潤が認められ、これらの細胞は従来報告されていたCCR4に加えてCXCR3を発現していた。NPC組織では、CCR4のリガンドであるTARC/CCL17とMDC/CCL22の発現は認められなかったため、制御性T細胞はCXCR3を介してNPC組織に誘導され腫瘍細胞に対する免疫反応の抑制に関与すると考えられた。

我々は、ATLではケモカイン受容体CCR4が高頻度・強陽性であること、CCR4発現はATLの高頻度皮膚浸潤を説明するとともに、ATLの起源がTh2あるいは制御性T細胞であることを示唆することを報告した(Blood, 2002)。このようなATLでのCCR4高頻度・高レベル発現は、ATLの発がん機構と転写レベルで密接に関係している可能性がある。そこで本研究では、ATLでのCCR4発現に関わる転写因子を明らかにし、それらのATL発がんへの関与を検討する。まずATL細胞におけるCCR4の転写開始部位を5'-RACEによってマップした。その結果、ATL細胞でのCCR4転写はエクソン1の上流と第1イントロン内の2領域で開始されており、それぞれ第1プロモーターと第2プロモーターと命名した。そこで第1プロモーター1-kbおよび第2プロモーター2-kbをルシフェレースレポータープラスミドにクローニングし、レポーターアッセイを行った。その結果、これらのプロモーターはATL細胞で強い転写活性を示した。そこで、それぞれのプロモーター領域を5'側から削っていってレポーターアッセイを繰り返し、発現に必要な領域を絞り込んだ。そしてこれらの必要領域に存在する候補エレメントに次々と点変異を導入し、必要エレメントの同定を行っている。第1プロモーターおよび第2プロモーターともにTh2分化に関わるGATA-3の認識配列が重要であることをみいだしたが、それ以外にも共同して作用するエレメントが存在することが明らかになってきた。現在、これらのエレメントに結合する転写因子の同定とクローニングを進めている。今後はCCR4の発現に関与することが明らかになった転写因子について、それらのATL発がんにおける役割を強制発現系やsiRNAによるノックダウン実験によって明らかにする。さらに、ATL細胞でのこれらの転写因子の構成的な発現および機能亢進のメカニズムを分子レベルで明らかにする。それによっていまだ不明なところの多い遺伝子レベルでのATLの発がん機構に関して新たな知見をもたらす。

1.マウス肺がんのリンパ節転移に対するCCR7の発現とCCL21/Secondary Lymphoid-tissue Chemokine (SLC)による運動能の亢進 本研究では、同様にC57BL/6マウスにリンパ節転移を引き起こす肺がん細胞(LLC)を用いて、CCL21及びCCR7とリンパ節転移との関連性について検討した。その結果、LLC細胞におけるCCR7の発現がmRNAレベルで確認され、運動能についてもCCL21の濃度に依存して亢進することが明らかとなった。更に、CXCR5、CXCR6の発現も確認され、それぞれの受容体に対するケモカインにより、濃度依存的に運動能の亢進が確認出来た結果から、CCR7以外にもリンパ節転移に関与する新たな受容体を同定するに到った。種々がん細胞のリンパ節転移は、リンパ管新生の惹起により亢進することが明らかとなってきている。(発表論文1.) 2.リンパ節転移性マウス肺がん細胞(LLC)に対するCCL21によるシグナル伝達系の解明 上記(1)でリンパ節転移性マウス肺がん細胞(LLC)にCCR7の発現が確認されたことで、次に、CCL21によるシグナル伝達系の解明を、行った。その結果、細胞増殖や生存に関与するシグナル伝達分子であるAkt及びErkのリン酸化の亢進が確認できた。今回、我々はAkt及びErk分子により、マウス結腸がんの肺転移が制御されている事を報告した。(発表論文2-3.) 3.がん細胞におけるケモカイン受容体、CCR7の発現亢進メカニズムの解明 現在までに、ケモカイン受容体の発現とがん転移の関連性に関する研究は、著しい知見の集積がなされている現在ではあるが、がん細胞におけるケモカイン受容体発現亢進のメカニズムは全く解明されていない。最近我々は、サイトカインががん転移を含むがん細胞の増悪化を引き起こすことを報告した。(発表論文3.)