A novel role for bone marrow-derived cells to recover damaged keratinocytes from radiation-induced injury.

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.

Affinity-coupled CCL22 promotes positive selection in germinal centres.

Antibody affinity maturation depends on positive selection in germinal centres (GCs) of rare B cell clones that acquire higher-affinity B cell receptors via somatic hypermutation, present more antigen to follicular helper T (TFH) cells and, consequently, receive more contact-dependent T cell help1. As these GC B cells and TFH cells do not maintain long-lasting contacts in the chaotic GC environment2-4, it is unclear how sufficient T cell help is cumulatively focused onto those rare clones. Here we show that, upon stimulation of CD40, GC B cells upregulate the chemokine CCL22 and to a lesser extent CCL17. By engaging the chemokine receptor CCR4 on TFH cells, CCL22 and CCL17 can attract multiple helper cells from a distance, thus increasing the chance of productive help. During a GC response, B cells that acquire higher antigen-binding affinities express higher levels of CCL22, which in turn 'highlight' these high-affinity GC B cells. Acute increase or blockade of TFH cells helps to rapidly increase or decrease CCL22 expression by GC B cells, respectively. Therefore, a chemokine-based intercellular reaction circuit links the amount of T cell help that individual B cells have received recently to their subsequent ability to attract more help. When CCL22 and CCL17 are ablated in B cells, GCs form but B cells are not affinity-matured efficiently. When competing with wild-type B cells in the same reaction, B cells lacking CCL22 and CCL17 receive less T cell help to maintain GC participation or develop into bone-marrow plasma cells. By uncovering a chemokine-mediated mechanism that highlights affinity-improved B cells for preferential help from TFH cells, our study reveals a principle of spatiotemporal orchestration of GC positive selection.

A CCR4 antagonist enhances DC activation and homing to the regional lymph node and shows potent vaccine adjuvant activity through the inhibition of regulatory T-cell recruitment.

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.

Reduced locomotor activity and exploratory behavior in CC chemokine receptor 4 deficient mice.

Chemokines and their receptors are key regulators of immune cell trafficking and activation. Recent findings suggest that they may also play pathophysiological roles in psychiatric diseases like depression and anxiety disorders. The CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are functionally involved in neuroinflammation as well as anti-infectious and autoimmune responses. However, their influence on behavior remains unknown. Here we characterized the functional role of the CCR4-CCL17 chemokine-receptor axis in the modulation of anxiety-related behavior, locomotor activity, and object exploration and recognition. Additionally, we investigated social exploration of CCR4 and CCL17 knockout mice and wild type (WT) controls. CCR4 knockout (CCR4(-/-)) mice exhibited fewer anxiety-related behaviors in the elevated plus-maze, diminished locomotor activity, exploratory behavior, and social exploration, while their recognition memory was not affected. In contrast, CCL17 deficient mice did not show an altered behavior compared to WT mice regarding locomotor activity, anxiety-related behavior, social exploration, and object recognition memory. In the dark-light and object recognition tests, CCL17(-/-) mice even covered longer distances than WT mice. These data demonstrate a mechanistic or developmental role of CCR4 in the regulation of locomotor and exploratory behaviors, whereas the ligand CCL17 appears not to be involved in the behaviors measured here. Thus, either CCL17 and the alternative ligand CCL22 may be redundant, or CCL22 is the main activator of CCR4 in these processes. Taken together, these findings contribute to the growing evidence regarding the involvement of chemokines and their receptors in the regulation of behavior.

CCR4 is critically involved in effective antitumor immunity in mice bearing intradermal B16 melanoma.

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.

C-C chemokine receptor type 4 antagonist Compound 22 ameliorates experimental autoimmune encephalomyelitis.

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.

Differential requirement for CCR4 and CCR7 during the development of innate and adaptive αßT cells in the adult thymus.

αßT cell development depends upon serial migration of thymocyte precursors through cortical and medullary microenvironments, enabling specialized stromal cells to provide important signals at specific stages of their development. Although conventional αßT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αßT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αßT cell lineages, notably the induction of Foxp3(+) regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αßT cells to the medulla is mediated by the chemokine receptor CCR7, how other T cell subsets gain access to medullary areas during their normal development is not clear. In this study, we show that combining a panel of thymocyte maturation markers with cell surface analysis of CCR7 and CCR4 identifies distinct stages in the development of multiple αßT cell lineages in the thymus. Although Aire regulates expression of the CCR4 ligands CCL17 and CCL22, we show that CCR4 is dispensable for thymocyte migration and development in the adult thymus, demonstrating defective T cell development in Aire(-/-) mice is not because of a loss of CCR4-mediated migration. Moreover, we reveal that CCR7 controls the development of invariant NKT cells by enabling their access to IL-15 trans-presentation in the thymic medulla and influences the balance of early and late intrathymic stages of Foxp3(+) regulatory T cell development. Collectively, our data identify novel roles for CCR7 during intrathymic T cell development, highlighting its importance in enabling multiple αßT cell lineages to access the thymic medulla.

Role of CC chemokine receptor 4 in natural killer cell activation during acute cigarette smoke exposure.

Cigarette smoke (CS)-induced lung injury involves innate immune responses. The activation of innate effector cells is thought to require cross talk with dendritic cells (DCs) and macrophages, but the mediators of interaction are unknown. One candidate, CC chemokine receptor 4 (CCR4), is expressed by innate and adaptive effector cells, and its ligands are produced by DCs and macrophages. Using flow cytometry and confocal microscopy, we defined innate responses of lung myeloid DCs, macrophages, and conventional natural killer (NK) cells in mice exposed to CS over 4 days and examined the contribution of CCR4 using CCR4 knockout (CCR4(-/-)) mice. CS affected populations differently, causing an increase in F4/80(+) macrophages, a reduction in parenchymal CD11c(+)CD11b(+)CD103(-) DCs, but no effect on mucosal CD11c(+)CD11b(-)CD103(+) DCs. CS also induced a population of primed/activated CD69(+) NK cells and bronchoepithelial expression of the stress-related NKG2D receptor-activating protein, retinoic acid early transcript 1. CS-exposed CCR4(-/-) mice were similar to controls regarding effects on DCs and macrophages but displayed substantially impaired NK priming/activation and reduced expression of transcripts for interferon gamma, CXCL10, and retinoic acid early transcript 1. Quantitative confocal microscopy revealed that lungs of CS-exposed CCR4(-/-) mice had significantly reduced contacts of NK cells with CD11c(+) cells. These findings demonstrate that acute CS exposure elicits NK cell responses and suggest that CCR4 promotes NK cell priming/activation by mediating contacts with sentinel cells in the lung.

Compartmentalized chemokine-dependent regulatory T-cell inhibition of allergic pulmonary inflammation.

BACKGROUND: Induction of endogenous regulatory T (Treg) cells represents an exciting new potential modality for treating allergic diseases, such as asthma. Treg cells have been implicated in the regulation of asthma, but the anatomic location in which they exert their regulatory function and the mechanisms controlling the migration necessary for their suppressive function in asthma are not known. Understanding these aspects of Treg cell biology will be important for harnessing their power in the clinic. OBJECTIVE: We sought to determine the anatomic location at which Treg cells exert their regulatory function in the sensitization and effector phases of allergic asthma and to determine the chemokine receptors that control the migration of Treg cells to these sites in vivo in both mice and human subjects. METHODS: The clinical efficacy and anatomic location of adoptively transferred chemokine receptor-deficient CD4(+)CD25(+) forkhead box protein 3-positive Treg cells was determined in the sensitization and effector phases of allergic airway inflammation in mice. The chemokine receptor expression profile was determined on Treg cells recruited into the human airway after bronchoscopic segmental allergen challenge of asthmatic patients. RESULTS: We show that CCR7, but not CCR4, is required on Treg cells to suppress allergic airway inflammation during the sensitization phase. In contrast, CCR4, but not CCR7, is required on Treg cells to suppress allergic airway inflammation during the effector phase. Consistent with our murine studies, human subjects with allergic asthma had an increase in CCR4-expressing functional Treg cells in the lungs after segmental allergen challenge. CONCLUSION: The location of Treg cell function differs during allergic sensitization and allergen-induced recall responses in the lung, and this differential localization is critically dependent on differential chemokine function.

The importance of CCR4 and CCR6 in experimental autoimmune encephalomyelitis.

Chemokine receptors (CCRs) play important roles in the pathogenesis of immune-mediated diseases, as well as in normal immune response. We examined the role of CCR6 and CCR4 in experimental autoimmune encephalomyelitis (EAE) by using CCR6(-/-)CCR4(-/-) double knockout (DKO) and single knockout mice. DKO mice developed less severe EAE and presented repressed recall response in the induction phase, especially in the activity of T helper 17 (Th17) cells. CCR6 expression in central nervous system (CNS)-infiltrated cells was diminished in DKO. Our results suggest that CCR6 and CCR4 were involved in a more rapid progression of EAE and that their regulation might be a therapeutic target of human inflammatory demyelinating diseases.

Regulatory T cells migrate to airways via CCR4 and attenuate the severity of airway allergic inflammation.

We have previously shown that regulatory T (Treg) cells that accumulate in the airways of allergic mice upregulate CC-chemokine receptor 4 (CCR4) expression. These Treg cells suppressed in vitro Th2 cell proliferation but not type 2 cytokine production. In the current study, using a well-established murine model of allergic lung disease or oral tolerance, we evaluated the in vivo activity of Treg cells in allergic airway inflammation with special focus on CCR4 function. We found that allergic, but not tolerant, mice treated with anti-CD25 Ab showed increased airway eosinophilia and IL-5- or IL-4-producing Th2 cells when compared with untreated mice. Notably, mice with CCR4 deficiency displayed an augmented airway allergic inflammation compared with wild-type or CCR2 knockout (KO) mice. The allergic phenotype of CCR4KO mice was similar to that observed in anti-CD25-treated mice. The exacerbated allergic inflammation of CCR4KO mice was directly associated with an impaired migration of Treg cells to airways and augmented frequency of pulmonary Th2 cells. Adoptive transfer of CD25(+)CD4(+) T cells expressing high levels of CCR4, but not CCR4KO CD25(+)CD4(+) T cells, attenuated the severe airway Th2 response of CCR4KO mice. Our results show that CCR4 is critically involved in the migration of Treg cells to allergic lungs that, in turn, attenuate airway Th2 activation and allergic eosinophilic inflammation.

Differing requirements for CCR4, E-selectin, and α4ß1 for the migration of memory CD4 and activated T cells to dermal inflammation.

CCR4 on T cells is suggested to mediate skin homing in mice. Our objective was to determine the interaction of CCR4, E-selectin ligand (ESL), and α(4)ß(1) on memory and activated T cells in recruitment to dermal inflammation. mAbs to rat CCR4 were developed. CCR4 was on 5-21% of memory CD4 cells, and 20% were also ESL(+). Anti-TCR-activated CD4 and CD8 cells were 40-55% CCR4(+), and ∼75% of both CCR4(+) and CCR4(-) cells were ESL(+). CCR4(+) memory CD4 cells migrated 4- to 7-fold more to dermal inflammation induced by IFN-γ, TNF, TLR agonists, and delayed-type hypersensitivity than CCR4(-) cells. CCR4(+) activated CD4 cells migrated only 5-50% more than CCR4(-) cells to these sites. E-selectin blockade inhibited ∼60% of CCR4(+) activated CD4 cell migration but was less effective on memory cells where α(4)ß(1) was more important. Anti-α(4)ß(1) also inhibited CCR4(-) activated CD4 cells more than CCR4(+) cells. Anti-E-selectin reduced activated CD8 more than CD4 cell migration. These findings modify our understanding of CCR4, ESL, α(4)ß(1), and dermal tropism. There is no strict relationship between CCR4 and ESL for skin homing of CD4 cells, because the activation state and inflammatory stimulus are critical determinants. Dermal homing memory CD4 cells express CCR4 and depend more on α(4)ß(1) than ESL. Activated CD4 cells do not require CCR4, but CCR4(+) cells are more dependent on ESL than on α(4)ß(1), and CCR4(-) cells preferentially use α(4)ß(1). The differentiation from activated to memory CD4 cells increases the dependence on CCR4 for skin homing and decreases the requirement for ESL.

Epicutaneous challenge of orally immunized mice redirects antigen-specific gut-homing T cells to the skin.

Patients with atopic dermatitis (AD) often suffer from food allergy and develop flares upon skin contact with food allergens. However, it is unclear whether T cells sensitized to allergens in the gut promote this skin inflammation. To address this question, we orally immunized WT mice and mice lacking the skin-homing chemokine receptor Ccr4 (Ccr4-/- mice) with OVA and then challenged them epicutaneously with antigen. Allergic skin inflammation developed in the WT mice but not in the mutants and was characterized by epidermal thickening, dermal infiltration by eosinophils and CD4+ T cells, and upregulation of Th2 cytokines. T cells purified from mesenteric lymph nodes (MLNs) of orally immunized WT mice transferred allergic skin inflammation to naive recipients cutaneously challenged with antigen, but this effect was lost in T cells purified from Ccr4-/- mice. In addition, the ability of adoptively transferred OVA-activated T cells to home to the skin following cutaneous OVA challenge was ablated in mice that lacked lymph nodes. These results indicate that cutaneous exposure to food antigens can reprogram gut-homing effector T cells in LNs to express skin-homing receptors, eliciting skin lesions upon food allergen contact in orally sensitized AD patients.

Role of CCL-2, CCR-2 and CCR-4 in cerulein-induced acute pancreatitis and pancreatitis-associated lung injury.

BACKGROUND AND AIMS: Acute pancreatitis is an inflammatory process of variable severity. Leucocytes are thought to play a key role in the development of pancreatitis and pancreatitis-associated lung injury. The interactions between inflammatory cells and their mediators are crucial for determining tissue damage. Monocyte chemoattractant protein-1 (or CCL-2), CCR-2 and CCR-4 are chemokines and chemokine receptors involved in leucocyte trafficking. The aim of the study was to evaluate the role of the CCL-2, CCR-2 and CCR-4 chemokine receptors in the pathogenesis of cerulein-induced pancreatitis and pancreatitis-associated lung injury. To address the role of CCL-2, CCR-2 and CCR-4 that attracts leucocytes cells in inflamed tissues, pancreatitis was induced by administering supramaximal doses of cerulein in mice that do not express CCL-2, CCR-2 or CCR-4. METHODS: The severity of pancreatitis was measured by serum amylase, pancreatic oedema and acinar cell necrosis. Lung injury was quantitated by evaluating lung microvascular permeability and lung myeloperoxidase activity. Chemokine and chemokine-receptor expression were quantitated by real-time PCR. The nature of inflammatory cells invading the pancreas and lungs was studied by immunostaining. RESULTS: The authors have found that pancreas CCL-2 and CCR-2 levels rise during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in CCL-2, whereas the deficiency in either CCR-2 or CCR-4 does not reduce the severity of both the pancreatitis and the lung injury. The amounts of neutrophils and monocyte/macrophages (MOMA)-2 cells were significantly lower in mice deficient in CCL-2 compared with their sufficient littermates. CONCLUSIONS: These results suggest that CCL-2 plays a key role in pancreatitis by modulating the infiltration by neutrophils and MOMA-2 cells, and that its deficiency may improve the outcome of the disease.