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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.

Recent advances in metagenomic analyses have made it easier to analyze microbiota. The microbiota, a symbiotic community of microorganisms including bacteria, archaea, fungi, and viruses within a specific environment in tissues such as the digestive tract and skin, has a complex relationship with the host. Recent studies have revealed that microbiota composition and balance particularly affect the health of the host and the onset of disease. Influences such as diet, food preferences, and sanitation play crucial roles in microbiota composition. The oral cavity is where the digestive tract directly communicates with the outside. Stable temperature and humidity provide optimal growth environments for many bacteria. However, the oral cavity is a unique environment that is susceptible to pH changes, salinity, food nutrients, and external pathogens. Recent studies have emphasized the importance of the oral microbiota, as changes in bacterial composition and balance could contribute to the development of systemic diseases. This review focuses on saliva, IgA, and fermented foods because they play critical roles in maintaining the oral bacterial environment by regulating its composition and balance. More attention should be paid to the oral microbiota and its regulatory factors in oral and systemic health.

This study assessed the somatosensory evoked potentials (SEPs) in dogs and cats to compare the effect of remifentanil on the action potentials evoked by peripheral noxious stimulation in the spinal cord. Five healthy dogs and five healthy cats underwent general anaesthesia induced with propofol and maintained with isoflurane. Each animals received all dosage of a constant-rate infusion of remifentanil at 0 (control), 0.25, 0.5, 1.0 or 2.0 μg/kg/min. The hair of the dorsal foot of a hind limb was clipped and an intraepidermal stimulation electrode that could selectively stimulate the nociceptive Aδ and C fibres was attached. An electrical stimulus was generated by a portable peripheral nerve testing device. The evoked potentials were recorded by two needle electrodes inserted subcutaneously in the dorsal midline between the lumbar vertebra: L3-L4 and L4-L5. Bimodal waveforms were obtained by electrical stimulation in control dogs and cats. The inhibitory effect of remifentanil was evaluated by comparing the changes in the N1P2 and P2N2 amplitudes. The N1P2 amplitude was depressed by remifentanil in a dose-dependent manner in dogs, but it showed no remifentanil-induced changes in cats. While the P2N2 amplitude was also depressed in a dose-dependent manner in dogs, it showed milder remifentanil-induced effects in cats. The N1P2 and P2N2 amplitudes observed herein are assumed to represent the evoked potentials derived from the Aδ and C fibres, respectively. Thus, the inhibitory effect of remifentanil on nociceptive transmission at the spinal cord was much weaker in cats, especially for transmissions possibly derived from Aδ fibres., Competing Interests: Declaration of Competing Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Oral functions are diverse and critical to human health. Therefore, insufficient secretion or poor quality of saliva, which is secreted into the oral cavity and plays various roles, could have a crucial influence on the oral microenvironment and be associated with systemic disease development. Here, we investigated the effects of food ingredients on saliva quantity and quality, including fermented ones. Through the in vitro submandibular glands' organ culture analyses, we found that "Yomo gyutto," fermented Japanese mugwort (Artemisia princeps), altered the expression of aquaporin-5, a water channel protein. We also found that Yomo gyutto increased saliva volume, along with the amount of α-amylase in mice, and caused changes in the oral microbiome composition of mice. These results suggested that by ingesting Yomo gyutto, we could directly and effectively manipulate the quantity and quality of saliva secreted from the salivary glands, potentially altering the oral microbiome composition for individual health., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Malignant features such as the acquisition of metastatic ability, stemness of cells, and therapeutic resistance of cancer cells are associated with epithelial-mesenchymal transition (EMT) accompanied by changes in motility and morphology. Recent reports implicated that the formation of polyploid giant cancer cells (PGCCs) in human malignancy correlated with the EMT processes. Chemokines are often involved in the regulation of cancer cell migration into tissues, and various types of human cancers exhibit enhanced expression of chemokine receptors, which could augment intrinsic potentials such as invasive activity, proliferating ability, and survival capacity in cancer cells. Nevertheless, the contribution of CCR3 in malignant cancer cells is controversial because it is a well-known primal receptor for the migration of eosinophils, one of the cells of the innate immune system. Here, we explored the blockage of chemokine receptor CCR3 in carcinoma cell lines and found that inhibition of CCR3 induced the formation of polyploid giant cells and stabilization of β-catenin via the PI3K/Akt/GSK-3β signaling pathway, which are processes associated with EMT. As a result of CCR3 inhibition, converted cells acquired enhanced mobile and proliferation abilities. In summary, these data indicate that modulation of the CCR3/PI3K/Akt/GSK-3β signaling pathway regulates polyploidization associated with the EMT processes., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

T helper 17 (Th17) cells express CC chemokine receptor 4 (CCR4) and secrete cytokines such as interleukin-17A (IL-17A) and granulocyte macrophage colony-stimulating factor (GM-CSF), while dendritic cells (DCs) produce CC chemokine ligand 22 (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 up-regulated 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., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The development of the parathyroid and the thymus from the third pharyngeal pouch depends on the activities of the Gcm2 and Foxn1 transcription factors, respectively, whose expression domains sharply demarcate two regions in the developing third pharyngeal pouch. Here, we have generated novel mouse models to examine whether ectopic co-expression of Gcm2 in the thymic epithelium and of Foxn1 in the parathyroid perturbs the establishment of organ fates in vivo. Expression of Gcm2 in the thymic rudiment does not activate a parathyroid-specific expression programme, even in the absence of Foxn1 activity. Co-expression of Foxn1 in the parathyroid fails to impose thymopoietic capacity. We conclude that the actions of Foxn1 and Gcm2 transcription factors are cell context-dependent and that they each require permissive transcription factor landscapes in order to successfully interfere with organ-specific cell fate., (© 2022. The Author(s).)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

The onset of lymphocyte development in the vertebrate primordial thymus, about 500 million years ago, represents one of the foundational events of the emerging adaptive immune system. Here, we retrace the evolutionary trajectory of thymopoiesis, from early vertebrates to mammals, guided by members of the Foxn1/4 transcription factor gene family, which direct the differentiation of the thymic microenvironment. Molecular engineering in transgenic mice recapitulated a gene duplication event, exon replacements, and altered expression patterns. These changes predictably modified the lymphopoietic characteristics of the thymus, identifying molecular features contributing to conversion of a primordial bipotent lymphoid organ to a tissue specializing in T cell development. The phylogenetic reconstruction associates increasing efficiency of T cell generation with diminishing B cell-generating capacity of the thymus during jawed vertebrate evolution., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Neutrophils are important phagocytic cells for host defense against pathogens. They are rapidly recruited to the site of infection, release antimicrobial peptides and cytokines, and engulf and kill microbes. Neutrophils also accumulate in allergic inflammatory sites. Here we characterized neutrophil accumulation in the nasal mucosa using a mouse model of allergic rhinitis, in which mice were sensitized by intraperitoneal injection of ovalbumin (OVA) and then challenged by intranasal administration of OVA or PBS. In the nasal mucosa of both PBS- and OVA-challenged mice, we found a cell subset expressing the eosinophil marker Siglec-F in the Ly-6G + neutrophil population. Morphological analysis of the sorted Ly-6G + Siglec-F + cells revealed that they were devoid of eosinophilic granules in the cytosol and were apparently neutrophils, but compared to conventional Ly-6G + Siglec-F - neutrophils, they had a more lobulated, "botryoid" nucleus. Siglec-F + neutrophils were barely found in the nasopharynx-associated lymphoid tissue, cervical lymph nodes, the spleen, or blood. Both Siglec-F + neutrophils and conventional neutrophils showed increased numbers in the nasal mucosa of OVA-challenged mice. Compared to conventional Siglec-F - neutrophils, Siglec-F + neutrophils exhibited an activated phenotype and enhanced effector functions. Taken together, our findings identify Siglec-F + neutrophils as a novel neutrophil subset with an activated phenotype that resides specifically in the nasal mucosa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The numbers of thymic epithelial cells (TECs) and thymocytes steadily increase during embryogenesis. To examine this dynamic, we generated several TEC-specific transgenic mouse lines, which express fluorescent proteins in the nucleus, the cytosol and in the membranes under the control of the Foxn1 promoter. These tools enabled us to determine TEC numbers in tissue sections by confocal fluorescent microscopy, and in the intact organ by light-sheet microscopy. Compared to histological procedures, flow cytometric analysis of thymic cellularity is shown to underestimate the numbers of TECs by one order of magnitude; using enzymatic digestion of thymic tissue, the loss of cortical TECs (cTECs) is several fold greater than that of medullary TECs (mTECs), although different cTEC subsets appear to be still present in the final preparation. Novel reporter lines driven by Psmb11 and Prss16 promoters revealed the trajectory of differentiation of cTEC-like cells, and, owing to the additional facility of conditional cell ablation, allowed us to follow the recovery of such cells after their depletion during embryogenesis. Multiparametric histological analyses indicate that the new transgenic reporter lines not only reveal the unique morphologies of different TEC subsets, but are also conducive to the analysis of the complex cellular interactions in the thymus.

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., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The ezrin-radixin-moesin (ERM) proteins are a family of membrane-associated proteins that link membrane proteins with actin filaments in the cell cortex and regulate many cellular processes, including cell shape determination, membrane transport, and signal transduction. Lymphocytes predominantly express two ERM members, ezrin and moesin. Mutations in the moesin gene in humans are associated with primary immunodeficiency with profound lymphopenia, and moesin-deficient mice exhibit a similar lymphopenia phenotype. In this study, we show that aging moesin-deficient mice develop a systemic lupus erythematosus-like autoimmune phenotype, which is characterized by elevated serum autoantibody levels and glomerulonephritis. Younger moesin-deficient mice exhibited elevated basal levels of several Ig isotypes and enhanced Ab affinity maturation upon immunization. Germinal center B cells and follicular helper T cells spontaneously accumulated in unimmunized mice, and CD8 + CD44 + CD122 + Ly49 + regulatory T (CD8 + Tregs) cells, which inhibit the expansion of follicular helper T cells, were severely reduced in these mice. Isolated CD8 + Treg cells from moesin-deficient mice showed impaired proliferation in response to IL-15, which was accompanied by defects in STAT5 activation and IL-15Rα internalization, suggesting that moesin plays a key role in IL-15-mediated signaling. These findings underscore the importance of moesin in IL-15-dependent CD8 + Treg cell homeostasis and, thus, the control of self-tolerance., (Copyright © 2017 by The American Association of Immunologists, Inc.)

This study was conducted to identify the brain regions corresponding to mechanical noxious stimulation in cats using functional magnetic resonance imaging (fMRI) and to investigate the modulatory effect of remifentanil on the activation of these regions. Six healthy cats were anesthetized using a constant-rate infusion of alfaxalone. Cats were allocated to one of three treatment groups: remifentanil 0 (saline), 0.25, and 0.5μg/kg/min. A 3.0-T MRI unit was used to collect fMRI data. During the fMRI scanning, mechanical noxious stimulation was applied by tail clamping. The brain regions activated by the stimulation were identified based on blood oxygenation level-dependent (BOLD) responses. The modulatory effects of remifentanil were evaluated using a region of interest (ROI) analysis comparing signal changes in each brain region. Increased activity from noxious stimulation was observed in the somatosensory area (the postcruciatus gyrus, the anterior part of the marginalis gyrus, and the anterior part of the ectomarginalis gyrus), the parietal association area (the middle part of the marginalis gyrus and the middle part of the ectomarginalis gyrus), the cingulate cortex, the hippocampus, and the cerebellum. The results of the ROI analysis indicated that activations in the somatosensory area, the cingulate cortex, the hippocampus, and the cerebellum were significantly modulated (P<0.05) by remifentanil. In cats, activation patterns evoked by mechanical noxious stimulation were observed in several brain regions thought to be involved in various aspects of pain processing, including sensory discrimination and integration, affect, and motor response. These brain responses were modulated by remifentanil., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The thymus is the site of T cell development in vertebrates. In general, the output of T cells is determined by the number of thymic epithelial cells (TECs) and their relative thymopoietic activity. Here, we show that the thymopoietic activity of TECs differs dramatically between individual mouse strains. Moreover, in males of some strains, TECs perform better on a per cell basis than their counterparts in females; in other strains, this situation is reversed. Genetic crosses indicate that TEC numbers and thymopoietic capacity are independently controlled. Long-term analysis of functional parameters of TECs after castration provides evidence that the number of Foxn1-expressing TECs directly correlates with thymopoietic activity. Our study highlights potential complications that can arise when comparing parameters of TEC biology across different genetic backgrounds; these could affect the interpretation of the outcomes of interventions aimed at modulating thymic activity in genetically diverse populations, such as humans.

Autoimmune and inflammatory conditions are frequent complications in patients with reduced numbers of T cells. Here, we describe a mouse model of thymic stromal dysplasia resulting in peripheral T-cell lymphopenia. In Foxn1:CFP-NTR transgenic mice, the bacterial nitroreductase enzyme is expressed in thymic epithelial cells and converts the prodrug CB1954 into a cytotoxic agent. This strategy enables titratable and durable destruction of thymopoietic tissue in early embryogenesis. Our results indicate that the resulting low levels of thymic capacity for T-cell production create a predisposition for the development of a complex autoimmune syndrome, chiefly characterized by inflammatory bowel disease and lymphocytic organ infiltrations. We conclude that the Foxn1:CFP-NTR transgenic mouse strain represents a suitable animal model to optimize established clinical protocols, such as thymus transplantation, to correct various forms of thymic dysplasia and to explore novel treatment options., (© The Author 2017. Published by Oxford University Press on behalf of The Japanese Society for Immunology.)

Obesity and type 2 diabetes mellitus (T2DM) are occurring at epidemic-like rates, and these epidemics appear to have emerged largely from changes in daily diet. In the present study, we compared effects of high-fat diet (HFD) and fructose-rich diet (FRD) in WBN/Kob-Lepr fa (WBKDF) rats that spontaneously develop obesity, dyslipidemia and T2DM. After a 4-week feeding of each diet, WBKDF-HFD and WBKDF-FRD rats exhibited aggravated obesity and dyslipidemia compared with WBKDF rats fed standard diet (STD). In contrast, hyperglycemia developed in WBKDF-STD rats was significantly inhibited in WBKDF-FRD rats, but not in WBKDF-HFD rats. The present study demonstrated that the 4-week feeding of HFD and FRD caused diet-induced obesity with a distinct phenotype in the glucose metabolism in WBKDF rats.

During nasal immune responses, lymphocytes activated in the nasopharynx-associated lymphoid tissue (NALT) are thought to traffic to the nasal mucosa. Here we found a prominent infiltration of CD4(+) memory T cells into the nasal mucosa in a mouse model of allergic rhinitis. CCR3 and CCR10 mRNA was increased in the NALT, and CCR3- or CCR10-expressing CD4(+) T cells were present in the nasal mucosa. CCL28, a chemokine ligand for CCR3 and CCR10, was upregulated in nasal epithelial cells. Our results suggest that memory CD4(+) T cells traffic to the nasal mucosa in a process that may involve CCL28 and its receptors CCR3 and CCR10., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The thymus is a lymphoid organ unique to vertebrates, and it provides a unique microenvironment that facilitates the differentiation of immature hematopoietic precursors into mature T cells. We subjected the evolutionary trajectory of the thymic microenvironment to experimental analysis. A hypothetical primordial form of the thymus was established in mice by replacing FOXN1, the vertebrate-specific master regulator of thymic epithelial cell function, with its metazoan ancestor, FOXN4, thereby resetting the regulatory and coding changes that have occurred since the divergence of these two paralogs. FOXN4 exhibited substantial thymopoietic activity. Unexpectedly, histological changes and a functional imbalance between the lymphopoietic cytokine IL7 and the T cell specification factor DLL4 within the reconstructed thymus resulted in coincident but spatially segregated T and B cell development. Our results identify an evolutionary mechanism underlying the conversion of a general lymphopoietic organ to a site of exclusive T cell generation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to investigate the effects of liraglutide, an analog of human glucagon-like peptide 1 (GLP1), on WBN/Kob-Lepr(fa) (fa/fa) rats, which spontaneously develop type 2 diabetes mellitus with pancreatic disorder and obesity. Male fa/fa rats (age, 7 wk) were allocated into 4 groups and received liraglutide (37.5, 75, 150 μg/kg SC) or saline (control group) once daily for 4 wk. All rats in the control group became overweight and developed hyperglycemia as they aged. Although the rats given liraglutide showed a dose-dependent reduction in food intake, no significant effects on body weight or fat content occurred. In the liraglutide groups, the development of hyperglycemia was suppressed, even as plasma insulin concentrations increased in a dose-dependent manner. Intravenous glucose tolerance testing of the liraglutide-treated rats confirmed improvement of glucose tolerance and enhanced insulin secretion. Histologic examination revealed increased numbers of pancreatic β-cell type islet cells and increased proliferation of epithelial cells of the small ducts in the liraglutide-treated groups. Although our study did not reveal a significant decrease in obesity after liraglutide administration, the results suggest a marked antidiabetic effect characterized by increased insulin secretion in fa/fa rats with pancreatic disorders.

WBN/Kob-Lepr(fa) (fa/fa) rats have been identified as a new animal model of type 2 diabetes (T2DM), as they are characterized by impaired pancreatic insulin secretion and severe insulin resistance. Our previous study demonstrated impaired insulin secretion and its involvement in hyperglycemia in fa/fa rats. The present study was aimed at elucidating the role of insulin resistance in the development and progression of diabetes in these animals. Troglitazone (TGZ) was used as an insulin sensitizer. Insulin resistance and insulin secretory capacity were measured by a homeostasis model assessment of insulin resistance and the area under the blood concentration-time curve for plasma insulin levels after intravenous glucose tolerance testing, respectively. The fa/fa rats exhibited marked insulin resistance between 5 and 11 weeks of age, compared with age-matched Wistar rats. The insulin secretory capacity of fa/fa rats was higher than that of Wistar rats at 5 weeks of age, but decreased by 50% between 9 and 11 weeks of age. The fa/fa rats were fed a standard diet, with or without 0.2% w/w TGZ, for 4 weeks. Treatment with TGZ significantly improved insulin resistance, hyperglycemia and hypertriglyceridemia in both prophylactic and therapeutic study groups. These results suggest that insulin resistance is markedly involved in the development and progression of T2DM in fa/fa rats.

The aim of this study was to investigate blood coagulation times in genetically obese rats and diet-induced obese (DIO) mice in order to clarify the relationship between visceral obesity and blood coagulation. WBN/Kob-Lepr(fa) (fa/fa) rats, a genetically obese model, exhibited a significantly shorter activated partial thromboplastin time (aPTT) and prothrombin time (PT) than age-matched Wistar rats. C57BL/6J mice fed a high-fat diet (60%), a DIO model, exhibited significantly shorter aPTT, PT and thrombin time than lean mice fed a standard diet. Higher body weight, visceral fat weight and insulin resistance were also shared by fa/fa rats and DIO mice. These results suggest that visceral obesity is related to accelerated blood coagulation in addition to disrupted metabolism of glucose and lipids.

Latent infection of human T-cell leukemia virus type 1 (HTLV-1) is considered to be preferentially associated with CCR4(+) CD4(+) T cells. Here we report that c-Maf, one of the critical transcription factors for Th2 differentiation, suppresses the transcriptional activity of HTLV-1 Tax by competing for CREB-binding protein. Notably, c-maf expression is selectively induced in a fraction of CCR4(+) CD4(+) T cells upon activation. Furthermore, c-Maf significantly decreases Tax-induced HTLV-1 envelope gp46 gene expression from an infectious HTLV-1 molecular clone and tax expression in a cell-free HTLV-1 infection system. Collectively, c-Maf may play a role in latent infection of HTLV-1 in CCR4(+) CD4(+) T cells by negatively regulating Tax activity., (© 2011 Japanese Cancer Association.)

CD4(+) T cells, particularly T helper (Th) 2 cells, play a pivotal role in the pathophysiology of allergic asthma. Suppressor of cytokine signaling (SOCS) proteins control the balance of CD4(+) T cell differentiation. Mice that lack SOCS3 in T cells by crossing SOCS3-floxed mice with Lck-Cre-transgenic mice have reduced allergen-induced eosinophilia in the airways. Here, we studied the effects of SOCS3 silencing with small interfering (si) RNA in primary CD4(+) T cells on Th2 cell differentiation and on asthmatic responses in mice. Th2 cells were generated from ovalbumin (OVA)-specific T cell receptor-transgenic mice in vitro and transferred into recipient mice. Transfection of SOCS3-specific siRNA attenuated Th2 response in vitro. Adoptive transfer of SOCS3-siRNA T cells exhibited markedly suppressed airway hyperresponsiveness and eosinophilia after OVA challenge, with a concomitant decrease in OVA-specific CD4(+) T cell accumulation in the airways. To investigate the mechanism of this impaired CD4(+) T cell accumulation, we inactivated SOCS3 of T cells by crossing SOCS3-floxed (SOCS3(flox/flox)) mice with CD4-Cre transgenic mice. CD4-Cre × SOCS3(flox/flox) mice exhibited fewer IL-4-producing cells and more reduced eosinophil infiltration in bronchoalveolar lavage fluids than control mice in a model of OVA-induced asthma. Expression of CCR3 and CCR4 in CD4(+) T cells was decreased in CD4-Cre × SOCS3(flox/flox) mice. CCR4 expression was also decreased in CD4(+) T cells after transfer of SOCS3 siRNA-treated T cells. These findings suggest that the therapeutic modulation of SOCS3 expression in CD4(+) T cells might be effective in preventing the development of allergic asthma.

Dendritic cells (DCs) express the immunoregulatory enzyme IDO in response to certain inflammatory stimuli, but it is unclear whether DCs express this enzyme under steady-state conditions in vivo. In this study, we report that the DCs in mesenteric lymph nodes (MLNs) constitutively express functional IDO, which metabolizes tryptophan to kynurenine. In line with a previous report that regulatory T cells (Tregs) can induce IDO in DCs via the CTLA-4/B7 interaction, a substantial proportion of the MLN DCs were located in juxtaposition to Tregs, whereas this tendency was not observed for splenic DCs, which do not express IDO constitutively. When CTLA-4 was selectively deleted in Tregs, the frequency of IDO-expressing DCs in MLNs decreased significantly, confirming CTLA-4's role in IDO expression by MLN DCs. We also found that the MLN DCs produced CCL22, which can attract Tregs via CCR4, and that the phagocytosis of autologous apoptotic cells induced CCL22 expression in CCL22 mRNA-negative DCs. Mice genetically deficient in the receptor for CCL22, CCR4, showed markedly reduced IDO expression in MLN-DCs, supporting the involvement of the CCL22/CCR4 axis in IDO induction. Together with our previous observation that MLN DCs contain much intracytoplasmic cellular debris in vivo, these results indicate that reciprocal interactions between the DCs and Tregs via both B7/CTLA-4 and CCL22/CCR4 lead to IDO induction in MLN DCs, which may be initiated and/or augmented by the phagocytosis of autologous apoptotic cells by intestinal DCs. Such a mechanism may help induce the specific milieu in MLNs that is required for the induction of oral tolerance.

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-kappaB site, while a mutant Tax selectively defective in NF-kappaB 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.)

Although CD4(+)CD25(+) regulatory T (Treg) cells are known to suppress Th1 cell-mediated immune responses, their effect on Th2-type immune responses remains unclear. In this study we examined the role of Treg cells in Th2-type airway inflammation in mice. Depletion and reconstitution experiments demonstrated that the Treg cells of naive mice effectively suppressed the initiation and development of Th2-driven airway inflammation. Despite effective suppression of Th2-type airway inflammation in naive mice, adoptively transferred, allergen-specific Treg cells were unable to suppress airway inflammation in allergen-presensitized mice. Preactivated allergen-specific Treg cells, however, could suppress airway inflammation even in allergen-presensitized mice by accumulating in the lung, where they reduced the accumulation and proliferation of Th2 cells. Upon activation, allergen-specific Treg cells up-regulated CCR4, exhibited enhanced chemotactic responses to CCR4 ligands, and suppressed the proliferation of and cytokine production by polarized Th2 cells. Collectively, these results demonstrated that Treg cells are capable of suppressing Th2-driven airway inflammation even in allergen-presensitized mice in a manner dependent on their efficient migration into the inflammatory site and their regulation of Th2 cell activation and proliferation.

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

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 kappaB. 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 (ATL) is a highly aggressive mature CD4+ T-cell malignancy that is etiologically associated with human T-lymphotropic virus Type 1 (HTLV-1). ATL is characterized by frequent infiltration of lymph nodes, spleen, liver, skin and gut. Previously, we and others have shown that the majority of ATL cases are strongly positive for CCR4, which may explain the frequent skin invasion of ATL. Here, we examined whether ATL cells express CCR9, which is involved in T-cell homing to the gastrointestinal tract. Human T cell lines carrying HTLV-1 consistently expressed CCR9 together with the HTLV-1-encoded transcriptional activator Tax. Although ATL cells freshly isolated from peripheral blood hardly expressed CCR9, ATL cells cultured for 1 day consistently expressed CCR9 in parallel with the upregulation of Tax. Induction of Tax by Cd2+ in JPX-9, a subline of Jurkat human T cell line carrying Tax under the control of metallothionein promoter, led to upregulation of CCR9. A luciferase reporter gene under the control of the CCR9 promoter was expressed by cotransfection of an expression vector for Tax or in Cd2+-treated JPX-9 cells. Furthermore, immunohistochemical staining demonstrated that ATL cells infiltrating gastrointestinal tract were frequently positive for CCR9. Collectively, CCR9 is inducible in ATL cells expressing Tax and may play a role in the gastrointestinal involvement of ATL., ((c) 2006 Wiley-Liss, Inc.)

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.

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 Galphai 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.

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 alpha4 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., (Copyright 2004 The American Association of Immunologists, Inc.)

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.

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.

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-1 alpha/CCL3, MIP-1 beta/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-kappa B 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-kappa B 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-kappa B 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.

We previously reported that mac25/angiomodulin (AGM), a 30-kDa secretory protein, is abundantly expressed in high endothelial venules (HEVs), which play a crucial role in lymphocyte trafficking to the lymph nodes and Peyer's patches. We report that mac25/AGM interacts preferentially with certain molecules that are expressed in or around HEVs. In particular, mac25/AGM interacted with not only the extracellular matrix proteins and glycosaminoglycans that are expressed in most blood vessels including HEVs, but also with some chemokines that are implicated in the regulation of lymphocyte trafficking, such as the secondary lymphoid-tissue chemokine (SLC; CCL21), IFN-gamma-inducible protein 10 (IP-10; CXCL10), and RANTES (CCL5). The binding of mac25/AGM to SLC and IP-10 was dose-dependent and saturable. The binding to IP-10 could be inhibited by SLC but not by a non-mac25/AGM-binding chemokine, EBI1-ligand chemokine (ELC; CCL19). Interestingly, mac25/AGM failed to interact with 18 other chemokines, suggesting that it binds to certain chemokines preferentially. Immunohistochemical analysis indicated that mac25/AGM colocalizes at least partially with SLC and IP-10 at the basal lamina of HEVs. Upon binding with mac25/AGM, SLC and IP-10 retained all their Ca(2+)-signaling activity in vitro, suggesting that mac25/AGM can hold and present chemokines in the basal lamina of HEVs. These results imply that mac25/AGM plays a multifunctional role, serving not only as an adhesion protein to interact with glycosaminoglycans and extracellular matrix proteins but also as a molecule to present chemokines so that lymphocytes extravasating through HEVs receive further directional cues subsequent to the luminal encounter with lymphoid chemokines.

Previous results have indicated that mac25/angiomodulin (AGM) is expressed in lymph node (LN) high endothelial venules (HEV), the specialized venules that support efficient lymphocyte transendothelial migration. How mac25/AGM's endothelial expression pattern is regulated in situ remains unknown. Here, we demonstrate that in mouse LN blood vessels, including HEV, mac25/AGM is localized, unlike previous reports, not to the luminal or lateral regions bordering the endothelial cells (EC), but exclusively to the basal lamina that is in direct association with EC. In the spleen, mac25/AGM was expressed in the vascular basal lamina, in direct association with smooth muscle cells and pericytes, but not with EC. In addition, we report herein that mac25/AGM is an inducible marker for activated EC. In inflamed tissues, mac25/AGM expression was strongly induced in the abluminal region of blood vessels. In vitro, mac25/AGM was readily induced in EC upon activation with pro-inflammatory cytokines such as tumor necrosis factor-alpha, indicating that mac25/AGM is an activated EC marker. mac25/AGM binds vascular endothelial growth factor and, together with its strict abluminal localization, it is suggested that mac25/AGM has a specific function(s) in the subendothelium of activated blood vessels such as capturing humoral factors produced in the vicinity of HEV.

Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L- and P-selectin and CD44. The binding of versican to L- and P-selectin was inhibited by CS B, CS E, and heparan sulfate (HS) but not by any other glycosaminoglycans tested. On the other hand, the binding to CD44 was inhibited by hyaluronic acid, chondroitin (CH), CS A, CS B, CS C, CS D, and CS E but not by HS or keratan sulfate. A cross-blocking study indicated that L- and P-selectin recognize close or overlapping sites on versican, whereas CD44 recognizes separate sites. We also show that soluble L- and P-selectin directly bind to immobilized CS B, CS E, and HS and that soluble CD44 directly binds to immobilized hyaluronic acid, CH, and all the CS chains examined. Consistent with these results, structural analysis showed that versican is modified with at least CS B and CS C. Thus, proteoglycans sufficiently modified with the appropriate glycosaminoglycans should be able to bind L-selectin, P-selectin, and/or CD44.